CA2177823C - Poly-beta-1-->4-n-acetylglucosamine - Google Patents

Abstract

A method of producing and purifying poly-.beta.-14-N-acetylglucosamine(p-GlcNAc)polysaccharide species and their derivatives is described. The polysaccharides produced by this method are free of proteins, and substantially free of single amino acids, and other organic and inorganic contaminants. These p-GlcNAc polysaccharides may be used commercially by the biomedical, pharmaceutical, and cosmetic industries in slow drug delivery systems, cell encapsulation systems, and treatments for the prevention of post-surgical adhesions. The figure shows the chemical structure of 100 % p-GlcNAc, wherein "n" is an integer from about 4,000 to about 150,000.

Description

~ WO95/15343 21 77823 PCrl[JS94113706 Poly~ 4 -N-Acetylqluco~m; nP
1. INTRnn~ TIoN
The present invention relates, f irst 1 to a 5 purified, easily produced poly-~-1~4-N-acetylglucoxamine (p-GlcNAc) polysaccharide species.
The p-GlcNAc of the invention is a polymer of high molecular weight whose con3tituent monosaccharide sugars are AttAch~-l in a ~ 4 conformation, and which lO is free of proteins, and substAnti~lly free of single amino acids, and other organic and inorganic ~-nnt ~min;lnt~ . In addition, derivatives and reformulations of p-GlcNAc are described. The present invention further relates to methods for the 15 purification of the p-GlcNAc of the invention from microalgae, preferably diatom, starting sources.
Still further, the invention relates to methods for the deriv~ti7at;r-n and reformulation of the p-GlcNAc.
Additionally, the present invention relates to the 20 uses of pure p-GlcNAc, its derivatives, and/or its ref ormulations .

2. BAC~9ROrlNn OF ~ NVENTION
There exists today an extensive literature on the 25 properties, activities, and uses of polysaccharides that consist, in part, of p-GlcNAc. A class of such materials has been generically referred to as "chitin", while deacetylated chitin derivatives have been referred to as "chito3an". When these terms were 30 first used, around 1823, it was believed that chitin and chitosan always occurred in nature as distinct, well-defined, unique, and invariant chemical species, with chitin being fully acetylated and chitosan being fully deacetylated compositions. It was approximately

3'i a century later, however, before it was discovered W095115343 2 1 7 7 8 2 3 PCTIUS94/13706 ~

that the terms 'Ichitin" and "chitosan" are, in fact, very ambiguous. Rather than re~erring to well-defined compounds, these terms actually refer to a family of compounds that exhibit widely di~ering physical and S chemical properties . ~hese dif ferences are due to the products ' varying molecular weights, varying degrees o_ acetylation, and the presence of cnnt~m; nAnts such as covalently bound, species-specific proteins, single amino acid and inorganic cl~nt~min~nts~ Even today, 10 the terms "chitin" and "chitosan" are used ambiguously, and actually refer to poorly defined mixtures of many diferent compounds.
For example, the properties of "chitins" isolated from conventional sources such as crustacean outer 15 shells and fungal mycelial mats are unpredictably variable. Such variations are due not only to species differences but are also due to varying envi. ~ t;?l and seasonal e~ects that determine some of the biochemical characteristic3 of the "chitin"-producing 20 gpecies. In fact, the unpredictable variability of raw material is largely responsible for the 610w growth of chitin-based industrie3.
No reports exist today in the 2~ i Pnt i f; c literature ~ cr;h;ng the isolation and prc~ tinn, 25 from material sources, of pure, fully acetylated p-GlcNAc, i~, a product or products llncnnt~m;n~ted by organic or inorganic impurities. While Mc~.achlan et al. (Mc~achlan, A.G. et al., 1965, Can. J. Botany 43:707-713~ reported the isolation of chitin, 30 subsequent studies have shown that the "pure~
substance obtained, in fact cnntz~;nPrl proteins and other cnnt~m;n~nt~.
Deacetylated and partially deacetylated chitin preparations exhibit potentially beneficial chemical 35 properties, such as high reactivity, dense cationic charges, powerful metal chelating capacity, the ability to covalently attach proteins, and solubility in many aqueous solvents. The unpredictable variability of these preparations, as described above, however, severely limits the utility of these heterogenous compounds. For example, the currently available "chitins" and "chitosans~ give rise to irreproducible data and to unacceptably wide variations in experimental results. Additionally, the available preparations are not sufficiently homogenous or pure, and the preparation constituents are not sufficiently reproducible for these preparations to be acceptable for use in applications, especially in medical ones. Thus, although extremely desirable, a true, purified preparations of chitin and chitosan, whose properties are highly reproducible and which are easily manufactured, do not currently exist.
3. SllMMARY OF T~T~ INVENTIQN
The present invention relates, irst, to an isolated, easily produced, pure p-GlcNAc species. The p-GlcNAc of the invention is a polymer of high r l ec~l Ar weight whose constituent monosaccharides are attached in a ,~-1~4 conformation, and which is free of 25 proteins, substAntiAlly free of other organic cl ntAm;nAnts, and substantially free of inorganic cont Am; nAnt ~ .
The importance of the present invention resides in the fact that the problem of unpredictable raw 30 material variability has been overcome. It is, for the first time, possible to produce, by simple means, and on a commercial scale, biomedically pure, p-GlcNAc of high molecular weight and consistent properties.
The material produced in the present invention is 35 highly crystalline and is produced from carefully -WO 95115343 2 1 7 7 8 2 3 PCT/US94113706 ~

controlled, aseptic cultures of one of a number of marine microalgae, preferably diatoms, which have been grown in a defined medium.
The present invention further describes derivatives and reformulations of p-GlcNAc as well as methods for the production of such derivatives and reformulations. Such derivatizations may include, but are not limited to polygl~ n~mi nP and its derivatives, and such reformulations may include, but are not limited to membranes, f;l -ntq, non-woven textiles, sponges, and three dimensional matriceY.
Still further, the present invention relates to methods for the purif ication of the p-GlcNAc of the invention from microalgae, preferably diatom, fiources.
~lrl;tinn;~lly, the present invention relates to the uses of the purified p-GlcNAc, its derivatives, and/or its reformulations. Among these uses are novel commercial applications relating to such industries as the biomedical, pharmaceutical, and cosmetic industries, all of which require starting materials of the highest desree of purity. For example, the p-GlcNAc materials of the invention may be formulated to exhibit controllable biodegradation properties, and, further, may be used as part of slow drug delivery systems, as cell ~n~-~rs~ tion systems, and as treatments for~the prevention of post-surgical adhesions .

4. BRIEF DESCRIPTION OF TTTF. FIçrr~F~
FIG. l. Chemical structure of 100% p-GlcNAc.
~n~ refers to an integer ranging from about 4,000 to about 150,000, with about 4,000 to about 15,000 being pref erred .

FIG. 2. Carbohydrate analysis of p-GlcNAc, Gas Chromatography-Mas~ Spectroscopy data. Solid squares represent p-GlcNAc purified using the acid treatment/neutralization variation of the

5 Chemical/Bio~ogical method, as described in Section 5.3.2, below.
FIG. 3A. Circular dichroism spectra of solid membranes of pure p-GlcNAc.
FIG. 3B. Circular dichroism spectra of solid membranes of Deacetylated p-GlcNAc. The disappearance of the 211 nm minimum and 195 nm maximum observed in pure p-GlcNAc (FIG. 3A) indicates complete 15 Deacetylation under the conditions used, as described in Section 5 . 4 below .
FIG. 4A. Infra-red spectra analyses of thin membranes of pure diatom p-GlcNAc prepared by the 20 mechanical force purification method, top, and the chemical/biological purification method, bottom.
FIG. 4B. Infra-red spectra analyses of two preparations of commercial "chitin" cast into 25 membranes according to the methods detailed in Section 5 . 5, below.
FIG. 4C. Infra-red spectra analyses of pure p-GlcNAc which was modified by heat denaturation (top) 30 and by chemical deacetylation (bottom), according to the methods detailed in Section 5 . 4, below.
FIG. 4D. Infra-red spectrum analysis of a p-GlcNAc membrane derived from the diatom ~h~ iosira 35 fluviatilis, using the chemical/biological WO 95115343 2 1 7 7 8 2 3 PCTIUS94/13706 ~

purification method, as detailed in Section 5.3.2, below .
FIG. 4E. Infra-red spectrum analysis of a p-S GlcNAc membrane prepared by the qn 1 CA 1 forcepurification method, as described in Section 5.3.1, below, following autoclaving.
FIG. 5A. NMR analysis of p-GlcNAc purified using 10 the chemical/biological purif ication method as described in Section S . 3 . 2, below. Chart depicting peak amplitudes, areas, and ratios relative to reference controls. Ratio of total areas of peaks.
FIG. SB . NMR analysis of p-GlcNAc purif ied using the ~h~qmi~Al/biological purification method as described in Section S . 3 . 2 . The graph depicts the ratios of total areas of peaks.
FIG. 6A-6B. TrAnc~csion electron mi-;~u~ phs (TEM) of a p-GlcNAc membrane prepared by the -- ~ n;~-Al force purification method as described in Section 5.3.1, below. Magnification: 6A: 4190x; 6B:
16, 250x.
FIG. 7A-7B. Transmission electron mi~;luyL~lJ,hs ~TEM) of a p-GlcNAc membrane by HF l .eai 1 as described in the ~licc~csion of the rh~ rAl/biologica purif ication method in Section 5 . 3 . 2, below.
Magnification: 7A: 5270x; 7B: 8150x.
FIG. 8A-8B. Transmission electron micrographs (TEM) of a p-GlcNAc membrane prepared by the acid Ll~ai t/neutralization variation of the ~-hq~nicAl/biological purification method, as described RECllFIED SHEET (RULE 91) ln Section 5.3.2, below. Nagnification: 8A: 5270x;
8B: 16, 700x.
FIG . 9A. ScAnn i n~ electron mi-,L O~L a~h depicting 5 a p-GlcNAc membrane prepared by the acid treatment/neutralization variation of the rhc~m;c:~l/biological purification method as described in Section 5 . 3 . 2, below.
Magnification: 200x.
FIG. 9B. Scanning electron mi-:LU~la~l~ depicting a p-GlcNAc membrane prepared by the acid treatment/neutralization variation of the chemical/biological purification method as described 15 in Section 5 . 3 . 2, below.
Magnif ication: lOOOx.
FIG. 9C. Sr~nning electron mi~LuyLaph depicting a p-GlcNAc membrane prepared by the acid 20 treatment/neutralization variation of the chemical/biological purification method as described in Section 5 . 3 . 2, below.
Magnif ication: 5 0 0 Ox .
FIG. 9D. Scanning electron mic-uyL~ depicting a p-GlcNAc membrane prepared by the acid treatment/neutralization variation of the rhPmic:~/biological purification method as described in Section 5 . 3 . 2, below.
30 Magnification: lO,OOOx.
FIG. 9E. Scanning electron micrograph depicting a p-GlcNAc membrane prepared by the acid treatment/neutralization variation of the RECrIFIED SHEET (RULE 91) -WO 95/15343 2 1 7 7 8 2 3 P,~/US94,l3706 ~

chemical/biological purif ication method as described in Section 5.3.2, below. Magnificatlon: 20,000x.
FIG. lOA-lOB. Sc~nnin~ electron mi~iLl~LClph6 of a 5 pure p-GlcNAc membrane made from material which waæ
initially ~L~duced using the cell dissolution/neutralization purification method described in Section 5 . 3, below, dissolved in dimethylacetamide/lithium chloride, and 10 reprecipitated in H20 into a mat, as described below in Section 5.5. M;-~n;fi~Ation: lOA: lOOOx; lOB: lO,OOOx.
FIG. llA-llB . S~Ann i ng electron micrographs of a deacetylated p-GlcNAc mat. Magnification: llA:
15 lOOOx; llB: lO,OOOx.
FIG. 12A-12B. Photographs of diatoms. Note the p-GlcNAc f iber6 extending from the diatom cell bodies .
FIG. 13. Diagram depicting some of the possible p-GlcNAc and deacetylated p-GlcNAc derivatives of the invention. (Adapted from S. Hirano, "Production and Application of Chitin and Chitosan in Japan", in "Chitin and Chito6an", 1989, Skjak-Braek, Anthonsen, and Sanford, ed6. Elsevier Science Publishing Co., pp . 37-43 ) FIG. 14. Cell viability study of cells grown in the plesel~ce or absence of p-GlcNAc membranes. Closed circle (-): cells grown on p-GlcNAc matrix; open circles tO): cells grown in absence of matrix.
FIG. 15A-15B. SEM mi~:Lo~ I.s of transformed mouse f ibroblast cells grown on p-GlcNAc membranes .
Magnification: 15A: lOOOx; 15B: 3000x.
REt,TlFlED SHEET (RULE 91) Wo 95/15343 2 1 7 7 8 2 3 PCr/USs4/13706 _ g _ FIG. 16A. Sc:~nnin~ eleetron mi~Lv~Lc~h (SEM) of a eollagen-only eontrol material pLeL,~red aeeording to the method deseribed, below, in Seetion 13.1.
Magnif ieation lOOx .

FIG. 16B. Seanning eleetron mi~Lvyl~h (SEM) of a eollagen/p-GleNAe hybrid material prepared aeeording to the method deseribed, below, in Seetion 13.1.
Ratio eollagen suspension:p-GleNAe suspension equals 10 3:1, with final ~vl.eel-LL~ tions of 7.5 mg/ml eollagen and 0. 07 mg/ml p-GleNAe. Magnifieation lOOx.
FIG. 16C. ~I~Ann;n~ eleetron mierograph tSEM) of a eollagen/p-GleNAe hybrid material prepared aeeording 15 to the method deseribed, below, in Seetion 13.1.
Ratio eollagen suspension:p-GleNAe suspension equals 1:1, with f inal l,VIIC~ L c,tions of 5 . O mg/ml eollagen and 0.12 mg/ml p-GleNAe. Magnifieation lOOx.
FIG. 16D. SeAnn;n~ eleetron mi~:LVyLCI~ll (SEM) of a eollagen/p-GleNAc hybrid material pL epa~ ed aeeording to the method deseribed, below, in Seetion 13.1.
Ratio collagen s-~apDnRinn:p-GlcNAc suspension equals 2:2, with final cc,l~cc..~L~tions of 10.0 mg/ml collagen 25 and 0.25 mg/ml p-GlcNAc. Magnification lOOx.
FIG. 16E. SeAnnin~ electron micrograph (SEM) of a collagen/p-GlcNAc hybrid material ~JL epar~d according to the method described, below, in Section 13.1.
30 Ratio eollagen suspension:p-GleNAc suspension equals RECrIFIED SHEET (RULE 91) Wo 95115343 2 1 7 7 8 2 3 Pcrrusg4rl3706 ~

1: 3, with f inal concentrations o 2 . 5 mg/ml collagen and 0.25 mg/ml p-GlcNAc. Magnification lOOx.
FIG . 17A. SEM of mouse 3T3 f ibrobla2it cells 5 cultured on the collagen-only control material of FIG.
16A, above. Magnification lOOx.
FIG 17B . SEM of mouse 3T3 f ibroblast cells cultured on the collagen/p-GlcNAc material of FIG.
10 16B, above. Magnification lOOx.
FIG . 17C. - SEM of mouse 3T3 f ibroblast cells cultured on the collagen/p-GlcNAc material of FIG.
16C, above. Magnification lOOx.
FIG . 17D . SEM of mouse 3T3 f ibroblast cells cultured on the collagen/p-GlcNAc material of FIG.
16D, above. Magnification lOOx.
FIG. 18. Transformed NMR data curves, used to obtain areas for each carbon atom and to then calculate the CH3 (area) to c-atom~area) ratios.
FIG. 19. Typical p-GlcNAc Cl3-NMR spectrum. The individual peaks L~Le:S~'lt the contribution to the spectrum of each unique carbon atom in the molecule.
FIG. 20. Transformed NMR spectrum data representing values calculated for CH3 (area) to C-atom(area) ratios. Top: Graphic depiction of data;
bottom: numerical depiction of data.
FIG. 21A-G. Three dimensional p-GlcNAc matrices produced in various solvents. Specifically, the p-GlcNAc matrices were produced in distilled water (FIG.

~ WO95/1~343 21 77823 PCr/13S94113?06 21A, FIG. 21D), 109~ methanol in distilled water (FIG.
21B), 2596 methanol in distilled water (FIG. 21C), 109~
ethanol in distilled water (FIG. 21E), 259~ ethanol in distilled water (PIG. 21F) and 40~ ethanol in 5 distilled water (FIG. 21G). MA~n;f;~Ation: 200x. A
6cale marking of 200 microns is indicated on each of these f igures .
FIG. 22A-G. Fibroblast cells grown on three 10 dimensional p-GlcNAc matrices prepared by lyophilizing p-GlcNAc in distilled water. Magnification: lOOx (FIGS. 22A, 22E), 500x (FIG. 22B), lOOOx (FIGS. 22C, 22F), 5000x (FIGS. 22D, 22G) . Scales marking 5, 20, 50, or 200 microns, as indicated, are ;nr~ d in each 15 of the figures.
FIG. 23. A typical standard curve obtained using the procedure described, below, in Section 18.1. A
standard curve such as this one was used in the 20 lysozyme-chitinase assay also described, below, in Section 18.1.
FIG. 24. p-GlcNAc lysozyme digestion data. The graph presented here depicts the At-l_ 1 Ation of N-25 acetylgl~l~ oci-m;n~ over time, as p-GlcNAc ~~ ' ldlles are digested with lysozyme. The graph compares the degradation rate of fully acetylated p-GlcNAc to partially (50%) deacetylated p-GlcNAc, and demonstrates that the degradation rate for the 30 partially deacetylated p-GlcNAc was subst~nt;Ally higher than that of the fully acetylated p-GlcNAc material .
FIG. 25. p-GlcNAc lysozyme digestion data. The 35 graph presented here depicts the A~ Ation of N-WO 95115343 2 1 7 7 8 2 3 PCT/US94/13706 ~

acetyl~ r~s~m;n~ over time, as p-GlcNAc membranes are digested with lysozyme. The graph compares the degradation rate of two partially deacetylated p-GlcNAc membranes (specifically a 25~ and a 50~
5 deacetylated p-GlcNAc membrane). The data demonstrate that the deg~tl~lti~n rate increases as the percent of deacetylation increases, with the ~ tion rate for the 50& deacetylated p-GlcNAc membrane being substantially higher than that of the 25~ deacetylated 10 p-GlcNAc membrane .
FIG. 26A-26E. p-GlcNAc in vivo biodegradability data FIG. 26A-26G depict rats which have had prototype 1 (fully acetylated p-GlcNAc) mem.brane 15 ~h~nm;n~1 ly implanted, as described, below, in Section 18.1. FIG. 26A shows a rat at day 0 of the implantation; FIG. 26B shows a rat at day 14 post-implantation; FIG 26C shows a rat at day 21 post-impl~nt~t;~ FIG. 26D-26E depict rats which have had 20 prototype 3A (lyophilized and partially deacetylated p-GlcNAc membrane) ~`~ ' n~l 1 y implanted, as described, below, in Section 18.1. FIG. 26D shows a rat at day 0 oE the implantation; FIG. 26E shows a rat at day 14 post-implantation.
FIG. 27. The graph depicted here illustrates data ~n~o~n;n~ the percent increase in tumor size of animala which either received no treatment (--) or received p-GlcNAc-lactate/5'Flurouracil (EU) (O), as 30 described, below, in Section 20.1.
FIG. 28. The graph depicted here illustrates data con~ l~n;n~ the perc~nt increase in tumor size of animals which either received p-GlcNAc-lactate alone -~ WO95/15343 2 1 7 7 8 23 PCTIUS94/13706 (--) or received p-GlcNAc-lactate/5 ' Flurouracil (FU) (O), as described, below, in Section 20.1 FIG. 29. The graph depicted here illustrates 5 data concerning the percent increase in tumor size of - animals which either received no treatment (--) or received p-GlcNAc-lactate/mitomycin (mito) (O), as described, below, in Section 20 . l .
FIG . 3 0 . The graph depicted here illustrates data rnn,-Prnin~ the percent increase in tumor size of animals which either received p-GlcNAc-lactate alone (--) or received p-GlcNAc-lactate/5' mitomycin (mito) (O), as described, below, in Section 20 . l .
FIG. 31 The bar graph depicted here illustrates the average percent change in tumor size per animal of animals treated with p-GlcNAc/5'FU high dose (bar l), p-GlcNAc/5'FU low dose (bar 2), p-GlcNAc membrane 20 alone (bar 3), and untreated (bar 4) . N=4 for bars l and 2, n=2 for bars 3 and 4.
5. DETATT.T'.n n~ RTpTIcN OF THE INVENTI3N
Presented below, is, f irst, a description of 25 physical t h~r~- t~ristics of the purified p-GlcNAc species of the invention, of the p-GlcNAc derivatives, and of their ref ormulations . Next, methods are ~ 5~rih~ for the purification of the p-GlcNAc species of the invention from microalgae, preferably diatom, 30 starting sources. Third, derivatives and reformulations of the p-GlcNAc, and methods for the prn~ 't i nn of such derivatives and re~ormulations are presented. Finally, uses are presented for the p-GlcNAc, p-GlcNAc derivatives and/or p-GlcNAc 35 refo,, l~t;nnR of the invention.

WO 95/15343 2 1 7 7 8 2 3 P~IUS94113706 ~

5 .1 l~ - Gl cNAc The p-Glc~c polysaccharide species of the 5 invention is a polymer of high molecular weight ranging from a weight average of about 800, 000 daltons to about 30 million daltons, based upon gel permeation chromatography measurements. Such a molecular weight range represents a p-GlcNAc 3pecies having about 4, 000 to about 150,000 N-acetylgl-~rn~m;nP monosaccharides attached in a ,~-1~4 configuration, with about 4,000 to about 15, 000 N-acetylglllrns;lm; n~ monosaccharides being preerred ~FIG. 1) .
The variability of the p-GlcNAc of the invention 15 is very low, and its purity is very high, both of which are evidenced by chemical and physical criteria.
Among these are chemical compo3ition and non-polysaccharide rnn~min~n~R First, chemical composition data for the p-GlcNAc produced using two 20 different purification methods, both of which are described in Section 5.3, below, is shown in Table I
below. As can be seen, the chemical composition of the p-GlcNAc produced by both methods i8, within the bounds of experimental error, the same as the formula 25 compositions o~ p-GlcNAc. Second, as is also shown in Table ~, the p-GlcNAc prQduced is free of detectable protein rr,n~Am;n~nts, is subst~n~;~lly free of other organic cnnt~m; n~n~R such as free amino acids, and is substantially free of: inorganic contaminants such as 30 ash and metal ions (the p-GlcNAc of the invention may contain up to about 0 . 05~ trace metals) . Further, the p-GlcNAc of the invention exhibits a very low percentage of bound water WO 95115343 2 1 7 7 8 2 3 PCrlUS94/13706 TA'8LE I
CHEMICAL ANAT~YSIS DATA (~ bY weiaht) Theoretical Values for Pu~e D-GlcNAc:
Carbon - 47 . 29 ~Iydrogen - 6 . 4 0 Nitrogen - 6 . 89 OxYgen - 3 9 . 41 10 Protein - o . oo ExDerimental Data on D-GlcNAc Mat8:
(Number of experimental batches for each membrane type being greater than 30 for each membrane type) MECT~ANT0'Ar~ FORCE ~ rl-/BIol,oGIt~Tl -Normalized 1 ~ Dev. Normalized I ~ Dev.
Carbon 47.21 + 0.08 -0.17 47.31 + 0.11 +0.04 E~ydrogen 6.45 0.08 +0.78 6.34 + 0.08 -0.94 Nitrogen 6.97 + 0.18 +0.87 6.94 + 0.16 +0.73 20Oxygen 39.55 + 0.36 +0.36 39.41 + 0.10 0.00 Average Values Average Values Protein 0 . 00 o . 00 Ash 1.30 0.98 Moisture 2 . 0 1. 2 25 1 Raw analytical data have bee~ normalized to account for ash and moisture content of the samples.
The pure p-GlcNAc of the invention exhibits a carbohydrate analysis profile 8ubst~n~;~11y similar to 30 that shown in FIG. 2. The primary monosaccharide of the pure p-GlcNAc of the invention is N-acetylgl~ m; n~ . Further, the pure p-GlcNAc of the invention does not contain the monosaccharide glucosamine .

WO95/~5343 21 77823 PCr/US9~113706 ~

The circular dichroism (CD~ and sharp infra-red spectra (IR) of the p-GlcNAc of the invention are shown in FIGS. 3A, and FIGS. 4A and 4D, respectively, which present analyses of material ~L~duced using the 5 methods described in Section 5 . 3, below. Such physical data corroborates that the p-GlcNAc of the invention is of high purity and crystallinity. The methods used to obtain the CD and IR data are described, below, in the Working Example in Section 6.
NNR analysis of the pure p-GlcNAc of the invention exhibits ~ pattern substantially similar to that seen in FIGS. 5A, 5B, 18A and 18B. Such an NNR
pattern indicates not only data which is consistent with the p-GlcNAc of the invention being a fully acetylated polymer, but also i' ~L~lLes the lack of contaminating organic matter within the p-GlcNAc species .
The electron micrographic ~-u~ LuLe of the p-GlcNAc of the invention, as produced using the methods described in Section 5 . 3, below and ~ ted in the Working Examples presented, below, in Section 8 and 9, i8 depicted in FIGS. 6A through FIG. 9E.
The p-GlcNAc of the invention exhibits a high degree of biocompatability . Bic ~ _t~bility may be determined by a variety of techniques, incl~ in~ but not limited to such procedures as the elution test, lAr implantation, or intracutaneou5 or systemic injection into animal subjects. Briefly, an elution test (U.S. Pharmacopeia XXII, 1990, pp. 1415-1497; U.S. Pharmacopeia XXII, 1991, Supplement 5, pp.
2702-2703) is designed to evaluate the biocompatability of test article extracts, and assays the biological reactivity of a r-r~-ol iAn cell culture line which is sensitive to extractable cytotoxic articles (such as, for example, the L929 cell line) in RECrIFIED SHEET (RULE 91) WO 95/15343 -- 17 -- PCrNSs4/l3706 response to the test article. The Working Example presented in Section 10, below, ~ ~Lcltes the high bi~ tability of the p-GlcNAc of the invention.
5 . 2 METHODS OF ~KODLIclN~i MIrRr~Ar~r SOURCES QF t~-GlcNAc 5 . 2 .1 MTrr~r,AT.r~T. SOURCES OF ~--GlrNAr The p-GlcNAc of the invention is produced by, and 10 may be purified from, microalgae, preferably diatoms.
The diatoms of several genuses and numerous species within such genuses may be utilized as p-GlcNAc starting sources. Each of these diatoms produce fiber6 ~ -sQ~ of p-GlcNAc which extend from their 15 cell bodies . See FIG . 12A-12B f or photographs of such diatoms. The diatoms which may be used as starting sources for the production of the p-GlcNAc of the invention include, but are not limited to members of the Coscinn~ cl~c genus, the Cyclotella genus, and the 20 Thalassiosira genus, with the Thalassiosira genus being pref erred .
Among the rosr~nn~l;ccl~c genus, the species of diato~ that may be used to produce the p-GlcNAc of the invention include, but are not limited to the 25 r~rnrinm~C and radiatus species. The diatoms among the Cyclotella genus which may be used include, but are not limited to the caspia, cryptica, and - ~ i n~ AnA
species. The Thalassiosira diatoms that may be utilized to produce the starting material for the p-30 GlcNAc of the invention include, but are not limitedto the nitzschoides, aestivalis, antarctica, ~3er~ ir~f-nc, ecc~--l Lica, floridana, fluviatilis, gr21vida, guillardii, hyalina, minima, nordenskioldii, oceanica, polychorda, pc~ nnAn~; rotula, tubifera, 35 tumida, and weissflogii species, with the fluviatilis and weissf logii species being preferred.
RECTIFIED SHEET (RLJLE 91~

WO 95/15343 2 t 7 7 8 2 3 PCrlUS941~3706 ~

Diatoms such as those described above may be obtained, for example, from the culture collection of the Bigelow ~aboratory for Ocean Sciences, Center for Collection of Marine Phytoplankton (McKown Point, West Boothbay Harbor, Maine, 04575).
5 . 2 . 2 METHODS ~OR GROWING DIATOMS
Any of the diatoms described in Section 5 . 2 . l, above, may be grown by utilizing, for example, the methods described in this section. New diatom cultures are initiated by inoculating, under sterile conditions, Nutrient Medium with an aliquot of a mature diatom culture. The Nutrient Medium must be free of all other microorganisms, th,or~fnre all materials, including water, organic components, and inorganic ~ ~ ^nts used in the preparation of the Nutrient Medium must be sterile. In addition, it is mandatory that all procedures involved in this operation be conducted under strictly sterile conditions, e., all containers, all transfers of substances from one vessel to another, etc. must be performed in a sterile environment. The quantity of Nutrient Medium to be prepared at one time should not exceed what is n~rr~s~ry to start a new culture. ~or example, Fernbach flasks which occupy approximately one square foot of surface may be used as vessels for the diatom cultures, and such vessels require one liter of Nutrient Medium for optimum growth of the diatom organism.
Preparation of the nutrient medium involves the following operations:
a) Acquisition and processing of seawater b) Preparation of distilled and deionized water .
c) Preparation of primary nutrient stocks WO 95/15343 2 1 7 7 8 2 3 PCTIUS9~113706 d) Preparation of nutrient working stocks e) Preparation of the f inal nutrient medium Filtered seawater may be obtained, for example, from the Marine Biology Laboratory (Woods Hole, 5 Massachusetts). Seawater cnnt~;n~rs should be stored - at 5 C. When required, the necessary volume of water may be filtered through a Buchner filtration unit, using a nitrocellulose filter membrane with 0.45 micron pore size (Millipore, Inc. ) . The seawater is 10 then sterilized by autoclaving at, for example, 121 C.
for 15 minutes per liter. On completion of the sterilization process, the capped are immediately cooled, preferably by transfer to a cold room capable of allowing the solutions to reach a temperature of 15 approximately 5 C. When it is to be used, solutions are allowed to reach room temperature.
Tap water is distilled and deionized using standard equipment and procedures, and collecte~ and ~tored in sterile, securely capped, preferably glas6, 20 rnnt~; nP~s .
~ isted below are formulas which may be followed in preparing the stock solutions necessary for the preparation of the Nutrient Medium. It is to be understood that while such formulas are to be used as 25 guides, it is intended that routine variations of such formulas which contribute to the preparation of a Nutrient Medium capable of sustaining microalgal diatom growth sllff;~ nt for the p-GlcNAc preparative processes described here also be within the scope of 3 0 the present invention .
I. Trace Metal PrimarY Stocks (TMPS) a. 39 mM CuS04' 5H20 (copper [II] sulfate pentahydrate) (9 . 8g copper [II] sulfate/L) Wo 95115343 2 1 7 7 8 2 3 PCTNS94/13706 b. 7.5 mM ZnSO; 7H20 (Zinc sulfate heptahydrate) (22g zinc sulfate/L) c. 42 mM CoClz 6H20 (Cobalt rII] chloride hexahydra t e ) ( 10 g cobalt [ I I ] chl ori de /L ) d. 91 mM MnCl,- 4H20 (Manganese [II]
chloride tetrahydrate) 18g - n~n~e [II] chloride/L) e . 26 mM NaMoO~ . 2H,O (Sodium molybdate dihydrate) 6 . 3g sodium molybdate/L) f. 153.5 mM H,SeO3 (Selenious acid) (12.9g selenious acid/L) .
Sterile filter each nutrient with a filter of no greater than O . 2mm pore size.
II. Vitamin Primarv Stocks (VPS) a. 1 mg/ml Vitamin B12 b. o.1 mg/ml Biotin Sterilc ~ilter both stocks with a filter of no greater than o . 2mm pore size .
III. Sorlium Salts Workinq Stock~ (SSWS) a . Sodium nitrate working stock: 0 . 88 M
(75 g NaNO3/L) b. Sodium phosphate monobasic monohydrate working stock: 36.2 mM NaHzPO~ HzO (5 g NaH,PO~ H20/L) c. Sodium metasilicate nonahydrate working stock: 0.11 M Na2SiO3 9H20 (30 g Na2SiO3 9H20/L) Sterile filter each of the SSWS with a filter of no greater than O . 2mm pore size .
IV. Trace Metal Workinq Stocks (TMWS) 11. 7 mM Na2EDTA (sthylenediamine Tetraacetic acid, disodium salt dihydrate) (4 . 36 g/L~
11.7 mM FeCl3 6HzO (Iron [III] chloride hexahydrate) (3.15 g/L) 1 ml/~ of each of the six primary trace metal stocks listed above Sterile f ilter with a f ilter of no greater than 0 . 2mm pore size. Note that the trace metal working stock 5 must be prepared fresh each time a new Nutrient Medium is assembled.
V. Vitamin Work;nq Stock (VWS) 1. O llg/ml Biotin (1. O ml primary Biotin 10 Stock/100 ml) 1. O llg/ml Vitamin B12 ( O .1 ml Vitamin B12 primary stock/100 ml) 20 mg of Th;~minf~ HCl (Thiamine hydrochloride/100 ml).
15 Sterile filter with a filter of no greater than 0.2mm pore size. Note that a new Vitamin ~orking Stock should be prepared fresh every time a new nutrient medium is being assembled.
Described below are techniques which may be followed for the preparation of Nutrient Medium and for diatom culturing. It is to be understood that, in addition to these techniques, any routine variation in the formulas and/or procedures described herein which result in a Nutrient Medium and in procedures capable of sustaining diatom growth sufficient for the preparative processes described herein is ; n~n~i~d to be wi~hin the scope of the present invention.
Nutrient Medium may be prepared, for example, as follows: To each liter of filtered and sterilized seawater may be added 1 ml of the NaNO3 working stock, 1 ml of the NaH2PO4 H20 working stock, 1 ml of the Trace Metal working stock, and 1 ml of the Na2SiO3 9H2O
working stock. Simultaneously with the addition of 35 Na2SiO3 9H2O, 2 mls of 1 N HCl may be added and the WO 95/15343 2 1 7 7 8 2 3 PCrlUS94113706 solution may be shaken to mix. Next, 1. 5 mls 1 N NaO~I
may be added and the solution may again be shaken to mix. Finally, O . 5 ml of the Vitamin working stock may be added.
In order to grow a new diatom culture, 7 ml of a mature culture, (having a cell density of approximately 1 x 105 cells/ml), may be transferred to a sterile ~nntA;n~ nntA;n;n~ 100 ml o~ sterile Nutrient Medium, which may be prepared according to the methods described above. The inoculated culture may then be incubated for 8 days under the following conditions:
Temperature: 20 degrees Centigrade Con3tant illumination.
Agitation: Gentle swirling of flasks once for two or three seconds every morning and every evening .
After 8 days of ;nrl1hat;nn, 80 ml of this incubated culture may be transferred, under sterile conditions, to 1000 ml of ~utrient Medium, which may, for example, be cnntil;n~rl in a 2.8 L Fernbach ~lask, protected by a cotton wool plug covered by cheesecloth. Such a culture may be allowed to incubate and grow to the desired cell density, or alternatively, may be used to inoculate new diatom cultures. Once a culture reaches a desired cell density, the culture' 3 p-GlcNAc f ibers may be harvested, and the p-GlcNAc of the invention may be purified, using me~hods such as those described below in Section 5.3, below.
Co2 may be dissolved in the culture solution in order to r-;nt~in a culture p~ of approximately 7 to 8, with approximately 7 . 4 being pref erred . The maintenance of such a neutral pH environment, greatly WO 95/15343 2 1 7 7 8 2 3 PCTIUS94/~3706 increa3es the p-GlcNAc yield that may be obtained from -~
each diatom culture.
5.3 METHODS FOR ISOLATION, PURIFICATION, AND
~ONc~;N1KATION OF ~-GlcNAc FIBBRS
Presented in this Section are methods which may be utilized for the preparation of p-GlcNAc fibers from diatom cultures such as those described, above, in Section 5 . 2 .
While each of the methods described below for the purification of p-GlcNAc from microalgae, preferably diatom, starting sources produces very pure, unadulterated, crystalline p-GlcNAc, each of the methods yields p-GlcNAc having specific characteristics and advantageous features. For example, the p-GlcNAc of the lnvention purified via the Mechanical Force method presented in Section 5 . 3 . l, below, produces a p-GlcNAc membrane that provides a superior substrate for the attachment of cells to the p-GlcNAc. The second method, described below in Section 5 . 3 . 2, the Chemical/Biological method, produces a much higher average yield than the average p-GlcNAc yield produced by the Merh~ni r~l Force method. Additionally, the acid treatment/
neutralization variation described as part of the Chemical/Biological method of Section 5.3.2, below, produces extremely long p-GlcNAc fibers, with some fibers being in excess of l00 llm, and of very high molecular weight, as high as 20-30 million daltons.

5 . 3 . l MBCHANICAL FORCE METHOD FOR PREPARATION
OF PURE ~-GlcNAc The p -GlcNAc f ibers may be separated f rom diatom cell bodies by subjecting the contents of the culture to an d~ L iate mechanical f orce . Such a mechanical force may include, but is not limited to, a shear Wo 95/15343 2 1 7 7 ~ 2 3 PCTIUS94113706 force generated by, for example, a colloid mill, an ultrasound device, or a bubble generator, or a cutting force generated by, for example, a Waring blender.
The resulting suspension of diatom cell bodies 5 and p-GlcNAc fibers are then segr~gated. For example, the suspension may be subjected to a series of centrifugation steps which segregate the p-GlcNAc f ibers f rom the cell bodies, yielding a clear supernatant exhibiting little, if any, visible lO flocculent material. A fixed angle rotor, and a temperature of about 10 C. are preferred for the centrifugation steps. The speed, duration, and total number of centrifugation steps required may vary ~r~nrl;ng on, for example, the speciiic centrifugation 15 rotor being used, but the determination of the values for such parameters will be apparent to one of ordinary r3kill in the art.
The p-GlcNAc fibers in the supernatant may then be ~r-n~-~nt~ated using techniques well known to those 20 of skill in the art. Such techniques may include, but are not limited to suction and filtration devices.
Finally, the c ~n~ ~nt~ated p-GlcNAc f ibers are washed with, for example, distilled-deionized water, HCl and ethanol, or other appropriate solvents, 25 preferably solvents, such as i31~)h.l1~, in which both organic and inorganic materi~ olve.
The Working Example presented in Section 7, below, demonstrates the use of this method for the purification of p-GlcNAc.

5 3 . 2 . C~EMICAL/BIO~OGICAl~ METHOD FOR
PURIFICATION OF D-GlcNAc In this method, p-GlcNAc fibers are separated f rom diatom cell bodies by sub; ecting them to chemical 35 and/or biological agents as described in more detail below .

WO 9~1~343 2 1 7 7 8 2 3 PC rNS94/13706 -- 2~ --Diatom cultures may be treated with a chemical capable of weaJ~ening diatom cell walls, which leads to a release of the p-GlcNAc fibers without altering their structure. Such a ~hf~m; ~ 1 may include, but i9 not limited to, hydrofluoric acid (HF).
Alternatively, a mature diatom culture may be treated with a biological agent capable of altering a biological process may be used to inhibit p-GlcNAc fiber synthesis, thus releasing the fibers already present. For example, such an agent may include, but is not limited to, polyoxin-D, an inhibitor of the enzyme N-acetylgl~ m; nyl-p-transferase~
The cell bodies and p-GlcNAc-~nnt;~;n;n~ fibers of diatom culture3 treated with a member of the above described chemical or biological agents are then segregated. For example, the contents of treated diatom cultures may be allowed to settle such that the ~ ntl~ntc of the cultures are allowed to form two distinct layers. The upper layer will contain primarily the p-GlcNAc fibers, while the bottom layer will contain the cell bodies. The upper p-GlcNAc fiber-~nt~;n;n~ layer may be ~ hr)n~ off, leaving behind the settled cellular material of the bottom layer .
The ~;rhtan~d off p-GlcNAc fiber-~ nt;l;n;n~ layer may then be further purified to remove protein and other unwanted matter by treatment with a detergent that will not damage the p-GlcNAc fibers. Such a detergent may include, but i8 not limited to, sodium dodecyl sulfate (SDS).
When acid treatment, such as HF treatment, 18 used to separate p-GlcNAc fibers from diatom cell bodies, a step may be included for the dispersal of the f ibers . Such a step may include, but is not limited to, the use of mechanical force for fiber Wo95115343 21 77823 PCrlUS94/13706 ~

disperaal, such as a step in which the f ibers are subjected to a Waring blender dispersal.
Alternatively, the acid-treated suspension may, in an optional step, be neutralized prior to further 5 purification by detergent treatment Such neutralization will, in general, change the pH of the suspension from appr~;r~tPly 1.8 to approximately 7.0, and may be accomplished by, for example, the addition of an appropriate volume of lM Tris (pH 8 . o ) lO or the addition of an appropriate volume of sodium hydroxide (NaOH) . Neutralization, in general, yields pure p-GlcNAc fibers of a substantially greater ~ength than the other purification methods discussed herein.
The purified p-GlcNAc fibers may then be 15 concentrated using techniques well known to those of skill in the art, such as by utilizing a suction and filtration device. Finally, the p-GlcNAc fibers are washed, in a series of steps with distilled-deionized water, HCl and ethanol, or other appropriate solvents, 20 preferably solvents, such as alcohols, in which both organic and inorganic materials dissolve.
The Working Example presented, ~elow, in Section 8 demonstrates the successful utilization of such a purif ication method .
5.4 7~RTVATIZATION OF ~-GlcNAc The pure, fully acetylated p-GlcNAc of the invention may be derivatized, by utilizing a variety of controlled conditions and procedures, into a large 30 range of different compounds. See FIG. 13 for a diagram depicting some of these compounds. Such derivatized compounds may include, but are not limited to, partially or completely deacetylated p-GlcMAc, which has been I '; f; Pfl via chemical and/or enzymatic 35 means, as described in further detail, below.

WO 95/15343 PCrlUS94113706 Additionally, p-GlcNAc, or its deacetylated derivative, may be derivatized by being sulfated, phosphorylated, and/or nitrated. Further, as detailed below, O-sulfonyl, N-acyl, O-alkyl, N-alkyl, deoxyhalogen, and N-alkylidene and N-arylidene and other derivatives may be prepared f rom the p-GlcNAc or deacetylated p-GlcNAc of the invention. The deacetylated p-GlcNAc of the invention may also be used to prepare a variety of organic salts and/or metal chelates. Further, the p-GlcNAc, or a derivative thereof, of the invention may have attached to it, either covalently or non-covalently, any of a variety of molecules. Still further, the p-GlcNAc of the invention, or a derivative thereof, may be subjected to controlled hydrolysis conditions which yield groups of molecules having uniform and discrete molecular weight characteristics.
One or more of the monosaccharide units of the p-GlcNAc of the invention may be deacetylated to form a poly-,B-1 ,4-N-glucosamine species. A poly-,~-1~4-N-glucosamine species of the invention in which each of the monosaccharide units of the poly-,~-1~4-N-acetylgl~ n~;lm;n~ species of the invention has been deacetylated wil have a molecular weight of about 640,000 daltons to about 24 million daltons, with about 640,000 daltons to about 2.4 million daltons being preferred. A species with such a molecular weight range represents a species having about 4000 to About 150, 000 gll~rn~:~m; nf~ monosaccharides covalently attached in a ,~-1~4 configuration, with about 4,000 to about 15, 000 glucosamine r n~crh~rides being - preferred. At least one of the monosaccharide units of the poly-,l~ 4-N-gluco6amine species may remain acetylated, with about 25~ to about 75" acetylation WO 95/15343 2 1 7 7 8 2 3 PCrNS94113706 being preferred, and about 309,~ acetylation being most pref erred .
The p-GlcNAc of the invention may be deacetylated by treatment with a base to yield gl-lrn.cF-mi nf~l with 5 free amino groups. This hydrolysia proces~ may be carried out with ~olutions of rnnr~n~rated sodium hydroxide or potassium hydroxide at elevated temperatures. To precisely control the extent of deacetylation and to avoid degradation of the main 10 carbohydrate chain of the polysaccharide molecule, however, it i3 preferable that an enzymatic ~L~cedu~ e utilizing a chitin deacetylase enzyme be u6ed for p-GlcNAc deacylation. Such a deacetylase enzymatic procedure is well known to tho3e of skill in the art 15 and may be performed as in (U. S . Patent No .
5,219,749), which ia incorporated herein, by reference, in its entirety.
One or more of the mono3accharide units of the p-GlcNAc of the invention may be derivatized to contain 20 at least one sulfate group, or, alternatively, may be phosphorylated or nitrated, aa depicted below:

o\
~OR~ H~\
~r or where, R and/or Rl, in place of a hydrogen, and/or R2, in place of -COC~3, may be a sulfate (-SHO3), a phosphate ( -P (OH) 2), or a nitrate ( -NOz) group .
Described below are methods by which such p-GlcNAc derivatives may be prepared. Before performing methods such as those described in this Section, it may be advantageous to first lyophilize, ~reeze in liquid nitrogen, and pulverize the p-GlcNAc starting material .
Sulphated p-GlcNAc derivatives may be generated, by, for example, a two step process. In the first step, O-carboxymethyl p-GlcNAc may be prepared from the p-GlcNAc and/or p-GlcNAc derivatives of the invention by, for example, utilizing techniques such as those described by Tokura et al. (Tokura, S. et al, 1983, Polym. J. 15:~85). Second, the sulfation step may be carried out with, for example, N, N-dimethyl-formamide-sulfur trioxide, according to techniques well known to those of skill in the art, such as are described by Schweiger ~Schweiger, R . G ., 1972, Carbohydrate Res. 21:219) . The resulting product may be; ~ as a sodium salt .
Phosphorylated p-GlcNAc derivatives of the invention may be prepared, for example, by 1~;1;7;ng techniques well known to those of skill in the art, such as those described by Nishi et al. (Nishi, N. et al., 1986, in "Chitin in Nature and Technology, WO 9~/15343 2 1 7 7 8 2 3 PCT~S94113706 ~

Muzzarelli et al., eds. Plenum Press, New York, pp.
297-299) . Briefly, p-GlcNAc/methi~n.o~--l fonic acid mixture may be treated with phosphorus pentoxide ( in an approximately 0 . 5 to 4 . 0 molar equivalent) with 5 stirring, at a temperature of about 0 C. to about 5 C. Treatment may be for about 2 hours. ~he resulting product may then be precipitated and washed using standard techniques well known to those of skill in the art. For example, the sample may be precipitated 10 with a solvent such as ether, centrifuged, washed with a solvent such as ether, acetone, or methanol, and dried .
Nitrated p-GlcNAc derivatives may be prepared by l~ti1;7in~ techniques well known to those of skill in 15 the art, such as those described by Schorigin and Halt (Schorigin, R and Halt, E:., 1934, Chem. Ber.
67:1712). Briefly, p-GlcNAc and/or a p-GlcNAc derivative may be treated with concentrated nitric acid to form a stable nitrated product.
One or more of the monosaccharide units of the p-GlcNAc of the invention may contain a sulfonyl group, as depicted beLow:
CH20S02~3 2 5 '/H `\
~ j _ ~H

30 where R3 may be an alkyl, an aryl, an alkenyl, or an alkynyl moiety. Such a derivative may be generated by well known methods such as the method described in '.
Kurita et al. (Kurita, K. et al., 1990, Polym. Prep [Am. Chem. Soc., Div. Polym. Chem.] 31:624-625).
3r; sriefly, an aqueous alkali p-GlcNAc solution may be Wo 95/15343 PCTNS94/13706 reacted with a chloroform solution of tosyl chloride, and the reaction may then be allowed to proceed smoothly at low temperatures.
One or more of the monosaccharides of the p-5 GlcNAc of the invention or its deacetylated derivative may contain one or more O-acyl groups, as depicted below:

o H /' h ";
~o NHoCR6 where R~ and/or Rs~ in place of hydrogen, may be an alkyl, an alkenyl, or an alkynyl moiety, and R6 may be 20 an alkyl, an alkenyl, or an alkynyl moiety. An example of such a derivative may be generated by well known methods such as those described by Komai (Xomai, T. et al., 19~6, in "Chitin in ~ature and Technology", Muzzarelli ~1.., eds., Plenum Press, New York, pp.
497-506). Briefly, p-GlcNAc may be reacted with any of a number of suitable acyl chlorides in methanesulfonic acid to yleld p-GlcNAc derivative~3 which include, but are not limited to, caproyl, capryl, lanroyl, or benzoyl derivatives.
- 30 One or more of the ~ A~rhArides of the deaceylated p-GlcNAc of the invention may contain an N-acyl group, as depicted below:

W095/15343 2 ~ 77823 PCT/US94/13706 ~

H,~
5 ~
H NH ~CR~
10 where R7 may be an alkyl, an alkenyl, or an alkynyl moiety. Such a derivatization may be obtained by ut;l;7;n~ techniques well known to those of 3kill in the art, such a6 the technique described in Hirano et al. (Hirano, S. et al., 1976, Carbohydrate Research 47:315-320), Deaeetylated p-GlcNAc is soluble in a number of aqueous solutions of organic acids. The addition of selected earboxylie anhydrides to sueh p-GleNAc-.-f.nt;.in;nS golutions, in aqueous methanolie aeetie 20 acid, results in the formation of N-acyl p-GlcNAc derivatives .
One or more of the ~q:~rt~h;~ides of the deacetylated p-GlcNAc of the invention or of its deacetylated derivative, may contain an O-alkyl group, 25 as depicted below:

H~
H2 "
or where R3 may be an alkyl, and alkenyl, or a alkynyl moiety. Such a derivatization may be obtained by using techniques well known to those of skill in the art. ~or example, the procedure described by Maresh et al. [Maresh, G. ~,., in "Chitin and Chitosan, Skjak-Braek, G. ~}1., eds., 1989, Elsevier pllhl;q~;ng Co., pp. 389-395). Briefly, deacetylated p-GlcNAc may be dispersed in dimethoxyethane (DME) and reacted with an excess of propylene oxide. The period of the reaction may be 24 hours, and the reaction takes place in an autoclave at 40 to 90 C. The mixture may then be diluted with water and filtered.
The DME may be removed by distillation. Finally, the end-product may be isolated via lyophilization.
One or more of the monosaccharide units of the p-GlcNAc of the invention may be an alkali derivative, as depicted below:
CH20N~I
~o 2 0 ~/H
~H

Such a derivative may be obtained by using techniques well known to those of skill in the art. ~or example, a method such as that described by Noguchi ~L-(Noguchi, J. et al., 1969, Kogyo Kagaku Zasshi 72:796-799) may be llt;1;7~-1. Briefly, p-GlcNAc may be - 30 steeped, under vacuo, in NaOH (4396, preferably) for a period of appr~nr;~t~ly two hours at about 0C.
- Bxcess ~aOH may then be removed by, for example, centrifugation in a basket centrifuge and by mechanical pressing.

Wo 95/15343 2 1 7 7 8 2 3 PCIIUS94/13706 ~

One or more of the monosaccharide units of the deacetylated derivative of the p-GlcNAc of the invention may contain an N-alkyl group, as depicted below: -where Rg may be an alkyl, an alkenyl, or an alkynyl moiety. Such a derivati~ation may be obtained by utilizing, for example, a procedure such as that of Maresh et al. tMaresh, G. et al., in "Chitin and Chitosan, " Skjak-Brack, G. et al., eds. 1989, Elsevier pllhl;~,h;ng Co., pp. 389-395), as described, above, for the production of O-alkyl p-GlcNAc derivatives.
One or more of the monosaccharide units of the deacetylated derivative of the p-GlcNAc of the invention may contain at least one deoxyhalogen derivative, as depicted below:

H/ o where R1o may be F, Cl, Br, or I, with I being preferred. Such a derivative may be obtained by using techniques well known to those of skill in the art.
For example, a=procedure such as that described by Kurita ~. ~Kurita, K. et al., l990, Polym. Prep.

~ W095115343 21 7 7 8 2 3 PCrNS94/13706 [Am. Chem. Soc. Div. Polym. Chem.] 31:624-625) may be utilized. Briefly, a tosylated p-GlcNAc is made to react with a sodium halide in dimethylsulfoxide, yielding a deoxyhalogen derivative. p-GlcNAc 5 tosylation may be performed by reacting an agueous - alkali p-GlcNAc solution with a chloroform solution of tosyl chloride. Such a reaction may proceed smoothly at low temperatures.
One or more of the monosaccharide units of the 10 deacetylated derivative of the p-GlcNAc of the invention may form a salt, as depicted below:

, . o H H
~ H o H H3N-OCOR,1 where Rll may be an alkyl, an alkenyl, or an alkynyl 20 moiety. Such a derivatization may be obtained by using technigues well known to those of skill in the ~ ==
art. For example, a procedure such as that described by Austin and Sennett (Austin, P.R. and Sennett, S., in "Chitin in Nature and Technology, ~ 1986, 25 Muzzarelli, R.A.A. ~L., eds. Plenum Press, pp. 279-286) may be ~t;1;7ed. Briefly, deacetylated p-GlcNAc may be suspended in an organic medium such as, f or example, ethyl acetate or isopropanol, to which may be added an ~ Liate organic acid such as, for 30 example, formic, acetic, glycolic, or lactic acid.
The mixture may be allowed to stand for a period of time (1 to 3 hours, for example). The temperature of reaction and drying may vary f rom about 12 to about 35 C., with 20 to 25C being preferred. The salts Wo95/15343 21 77823 PCr/US94113706 ~
may then be separated by filtration, washed with fresh medium, and the residual medium evaporated.
One or more of the monosaccharide units of the deacetylated derivative of the p-GlcNAc of the 5 invention may form a metal chelate, as depicted below:

~
`"~`

H HNH
X

where R1~ may be a metal ion, particularly one of the transition metals, and X is the dative bond established by the nitrogen electrons present in the amino and substituted amino groups present in the 20 deacetylated p-GlcNAc.
One or more of the nsa~-h~ride units of the deac:etylated derivative of the p-GlcNAc of the invention may contain an N-alkylidene or an N-aryl idene group, as depicted below:

/ H
O
3 0 H NHCR1a where Rl3 may be an alkyl, an alkenyl, an alkynyl, or an aryl moiety. Such a derivatization may be obtained by using techniques well known to those of skill in 35 the art. For example, a procedure such as that described by Hirano et al. (~irano, S. et al., 1981, Wo 95/15343 2 1 7 7 8 2 3 PCr/US9~/13706 J. Biomed. Mat . Res . 15: 903-gll) may be utilized.
Briefly, an N-substitution reaction of deacetylated p-GlcNAc may be performed with carboxylic anhydrides and/or arylaldehydes to yield acyl- and/or arylidene 5 derivatives.
Further, the p-GlcNAc of the invention, or its deacetylated derivative, may be subjected to controlled hydrolysis conditions, which yield groups of molecules having uniform, discrete molecular weight lO and other physical characteristics. Such hydrolysis conditions may include, for example, treatment with the enzyme, lysozyme. p-GlcNAc may be exposed to lysozyme for varying periods of time, in order to control the extent of hydrolysis. In addition, the 15 rate of hydrolysis may be controlled as a function of the extent to which the p-GlcNAc that is being lysozyme treated has been deacetylated. Deacetylation conditions may be as described earlier in this Section. The more fully a p-GlcNAc molecule has been 20 deacetylated, the more fully the molecule will be hydrolyzed. Changes in physical characteristics, in addition to the lowering of molecular weight, may be elicited by hydrolysis and/or deacetylation treatments. Extensive hydrolysis causes liquefication 25 of the p-GlcNAc. The results of a hydrolysis/deacetylation procedure are presented below in the Working Example of Section 9, below.
Further, heat denaturation may function to modify the crystalline structure o~ the p-GlcNAc. Such a 30 modification of the p-GlcNAc product crystalline structure may advantageously affect, for example, the reactivity of the p-GlcNAc.
Further, a variety of molecules may be covalently or non-covalently functionally attached to the 35 deacetylated derivatives of the p-GIcNAc of the Wo9~/15343 21 77823 PCrtUSg~tl3706 invention. Such molecules may include, but are not limited to such polypeptides as growth factors, such as nerve growth factor, proteases, such as pepsin, hormones, or peptide recognition sequences such as RGD
sequences, f ibrDnectin recognition sequences, laminin, integrins, cell adhesion molecules, and the like CoYalent attAr? t of molecules to the exposed primary amines of deacetylated p-GlcNAc may be accomplished by, for example, chemical attachment utilizing bi-functional cross-linking reagents that act as specif ic length chemical 6pacers . Such techniques are well known to those of skill in the art, and may resemble, for example, the methods of Davis and Preston (Davis, M. and Preston, J.F. 1981, Anal. Biochem. 116:404-407) and Staros et al. (Staros, J. V. et al., ~1986, Anal. Biochem. 1~6:220-222) -Briefly, carboxylic residues on the peptide to be attached to the deacetylated or partially deacetylated p-GlcNAc of the invention may be activated and then crosslinked to the p-GlcNAc. Activation may be accomplished, fDr example, by the addition of a solution such as carbodiimide EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) tD a peptide solution in a phosphate buffer. Preferably, this solution would additionally contain a reagent such as sulpho-NHS ~N-hydroxysulphos~lrc;nimide) to enhance rrllrl inr. The activated peptide may be crosslinked to the deacetylated p-GlcNAc by mixing in a high pH
buffer, such as c;2rh-~n~te buffer (p~ 9.0-9.2).
Alternatively, such molecules such as those described above may be non-covalently attached to deacetylated p-~lcNAc using techniques well known to '~
those of skill in the art. For example, a molecule or molecules of choice may be mixed with a deacetylated p-GlcNAc solution prior to lyophilization.

Wo95/15343 2 1 77823 PCr/US94/13706 Alternatively, hybrids comprising p-GlcNAc and/or p-GlcNAc derivatives may be formed. Such hybrids may contain any of a number of natural and/or synthetic -materials, in addition to p-GlcNAc and/or p-GlcNac 5 derivatives. For example, hybrids may be formed of - p-GlcNaC and/or p-GlcNac derivatives plus one or more extracellular matrix (ECM) f-~ n~nt c: . Such ECM
components may include, but are not limited to, collagen, fibronectin, glycosaminoglycans, and/or l0 peptidoglycans. Hybrids may also be formed of p-GlcNAc and/or p-GlcNAc derivatives plus one or more :~
synthetic materials such as, for example, polyethylene. Such a p-GlcNac/polyethylene or p-GlcNac derivative/polyethylene hybrid may be made by 15 thermally linking the hybrid components via, for example, autoclaving.
Additionally, an iodo-p-GlcNAc derivative may be copolymerized with, for example, styrene, for the manufacture of novel plastic materials. Iodo-p-GlcNAc 20 can be prepared by a process similar to that described by Kurita and Inoue (Kurita, K. and Inoue, S., 1989, in "Chitin and Chitosan", Skjak-Braek et al., eds., Elsevier Science Publishing Co., Inc., p. 365), via tosylation and iodination of p-GlcNAc. The iodo 25 derivative of p-GlcNAc can then be dispersed in nitrobenzene and reacted with styrene, with tin (IV) chloride being used as a catalyst.
In the case of a collagen/p-GlcNAc hybrid, briefly, a p-GlcNAc suspension and a collagen 30 suspension may be mixed and lyophilized, and crosslinked, preferably dehydrothermally crosslinked.
The collagen species of such hybrids may be native or synthetic, and may be of human or non-human, such as bovine, for example, origin. p-GlcNAc/collagen and/or 35 p-GlcNAc derivative/collagen hybrid materials exhibit WO 95/15343 2 l 7 7 8 2 3 PCT/US94/13706 uniform properties, and form a porous matrix that may act, for example, as an efficient three-dimensional matrix for the att ~rl t and growth of cells. The Working Example presented in Sectior, 13, below demonstrates the formation, properties and usefulness of such a p-GlcNAc/collagen hybrid.
Hybrids compri3i~g rr~hi n~t i ~n~ of deacetylated p-GlcNAc and such compound3 a3, for example, heparin, sodium alginate, and caLb~,~y, thyl p-GlcNAc may be formulated u3ing technique3 3uch as those described herein . Such combinations may be f ormed or ref ormed into, for example, membrane3 and fiber3.
Complexes of deacetylated p-GlcNAc with polyanions such as, for example, polyacrylic acid or pectin, possessing both positive and negative charges, may be formulated. The formation of 3uch complexes may be accomplished ~r~r~l;n~ to a method similar to that described by Mireles et al . (Mireles , C . et al ., 1992, in "Advances in Chitin and Chitosan", srine~
C.J. et al ., eds ., Elsevier Pllhl; ~h~s, 3.td. ) .
Deacetylated p-GlcNAc and polyacrylic acid, carrageenan or pectin, for example, are dissolved in HCl and NaCl, respectively, and the reactant solutions, with equal pH, are mixed. This operatio~
produces ef fective florrlll At; ns molecules possessing both positive and negative characteristics, useful, for example, in the proce33ing of waste water3.
5 . 5 ~ ~u~-ls~ATIONS
The p-GlcNAc of the invention, a~ well a3 it3 deacetylated derivative3 and/or their derivat;7~t;t~nf~, such as those described, above, in Section 5.4, may be dissolved and subsequently reformulated into a variety of shapes and conf igurations .

Solution of the p-GlcNAc of the invention can be achieved by treatment with dimethyl acetamide ~DM~) /lithium chloride. p-GlcNAc may be readily dissolved by stirring in a DMA solution containing 5%
LiCl (by weight of the DMA) . Water soluble p-GlcNAc -~
derivatives, such as p-GlcNAc salt3, may be dissolved in water. P-GlcNAc which ha8 been at least about 75~6 deacetylated may be put into solution in, for example, a mild acidic solution, such as 1~ acetic acid.
p-GlcNAc derivatives that are water-insoluble may be put into solution in organic solvents.
Derivativization of p-GlcNAc in DMA:LiCl with phenyl isocyanates may be used to produce ~ ;~h~n; 1 Ateg . Further, derivatization of p-GlcNAc in DMA:LiCl with toluene-p-sulphonylchloride may be used to produce toluene-p-sulfonate.
The p-GlcNAc of the invention, its deacetylated derivatives, and/or their derivatizations in solution may then be precipitated and refu~ 1 At~d into shapes which include, but are not limited to, mats, strings, ropes, microspheres, microbeads, membranes, f ibers, powders, and sponges. Further, ultrathin (i e., less than about 1 micron thick) uniform membranes may be f ormulated .
Such reformulations may be achieved, by, for example, taking advantage of the fact that pure p-GlcNAc is insoluble in solutions such as water and alcohol, preferably ethanol. Introduction, by conv.snt;~-n~l means, such as by injection, for example, of the p-GlcNAc-~ nt~;n;ng DMA/LiCl mixture into such a water or alcohol, preferably ethanol, solution will bring about the reprecipitation, and therefore reformulation, of the dissolved p-GlcNAc. Such a pure p-GlcNAc ref~,L 1~t;on is demonstrated in the Working 35 Example presented, below, in Section 11. In the case WO 95~15343 2 1 7 7 8 2 3 PCr~S9~/13706 ~

of water soluble p-GlcNAc derivatives, reformulations may be achieved by reprecipitating in such organic solvents as, for example, ethyl acetate or isopropanol. Reformulations of p-GlcNAc which has 5 been at least about 75% deacetylated may be achieved by reprecipitating in an ~1 k~l i n~ solution. Water-insoluble p-GlcNAc derivatives may be reformulated by reprecipitation is aqueous solutions, such as, for example, water.
Deacetylated p-GlcNAc, in conjunction with oxidized celI~lose, may be formulated to produce p-GlcNAc/cellulose hybrid materials improving the wet-strength of paper products. An oxidized cotton substrate can be approached closely by the 15 deacetylated p-GlcNAc chain which has a flat ribbon-like shape, similar to that of cotton. Such proximity maximizes the contribution of the ver der Waals forces to the forces promoting adsorption, thus enhancing the wet-strength properties of the hybrid p-GlcNAc-0 cellulose materials.p-GlcNAc membranes and three dimensional p-GlcNAc matrices may be produced via methods which provide for the formation of controlled average pore sizes within either the membranes or the matrices. Pore size can 25 be controlled in membranes and matrices by varying the amount o~ p-GlcNAc material used, and by the ~ liti~ln of certain solvents such as methanol or ethanol, with ethanol being preferred, in specific amounts, ranging from about 5~ to about 40~, prior to the formation of 30 membranes and/or matrices. In general, the greater the percentage of solvent, the smaller the average pore size formed will~be. The Example presented, below, in Section 15, demonstrates the synthesis and characterization of such porous p-GlcNAc structures.

~ WO95115343 21 77823 PCrlUS94113706 5 . 6 US~:S
The p-GlcNAc of the invention, as well as its deacetylated derivatives and their derivatizations, such as those described, above, in 5 Section 5.4, and reformulations, such as those described above, in Section 5.5, have a variety o~
uses For example, the ~on-toxic, non-pyrogenic, biodegradable, and biocompatible properties of the molecules of the invention, in addition to the 10 advantageous properties o~ the p-GlcNAc and its derivatives, as described herein, lend themselves to applications in such diverse fields as agriculture, cosmetics, the biomedical industry, animal nutrition and health, and the food, chemical, photographic, and 15 pharmaceutical industries.
5.6.1 BIQMT'nI~T. TT.qT~.q QF ~-GlrN~r MpT~RTZ~T..q 5 . 6 .1.1 DRUÇ _T~MOBIT-T7:~TI0~/D~T TVET~Y UqT~q Biomedical u~es of p-GlcXAc material may include, 20 for example, enzyme and/or drug 7ation/delivery methods. ~or example, the p-GlcNAc of the invention or its derivatives, may have peptides of interest ~growth factors, for example) covalently attached to them, as described, above, in 25 Section 5.4. Peptide-cr~n~A;n;n~ p-GlcNAc may be administered to a patient using ~tandard procedures well known to those of skill in the art, which include, but are not limited to injection, implantation, arthroscopic, laparoscopic or similar 30 means. Upon introduction of the peptide-r- nt;~in;ng p-GlcNAc into a patient, the p-GlcNAc of the invention - biodegrades, such that the attached peptides are gradually released into the bloodstream of the patient, thus providing a method for controlled drug 35 delivery.

W095/15343 _ 44 _ PCrll~S94/13706 Deacetylated or partially deacetylated p-GlcNAc species may be produced having a predictable rate of biodegradability. For example, the percentage of deacetylation affects the rate at which the p-GlcNAc 5 species degrades. Generally, the higher the percentage of deacetylation, the faster the rate of biodegradability and resorption will be. Thus, the degree of p-GlcNAc biodegradability and the in vivo rate of resorption may be controlled during the p-10 GlcNAc ' s production . Examples of the production andcharacterization of such p-GlcNAc materials are presented in Section 18, below. p-GlcNAc materials having such controllable biodegradability rate3 may be formulated into membranes, gels, sponges, 15 microspheres, fibers, and the like. These p-GlcNAc products adhere and mold to tissues, both soft and hard tissues, in the human body with no need for suturing. The p-Glc~ac materials may, for example, be applied during general or minimally invasive surgery, 20 such as laparoscopic surgery.
p-GlcNAc materials having a controllable rate of biodegradation may be useful, for example, to promote hemostasis in bleeding tissues, organs and blood vessels, to provide periodontal barriers for the 25 separation of soft and hard tissue during the repair process following periodontal surgery, to provide surgical space f illers, to promote sof t tissue augmentation, particularly in the skin for the purpose of reducing skin wrinkles, and as urinary srh i nct-30 au. ~tion, for the purpose of controlling;nrt~nt;n~nce. The Example presented in Section 19, below, demonstrates the use of such p-GlcNAc materials in one such application, namely, to promote hemostasis .

Wo 95/15343 2 1 7 7 ~ 2~ PCT/US94/13706 -- 4~ --In addition, the molecules of the invention may serve as slow release drug delivery vehicles wherein the drug of interest has been encapsulated by the p-GlcNAc, or a derivative thereof. A drug/p-GlcNAc 5 encapsulation may be produced, for example, by - following a modification of the acid treatment/neutralization variation of the chemical/biological purification method presented, above, in Section ~ . 3 . 2 . Rather than raising the pH
of the p-GlcNAc solution to approximately neutral pH
range ( i . e ., approximately 7 . 4 ), one may create a basic pH environment, by raising the pH to approximately 9 . 0 after the purification of the p-GlcNAc is completed. At a more basic pH, the structure of the p-GlcNAc of ~the invention, or a derivative thereof, assumes a more three dimensional or "open" configuration. As the pH is lowered, the molecule's configuration reverts to a more compact, ~closed" configuration. Thus, a drug of interest may be added to a p-GlcNAc at a high pH, then the pH of the p-GlcNAc/drug suspension may be lowered, thereby " trapping" or encapsulating the drug of interest within a p-Glc~Ac matrix.
Such p-GlcNAc Pn~-~rAl1l ~tions may be administered to a patient using standard techniques well known to those of skill in the art, 80 that, upon administration, the encapsulated drug is slowly released into the system of the patient as the p-GlcNAc of the ~n~rsl~l~tion degrades.
p-GlcNAc-based gels and membranes have a variety of applications as therapeutic drug delivery systems.
Such applications include, for example, anti-tumor drug delivery systems. The drug delivery systems described herein are feasible for use with any anti-tumor drug. Such drugs are well known to those of ~~

WO 95/15343 2 1 7 7 8 2 3 PCT~s94~l3706 ~

3kill in the art, and may be formulated into p-GlcNAc gels or membranes, for example, 80 as to provide site-specif ic slow-release delivery directly to the tumor or to the region vacated by the tumor following 5 surgery. Such an immobilized slow-release p-GlcNAc drug product can act as an important initial defensive procedure after surgery. Such p-Glc~Ac anti-tumor drug delivery systems are particularly useful in treating tumors which are totally or partially 10 inaccessible through surgery, such as, for example, is the case with certain brain tumors.
Additional targets for p-GlcNAc anti-tumor systems include, but are not limited to, skin, GI
tract, pancreatic, lung, breast, urinary tract and 15 uterine tumors, and HIV-related Kaposi~s sarcomas.
Antitumor drugs that are r~ t; on enhancers are preferred or instances in which radiation therapy treatment is to be prescribed, either in lieu of, or ~ollowing surgery. Examples of such drugs include, 20 for example, 5'-fluorouracil, mitomycin, cis-platin and its derivatives, taxol, adriamycin, actinomycin, bleomycins, daunomycins, and methamycins.
Dose ranges for anti-tumor drugs may be lower than, equal to or greater than the typical daily doses 25 prescribed for systemic treatment of patients. Higher doses may be tolerated in that the drugs are delivered locally at the site of a tumor. Other tissues, there~ore, including blood cells, are not as readily exposed to the drugs. Do8e8 of 8uch drugs are well 30 known to those o skill in the art, and may, alternatively, routinely be deter~ined using standard techniques well known to those of skill in the art, -~
such as, for example, are described, below, at the end of this Section.

Wo 95/15343 2 1 7 7 8 2 3 pCT/US94/13706 The p-GlcNAc/drug delivery systems of the invention may, additionally, be u3ed for the treatment of infections. For such an application, antibiotics, either water soluble or water insoluble, may be 5 immobilized/formulatea in p-GlcNAc based materials, - such as, for example, gels and membranes. Antibiotics are well known to those of skill in the art, and include, for example, penicillins, cephalosporins, tetracyclines, ampicillin, aureothicin, bacitracin, 10 chlc~. ,h~nicol~ cycloserine, erythromycin, gentamicin, gramacidins, kanamycins, neomycins, streptomycins, tobramycin, and vancomycin. Doses of such drugs are well known to those of skill in the art, and may, alternatively, routinely be determined 15 using standard techniques well known to those of skill in the art, such as, for example, are described, below, at the end of this Section.
Such p-GlcNAc antibiotic products may be used to treat bacterial infections that occur either 20 externally, e.q., on skin, scalp, dermal ulcers or eyes, or internally, ~[, localized infections of the brain, muscles, abdomen. A L~ ;n~nt application is for treatment of HIV-related ~ " Lullistic infections.
The p-GlcNAc/drug delivery systems of the 25 invention may be formulated with anti-inflammatory drugs to control dysfunctional activity of the inf lammatory and immune processes . For example, p-GlcNAc may be formulated with non-steroidal anti-infli tory drugs (NSAIDs) and used to the reduction 30 of local pain nd inflammation induced by diseases such as Rheumatoid arthritis, osteoarthritis and systemic lupus, to name a few. The localized delivery of such NSAIDs using the p-GlcNAc gel or membrane/drug delivery systems of the invention may serve to reduce 35 NSAID side effects, which may include gastric WO 95/15343 2 1 7 7 8 2 3 PCTIUSg4/13706 ~

irritation, azotemia, platelet disfunction and liver function abnormalities. ~SAIDs are well known to those of skill in the art and include inhibitors of cycloxygenase, such as aspirin, etodolac, fenoprofen 5 and naproxen. Other anti-inflammatory drugs may be utilized as part of the p~GlcNAc/drug delivery systems of the invention, such as, for example, inhibitors of lipid inf lammatory mediators, such as leucotrienes .
Doses for such dru~s are well known to those of skill lO in the art, and may, alternatively, routinely be determined using standard techniques well known to those of skill in the art, such as, for example, are described, below, at the end of this Section.
The p-GlcNAc/drug delivery systems of the 15 invention may additionally be formulated with ~nt; f~ln~:ll agents, using techniques described above, for the treatment of specific fungal diseases.
Antifungal agents are well known to those of skill in the art, and may include, for example, amphotericin, 20 anisomycin, antifungone, blastomycin, griseofulvins, and nystatin. Doses of such drugs are well known to those of skill in the art, and may, alternatively, routinely be determined using standard techniques well known to those of skill in the art, such as, for 25 example, are described, below, at the end of this Section .
The p-GlcNAc/drug delivery systems of the invention may also be formulated with antiprotozoal agents, using techniques descrihed above, for the 30 treatment of specific protozoal infections.
Antiprotozoal agents are well known to those of skill in the art, and may include, for example, ~nt;~ ohin~ -antiprotozin, monomycin, paromomycin and trichomycin.
Doses of such drugs are well known to those of skill 35 in the art, and may, alternatively, routinely be WOgS/1~343 21 7 7 823 PCT/US94ll3706 determined using standard techniques well known to those of skill in the art, such as, for example, are described, below, at the end of this Section.
The p-GlcNAc drug delivery 3ystems of the 5 invention may be formulated with spermicidal - compounds, using techniques such as those described, above, to produce effective contraceptives.
Appropriate spermicides are well known to those of skill in the art. Doses of such spermicides are well 10 known to those of skill in the art, and may, alternatively, routinely be determined using standard techniques well known to those of skill in the art, such as, for example, are de6cribed, below, at the end of this Section.
The p-GlcNAc drug delivery 3ystems of the invention may, still further, be formulated using therapeutic protein agents. Such formulations may be produced using, for example, techniques such as those described above, By utilizing such p-GlcNAc 20 therapeutic protein systems, it is possible to deliver specific proteins directly to desired target sites and to effect slow release of the proteins at such sites, ~xamples of possible proteins include, but are not limited to insulin, monoclonal antibodies, breast 25 cancer immunotoxin, tumor necrosis factor, interferons, human growth hormone, l~,, h~kin~, colony stimulating factor, interleukins and human serum albumin. Doses of such therapeutic protein agents are well known to those of skill in the art, and may, 30 alteratively, routinely be detPrm;n~l using standard te~hn;qn~ well known to those of skill in the art, - such as, for example, are described, below, at the end of this Section.
~ecause the p-GlcNAc materials of the invention 35 are themselves i sutral, in that they do not Wo 95/1~343 `' 2 1 7 7 8 2 3 PCT/US94/13706 ~
-- 5~J -elicit an immune response in humans, such p-GlcNAc devices, as described above, comprising p-GlcNAc membranes, 3D porous matrices and/or gels that harbor immobilized drugs, may deliver such drugs in a manner 5 that there is no immune response. Gertain additional materials, such as natural alginatec and synthetic polymers, may be used in some cases to construct such devices in combination with the p-GlcNAc material.
The therapeutically effective doses of any of the lO drugs or agents described above, in conjunction with the p-GlcNAc-based systems described herein, may routinely be determined using techniques well known to those of skilI in the art~ A "therapeutically effective" dose refers to that amount of the compound 15 sufficient to result in amelioration of symptoms of the processes and/or diseases described herein.
Toxicity and therapeutic efficacy of the drugs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, 20 e.~., for det~rrn;n;ns the LDso (the dose lethal to 50S
of the population) and the EDs~ (the dose therapeutically ef fective in 50'c of the population) .
The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as 25 the ratio ~Dso/EDso~ , ol~nr~c which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue 30 in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage f or use in humans . The dosage of such 35 compounds lies preferably within a range of ~ WO95/15343 2 1 7 7 8 2 3 PCr/US94/13706 circulating r~nr~n~rations that include the ED50 with little or no toxicity. The dosage may vary within this range ~l~rPn~;ng upon the do~age form employed and the route of administration llt;li7F'fl, For any 5 compound used in the method of the invention, the - therapeutically effective dose can be estimated initially from cell culture assay3. A dose may be formulated in animal models to achieve a circulating plasma rr,nr.~n~ration range that includes the IC50 10 ( i . e ., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine u6eful doses in humans. Levels in plasma may be measured, for 15 example, by high performance liquid chromatography.
5 . 6 .1. 2 . P-GlcNAc ~RT.T, ENCAPSIJLATION TT~::R.'~
p-GlcNAc encapsulated cells may be formulated, and such p-GlcNAc encapsulated cells may be 20 administered to a patient, via standard techniques well known to those of skill in the art. See, for example, the administration techniques described, above, in Section 5.6.1.1. Alternatively, see, for example , Aebisher et al . (Aebisher , P . et al ., in 25 'lF~ln(~ t;ll ~ of Animal Cell Encapsulation and Immobilization", 1993, CRC Press, pp. 197-224), which is incorporated herein by reference in its entirety.
Cells may be encapsulated by, on, or within p-GlcNAc or partially deacetylated p-GlcNAc membranes, three 30 dimensional p-GlcNAc porous matrices, or p-GlcNAc gels .
Three dimensional matrices can be seeded with cells and used in certain applications without further l~nr;-r5l~l ation . Alternatively, cells can be 35 encapsulated into microspheres or droplets of p-W0 9511~343 ~ 2 1 7 7 8 2 3 PCT/US94/13706 ~

GlcNAc-based polymer gels such as, for example, a p-GlcNAc-lactate po~yelectrolyte polymer (a polycationic polymer). Gels, droplets or microspheres into which cells have been encapsulated may then be coated with a 5 second polyelectrolyte of opposite charge (e.cr., with a polyanion, such as an alginate) to form an outer capsule which provides immuno- isolation for the encapsulated cells, thus reducing the risk of immune rejection by the host organism.
Additionally, cells entrapped in p-GlcNAc gels, three dimensional p-GlcNAc matrices, or both, can be loaded into thermoplastic capsules in yet another method of formulation. Thermoplastic-based capsules can also be utilized to provide immuno-protection for 15 implanted cells in a host organism. Such thermoplastic capsules are made of materials such as hydroxyethyl methylacrylate-methylmethacrylate copolymer (HEMA-MMA) . Thermoplastic-derived microcapsules are formed, for example, by the 20 coextrusion of a solution of HEMA-MMA in polyethylene glycol and the cell-cnntA;n;n~ p-GlcNAc matrix and/or gel medium, into an r~ U~L iate organic solvent such as h~ rAn~. See, for example, the method described by Aebisher et a~ (Aebisher, P. et al., in 25 ~Fundamentals of Animal Cell ~nrArs11l Ation and Immobilization", 1993, CRC Press, pp. 197-224).
The p-GlcNAc cell .on~-Ar5~1 Ations have a variety of appl;cRt;r~nR. First, they may be utilized for the delivery of therapeutic compounds, synthesized and 30 secreted by cells ~tt~A~ch~d to and encapsulated in the membranes, matrices or gels. For example and not by way of limitation, the p-GlcNAc/cell encaps~ t; ~nR
may be used for delivery of insulin in the treatment of 1; Ahet~R, nerve growth factor for the treatment of 35 ~17h~i -r~8 disease, factor VIII and other clotting ~ WO gs115343 2 1 7 7 8 2 3 PCrlUS94/13706 factors for the treatment of hemophilia, ~ pAm;n~ for the treatmént of Parkinson' s disease, .-nkf~rhAl ins via adrenal chromaffin cells for the treatment of chronic pain, dystrophin for the treatment of muscular 5 dystrophy, and human growth hormone for the treatment - of abnormal growth.
Further, because the p-GlcNAc materials of the invention are themselves; nnn~utral, as they do not elicit an immune response in humans, it is possible to lO engineer and construct devices consisting of p-GIcNAc membranes, three-dimensional porous p-GlcNAc matrices and/or p-GlcNAc gels that harbor attached cells which can deliver cell-based therapeutics in a manner such that the cells are immuno-isolated, i.e., no anti-cell 15 host immune response is elicited. Certain additional materials, such as, for example, natural alginates and synthetic polymers, may be used to construct such devices in addition to the p-GlcNAc material itself.
p-GlcNAc/cell encapsulation compositions may 20 additionally be utilized for the delivery of cells to seed tissue regeneration. Applications of specific cell types encapsulated for the seeding of cell growth leading to tissue regeneration at the site of an injury may include, but are not limited to 25 regeneration of skin, cartilage, nerves, bone, liver, and blood vessels. ~he tissue regeneration applications of cells encapsulated in p-GlcNAc materials are advantageous, in part, because of the ability of the p-GlcNAc material to adhere to injured 30 tissue, to provide a substrate for 1 i An cell growth, and to undergo bioresorbtion co; nc; IS-~nt with the growth of new healthy tissue during the tissue regeneration process at the site of injury. Examples include, but are not limited to the regeneration of WO 95/15343 ~ 2 1 7 7 8 2 3 P~:T/US94/13706 ~

6kin, bone, cartilage, liver, te~don, and ligament tissues .
5 . 6 .1. 3 UTILIZING p-GlcNAc MATERIAL5 FOR THE
~;v~;N ! lON OF POST SURGICAL AnT~ION5 Additionally, p-Glc~Ac membranes may be used to provide a biodegradable, biocompatible mechanical barrier to prevent post-surgical adhesions. The Example presented in Section 17, below, demonstrate such a p-GlcNAc application. Solid p-GlcNAc or p-GlcNAc derivatives formulated into membranes or sponges may be utilized for such an application.
Pre~erred membranes are thin, generally less than about 1 mm in thickness. Preferable p-GlcNAc derivatives are- p-GlcNAc derivatives which have been about 50-809~ deacetylated. Such p-GlcNAc derivatives will generally~be resorbed approximately 7-21 days post implantation.
Liquid p-GlcNAc derivatives are also suitable for use in the preYention of post surgical adhesions.
Preferable liquid p-GlcNAc derivatives for such an application are deacetylated p-GlcNAc salt derivatives and carboxymethyl p-GlcNAc derivatives. A p-GlcNAc derivative which i8 particularly preferred for the prevention of post surgical adhesions is a p-GlcNAc-lactate derivative, especially a p-GlcNAc-lactate gel derivative. Such p-GlcNAc-lactate derivatives may be formulated using propylene glycol and water, as, for example, described in Section 17.1. p-GlcNAc-lactate 3 0 derivatives may be produced having high and low viscosities, which allows for the ability to tailor the p-GlcNAc used to the specific indication of interest. For example, it may be useful to use a p-GlcNAc product having a lower viscosity for delivery through a syringe or via a spray, while it may be desirable to use a p-GlcNAc product having a higher WO 9SIIS343 2 1 7 7 ~ 2 3 PcTiusg~/l37n6 viscosity, and therefore greater lubrication properties, when the indication is an ortl~np~ one.
For the prevention of post surgical adhesions, solid p-GlcNAc formulations are suitable for clearly 5 circumscribed wound sites. Such p-GlcNAc formulations - should be applied following the surgical procedure and the material should completely cover the tr;~ t; 7F.~
tissue. It can be applied either in conjunction with either general or m;n;r-1 ly invasive (e.g., laparoscopic) surgical procedures. The solid p-GlcNAc formulations can be cut and applied using standard surgical procedures and instrumentation well known to those of skill in the art.
The liquid p-GlcNAc formulations can be applied, for the prevention of post surgical adhesions, in larger areas prone to form such postoperative adhesions. The p-GlcNAc-lactate gel, for example, can be applied before the surgical procedure to provide additional lubrication and thus reduce the amount of traumatized tissue. Alternatively, the liquid p-GlcNAc f~l l~t;nn, such as p-GlcNAc-lactate, can be applied following the surgical procedure to form a physical barrier to prevent postoperative adhesion f ormation .
The p-GlcNAc material can be painted, sprayed or dropped from a syringe device onto the wounded site.
In laparoscopic procedures, low viscosity materials can, for example, be delivered with standard suction irrigation devices. ~igher viscosity materials will 3 0 require pressure to reach its target . The pressure can be provided by a compressed gas powered pi3ton or a syringe type device.
The amount of liquid p-GlcNAc f~JLI 1~t;nn, such as the p-GlcNAc-lactate gel f~ tion, required for prevention of post surgical adhesions is proportional WO 95~1~343 2 1 7 7 8 2 3 PCrlUS94113706 to the extent of the traumatized tissue The p-GlcNAc material administered should be applied in the range of O .1 ml to 1. 5 ml per sq. cm of surface area .
5 5.6.1.4 O~TET` BIoM~nIcAL USES OF T~-GlcNAc r~A~T~T,C
Other biomedical uses of p-GlcNAc materials include, for example, the use of such materials as cell culture substrates. For example, as shown in the Working Example presented in Section 12, below, the p-10 GlcNAc of the invention acts as a very ef f icientsubstrate for mammalian cells grown in culture.
Further, three dimensional configurations of p-GlcNAc may be used as a medium components which will allow three dimensional cell culture growth.
The cell substrate capabilities of the p-GlcNAc of the invention may also be utilized in vivo. Here, the p-GlcNAc of the invention, or a derivative thereof, as described herein, may act to facilitate tissue regeneration (e.~., regeneration of connective tissue covering teeth near the gum line, vascular grafts, ligament, tendon, cartilage, bone, skin, nerve tissues) . The p-GlcNAc molecules of the invention may, therefore, for example, have extensive plastic surgery applications.
Deacetylated p-GlcNAc is preferred for use as a sealant of vascular grafts. Deace~ylated p-GlcNAc derivatives such as N-carboxymethyl and N-carboxybutyl deacetylated p-GlcNAc are preferred as tissue regeneration reagents. N-carboxymethyl deacetylated p-GlcNAc may, for example, be inoculated into the cornea to induce neovascularization.
Further bi, ~ 71 applications of the p-GlcNAc of the invention or of its derivatives, as described herein, may involve the molecules ' use in wound ~ Wo 95115343 2 1 7 7 8 2 3 p~T/US94/l3706 dressing, wound healing o;n nt~, and surgical sutures, sponges, and the like.
Still further, such molecules may be used, for example, in the treatment of osteoarthritis, in the r~ t; ~n of blood serum cholesterol levels, as anti-viral agents, as anti-bacterial agents, as torg, ag anticoagulants, as dialysis and ultrafiltration membranes, as anti-tumor agents, as contact lens material, and as oral adsorbents for iremic toxins when administered to kidney failure patients. Microcrystalline p-GlcNAc suspensions or water soluble p-GlcNAc derivatives are preferred for the treatment of arthritis, by, for example, injection directly into arthritic j oints .
p-GlcNAc has additional applications as a rmPnt of artificial or donor skin. For example, p-GlcNAc, preferably as non-woven p-GlcNAc films, may be applied to split th;~-kn~ skin donor sites, over, for example, donor dermis.
Deacetylated p-GlcNAc to which a protease, such as pepsin, has been attached may be used for the controlled digestion of proteins in contact with such p-GlcNAc/protease compounds.
Certain derivatizations of the p-GlcNAc of the invention, or of its derivatives, may be preferred for specific applications. (Derivatizations are described in Section 5 4, above. ) For example, sulfated, phosphorylated, and/or nitrated p-GlcNAc derivatives may be preferred as anticoagulants or as lipoprotein lipase activators. N-acyl p-GlcNAc derivatives may also be preferred for anticoagulants, in addition to being preferred for, for example, use in production of ~:~
artificial blood vessels, anti-viral compounds, anti-tumor (specifically, cancer cell aggregating compounds), dialysis and ultrafiltration membranes, WO 9511~343 2 1 7 7 8 2 3 PCrlUSg4/13706 and in the proauction of controlled release drug de~ivery systems. O-alkyl p-GlcNAc and its deacetylated derivatives may also be preferred in the production of controlled release drug delivery 5 systems. N-alkyl p-GlcNAc derivatives may be pref erred a3 anti -bacterial agents . Oxido deaminated derivatives may be preferred as anti-cancer agents, specifically their use in conjunction with immunotherapy for cancer cells. Deacetylated p-GlcNAc l0 derivatives may be pref erred as wound healing agents .
N-alkylidene and N-arylidene p-GlcNAc derivatives may be preferred for the enzyme; hi 1 i 7~tion applications .
5 . 6 . 2 AGRICULTURA~ USE:S OF ~-GlcNAc MAT~IALS
The p-Glc~Ac of the invention or it3 derivatives may be used in various agricultural applications, as well. Such applications include, but are not limited to insecticide, fungicide, bactericide, and nematocide 20 applications. N-ca,l.J,-y, -thyl deacetylated p-GlcNAc derivatives are preferred ior use as ef fective bacteriostatic reagents. N-alkyl p-GlcNAc derivatives may be preferred i~or ~ungicide applications.
iti~n~lly, the molecules of the invention may be 25 used in various soil treatment applications, including, but not limited to, fertilizer compositions. Further, controlled release of agrochemicals may be achieved by entrapping such chemicals via the immobilization, encapsulation, and 30 other methods described, above, in this Section.
Additionally, analogs of, for example, Rhizobium nodulation factors and/or nitrogen f;~ tj~n in~
may be immobilized onto, and administered via, the p-GlcNAc and/or p-GlcNAc derivatives of the invention.

5.6.3 N[JTRITION/FOOD lNl~USl'~Y USES OF p-GlcNAc MATT~'R T RLS
The p-GlcNAc of the invention and its derivatives as described herein additionally have applications in 5 the fields of animal and human nutrition. For - example, the molecules of the invention may be used as feed ingredients Techniques such as those described, above, in this Section, may be used in the production of controlled release products in animal systems.
lO Additionally, the biomedical applications described above may be utilized in animal systems by incorporating routine modifications well known to those of ordinary skill in the art.
Food industry applications of the p-GlcNAc of the 15 invention and of its derivatives, as described herein, may include, but are not limited to anticholesterol ( i . e ., hypocholesterolemic compounds ), f at -binding compounds, emulsifiers, carriers, preservatives, seasonings, and food texturizers, in addition to fruit 20 coatings, and food packaging products.
5 . 6 . 4 COSMETIC USES OF T~-GlCNAC MATERI.
Cosmetic applications of the p-GlcNAc of the invention may include, but are not limited to, the 25 production of products for hair and skin care. Skin care products may include! for example, cosmetics l~t;l i~in~ deacetylated p-GlcNAc salts, carboxymethyl p-GlcNAc-rnnt~;n;ns products, and cosmetic packs ~ nt~;n;nS deacetylated p-GlcNAc and such derivatives 30 a8 l1Y-1L~ Y~L~Y1-, N-succinyl-, and quaternary p-GlcNAc derivatives. Hair products may include, for - example, carboxymethyl p-GlcNAc~ nt~;nin~ products, and film-forming p-GlcNAc derivatives.

WO 95l~5343 ~ 2 l 7 7 8 2 3 PCTIUS94/13706 ~

5.6.5 CHEMICAL ~ N~ltTr~G APPLICATIONS OF p-GlcNAc MATERIALS =
The p-GlcNAc of the invention and its derivatives have a variety of applications that are useful in the 5 ~h~mi r:3l engineering industry. For example, p-GlcNAc may be u3ed a3 a coupling agent ~or adhesion of metals to polymers, membranes formed by glycol p-GlcNAc may be used in ~ 1 ;n~tion applications, and membranes formed by other p-GlcNAc derivatives may be used for 10 transport of halogen ions. Other applications may include the production of flame retardants, and the manufacture oi~metal chelating compounds and compounds capable of removing trace and heavy metals from liquids as well as water-soluble industrial 15 pollutants, such as PCss, for example. p-GlcNAc and/or p-GlcNAc derivatives may be used in photographic applications. For example, the ability of p-GlcNAc and/or p-GlcNAc derivatives to chelate metals, such as silver halides, may be utilized by 20 contacting photographic solutions to recast mats, such a3 thin membranes, of p-GlcNAc and/or p-GlcNAc derivatives .

6. EX~MPLE: PHYSICAL CHARACTERIZATION OF PURE
PREPARATIONS OF D-GlcNAC
Presented in this Example, are circular dichroism (CD) and infra-red spectra (IR) analyses of p-GlcNAC
and deacetylated p-GlcNAC membranes.
3 0 6 .1 MATERIALS AND METHODS
~-GlcNAC and commercial "chitin" ~rel~arations:
The p-GlcNAc used in the CD studies was prepared using the Mechanical Force purification method described, above, in Section 5.3.1.

WOgS115343 PCrlUS94113706 Commercial "chitin" was purchased from NovaChem, Ltd., PO Box 1030 Armdale, Halifax, Nova Seotia, Canada, B3L 4K9.
The p-GlcNAC ' dl~e:s used in the IR studies 5 were prepared by either the Me- h~n' r;il Force - purification method as described, above, in Section 5.3.1, or by the Chemical/Biological purification method, as described, above, in Section 5.3.2, as indicated .
The commercial "p-GlcNAc" preparations were cast into membranes by dissolving in a dimethylacetamide solution ~ ntil;nin~ 59~ lithium chloride, and layering onto distilled, deionized water until membranes precipitated .
p-GlcNAC derivatives and treatments;The Deacetylated p-GlcNAC used in both the CD and IR
studies was prepared by treatment of the p-GlcNAC with 50~ NaOH at 60 C. for 2 hours. The heat-denatured p-GlcNAC membranes used in the IR studies were modif ied 20 by boiling in 0.2mM EDTA for 3 minutes. Autoclaved p-GlcNAc was autoclaved or 3 0 minutes at 122 C .
CD t,~hn;~rues: Solid state CD techniques were carried out essentially according to Domard (Domard, A., 1986, Int. J. Macromol. 8:243-246).
6 . 2 RESUI,TS
6.2.1 CD ANALYSIS
In the CD spectra obtained f rom untreated p-GlcNAc (FIG. 3A), the expected n-7r and 7r-7r* optieally 30 active electronic transitions (220-185nM) were observed due to the preseIlce of the carbonyl group in the acetyl moiety of p-GleNAc are present. Such peaks are eompletely absent in the CD speetrum obtained from the deaeetylated p-GleNAe produet, as shown in FIG.
35 3B.

WO 95115343 2 1 7 7 8 ~ 3 PCTIUS94113706 6 . 2 . 2 IR SPECTRA ANALYSIS
The IR spectra obtained in this study are consistent with the chemical structure of p-GlcNAc.
Additionally, ~he sharp rl~finiti~n of each IR peak is 5 indicative of the presence of an ordered and regular (i.e, pseudocrystalline) structure in the p-GlcNAc fibers. See FIG. 4A for the IR spectrum of p-GlcNAc purified via the Mechanical Force purification method, and FIG. 4D for the IR spectrum of p-GlcNAc purified lO via the Chemical/Biological method. For comparison, see FIG. 4B, which demonstrates the IR spectrum of a commercial "chitin" preparation.
The IR spectrum obtained from the autoclaved p-GlcNAc material ~FIG. 4E) does not differ visibly from 15 the IR spectrum observed in FIG. 4A. This data indicates that the p-GlcNAc material may be sterilized by autoclaving with no loss of polymer structure.

7. EXAMPLE: PURIFICATION OF p-GlcNAC USING THE
MECHANICAL FORCE PURIFICATION METHOD
In this section, p-GlcNAC was purified using the Mechanical Force technique described above, in Section 5.3 .l.
7 . l MATERIALS AND METHODS/RESULTS
Diatom culture conditions: The diatom species Thallasiosira fluviatilis was grown in culture according the procedures described, above, in Sections 5.1 and 5.2.
3 0 SEM Procedures: The SEM techniques used here are as those described, below, in Section 12 . l .
p-GlcNAc Purification Procedure:p-GlcNAC was purified from ~he diatom culture by utilizing the Mechanical Force technique described above, in Section 5 3.1. Specifically, the p-GlcNAc fibers were separated from the diatom cell bodies by subjecting WO 95/15343 2 1 7 7 8 2 3 PCr~S94/13706 the contents of the culture to three short bursts of top speed mlxing motion in a Waring blender. Total time of the three bursts was about one second. The resulting suspen3ion was centrifuged at 3500 rpm in a 5 Sorvall GS-4 fixed angle rotor, for 20 minutes at about 10C. The supPrnAtAnt was ~.orAn~ , and centrifuged again, this time at, 4000 rpm in a Sorvall GS-4 fixed angle rotor for 20 minutes at about 10C.
Once again, the supernatant was decanted and 10 centrifuged at 4000 rpm at 10 C. The final 13upernatant of the third centrifugation was clear, with little, if any, visible flocs floating in the liquid. The clear supernatant was decanted into a Buchner filtration unit equipped with nitrocellulose 15 with 0 . 8~m pore size, suction was then applied and the liquid was filtered from the fiber suspension, allowing the fibers to be collected onto the membrane.
The collected f ibers were washed with 1 liter of di6tilled, deionized H2O at 70 C. When almost all of 20 the water had been drained, fibers were washed, with suction, with 1 liter of 1 N HCl at 70C. When most of the acid solution had been drained, the fibers were washed with 1 liter of distilled, deionized H2O at 70C, using suction. When most of the wash water had 25 been drained, the fibers were washed with 1 liter of 95~ ethanol at room temperature, and vacuum was applied. The filter membrane on which the white fiber membrane had been collected was then removed from the filtration unit and the membrane and its membrane 30 support was dried in a drying ove~ at 58OC for 20 minutes, af ter which the membrane and its support was - placed in a desiccator for 16 hours.
Following this purification procedure, the yield of p-GlcNAc from a 1000 ml culture was 6 . 85 milligrams 35 per liter of diatom culture. SEM photographs of the WO95/1S343 2 1 7 7 8 2 3 PcrlllS94/13706 membrane formed by the collection of the p-GlcNAC
~ibers via this technique is shown in FIG. 6A-6B.

8. EXAMPLE: PURIFICATION OF p-GlcNAC USING THE
BIOLOGICAL/CHEMICAL PURIFICATION
S MT~'TllOD
In this section, p-GlcNAC was purified using two of the ~h~mirAl/Biological terhn;q~ C described above, in Section 5.3.2. Briefly, p-GlcNAC was purified via 10 HF LLe:ai t, in one case, and via acid LL~aL L/neutralization in the second case.
8 .1 MATT`RTAT C AND ~ETHODS/RESULTS
Diatom rllltllre conditions: The diatom species 15 Thallasiosira fluviatilis was grown in culture according the pL-)ce-luL~s described, above, in Sections 5.1 and 5.2.
SEM ,~L~ceduL~8 The techniques utilized in this study were as described, below, in Section 12.1.
Purification l,LoceduLe: First, p-GlcNAC was purified by HF treatment, the results of which are shown in FIG. 7A-7B. Specifically, under a fume hood, 2 . 42 ml of a 49% t29N) HF solution was added to the diatom contents of the culture, at room t~ clLuLe, for each 1000 ml of the volume of the original cell culture, resulting in a O . 07 M HF solution. The mixture was then shaken vigorously for about 30 seconds, causing persistent foam to appear over the liquid. The container was allowed to stand undisturbed for 5-6 hours to allow heavy particulates to settle. At the end of this time, a layer o~ foam had formed, while the liquid itself was divided into two strata: f irst, a narrow, very dark green layer resting on the bottom of the container below a second, much lighter colored grayish-green and murky phase which represented perhaps 85-909~ of the total volume REt~lFlED SHEET (RULE 91~

WO 95115343 2 1 7 7 8 2 3 PCT~S94,l3706 of liquid The foam layer was carefully siphoned off, using a capillary glass tube and vacuum suction. The grayish cloudy supernatant was then siphoned of f, with - care being taken to not disturb the dark bottom layer, 5 which consisted mainly of settled cell bodies, and was transferred to a separate plastic container. The grayish cloudy supernatant was allowed to stand undisturbed for an additional 16 hours. The liquid was initially almost colorless, light grey, but not transparent. After~16 hours settling time, a small amount of foam remained on top of the main body of liquid and a small amount of green matter had settled on the bottom of the container. The liquid was lighter in color, but still not transparent. The foam on top of the liquid was siphoned off as before. The main body of liquid was then carefully siphoned off, leaving behind the small amount of settled green material at the bottom of the c~ntAin~r. The liquid which had thus been isolated, contained the majority of the p-GlcNAc fibers and some impurities.
To remove proteins and other unwanted matter liberated by the diatoms during the preceding steps in the procedure from the fiber-~-~nt~;nin~ liquid, the suspension of fibers and cell remnants was washed with sodium dodecyl sulfate ~SDS). Specifically, the necessary volume of a 20~ SDS solution was added to make the final concentration of the liquid 0.5~ SDS by volume. The container holding the liquid was sealed, secured in a horizontal position on a shaking machine, and agitated for 24 hours at about 100 shakes a minute. Soon after shaking began, large flocs of white p-GlcNAc fibers appeared in the suspension, and a considerable amount of foam accumulated in the head space of the ~ nt;l;n~rs At the end of the SDS
washing, the contents of the ,~nt~in~rs were WO 9S/1~343 2 1 7 7 ~ Z 3 PCT/US94/13706 transferred to Buchner filtration equipment equipped with a o . 8 ~m (Supor Filter, Gelman) filter membrane .
The liquid was filtered with suction, and the p-GlcNAc fibers in the liquid were collecte,d on the filter 5 membrane.
The p-GlcNAc fibers collected on the filter membrane were then washed further. First, the fibers were washed with hot (70 C. ) distilled, ~ n;
using three times the volume of the original 10 suspension. With a water jet using distilled, deionized H20, the white fiber clumps collected on the filter membrane of the Buchner filter were transferred to a waring blender, and the f iber clumps were disintegrated w1th about 10 short mixing bursts. The 15 suspension of disintegrated fibers was transferred to a Buchner filter funnel equipped with a nitrocellulose filter membrane as described above, and the liquid was removed under suction. The collected fibers were washed with loO0 ml of hot ~70C) lN HCl solution, and 20 subsequently further washed with 1000 ml hot (70C) distilled, deionized H20. Finally, the ~ibers were washed with 1000 ml 95~ ethanol at room temperature, and filtered to dryness. The fiber m_..l,Lc..l~ and the f ilter membrane supporting the f iber membrane were 25 then dried in a drying oven at 58C for 20 minutes.
The membrane and membrane support was then placed in a desiccator for 16 hours. The membrane was then carefully detached from the filter membrane.
Second, p-GlcNAc was purif ied by using the acid 30 treatment/neutralization method described, above, in Section 5 . 3 .2. Specifically, the p-GlcNAc was processed as described earlier in this Section, until prior~to the SDS wash step, at which point the solution was neutralized to a pH of approximately 7 . o 35 by the addition of a 2 . 9M Tris solution. The p-GlcNAc -Wo gs/ls343 2 1 7 7 8 2 3 PCT/US94ll3706 yield from thi5 purification lloc~-luL~ was 20.20 milligrams per liter of diatom culture. On average, approximately 60 milligrams per liter diatom culture are obtained. SEN mi~ yl~pl-s of ~ ~ ~nes formed 5 during the purification pl~,~e-luLc: are shown in FIGS.
8A-8B and 9A-9E.

9 . T~YAMPT T~: D - Gl cNAr r~T A~'T TYT A'rION
A p-GlcNAc membrane was ~u~ l in a solution

10 containing 50~ NaOH. The suspension was heated at 80C
for 2 hour6. The resulting deacetylated ~ al~e was dried and studied by crAnning electron microscopy, as shown in FIG. llA-llB.
lS 10. R~AMPLE: P-GlcNAc BIOCOMPAl`TRTT~TTY
In this Example, it i6 d' Ll~ted that the p-GlcNAc of the invention exhibits no detect~hle biological reactivity, as as6ayed by elution tests, i..L~ - lAr implantation in rabbits, intracutaneous 20 injection in rabbits, and systemic injections in mice.
10 . 1. MAT~RTAT C AND ~T ~H~n~
10.1.1. ET,UTION TEST
Conditions for the elution test conformed to the 25 specifications set forth in the U.S. Pharr~~opeiA
XXII, 1990, pp. 1415-1497 and to U.S. Pharr--Op~iA
XXII, Supplement 5, 1991, pp. 2702-2703.
CQ11 cult~re: Mouse fibroblast L929 cell line (American Type Culture Collection Rockville, ND; ATCC
3 No. CCLl; NCTC clone 929) was utilized. A 24 hour confluent monolayer of L929 cells was propagated in - complete Ninimum Essential Nedium tNEN).
D-GlcNAc: a solid membrane of p-GlcNAc which had been prepared according to the Mechanical Force method of purif ication described, above, in Section 5 . 3 .1, RECTIFIED SHEET (RULE 91) WO 95/1~343 2 1 7 7 8 2 3 PCr/US94/13706 0 was extracted in 20 ml serum-supplemented MEM as per U.S. Pharmacopeia XXII (l990) requirements.
Controls- Natural rubber was used as a positive control, and s;licf~n~o was used as a negative control.
5 Controls were tested in the same manner as the test article, p-GlcNAc.
Extracts: Extracts were prepared at 37OC, ln a humidi~ied atmosphere containing 5~ carbon dioxide, for 24 hours. Extracts were evaluated for a change in lO pH, and adjustments were made to bring the pH to within +/- 0 . 2 pH units of the original medium.
Adjustments were made with HCl lower extract pH on with NaHCO3 to raise the extract pH Extracts were sterile filtered by passage through a 0 . 22 micron 15 filter, prior to being applied to the cell monolayer.
Dosinq: 3 mls of p-GlcNAc or control extracts were used to replace the ~-int~n;~nce medium of cell cultures. All extracts were tested in duplicate.
Evaluatior~ Criteria: Response of the cell 20 monolayer was evaluated either visually or under a microscope . The biological reactivity, i . e ., cellular degeneration and/or malformation, was rated on a scale of 0 to 4, as shown below. The test system is suitable if no signs of cellular reactivity (Grade o) 25 are noted for the negative control article, and the positive control article shows a greater than mild reactivity (Grade 2). The test article (~,~, p-GlcNAc) meets the biocompatibility test if none of the cultures treated with the test article show a greater 30 than mild reactivity.
Grade Reactivity Description of Reactivity Zone 0 None Discrete intracytoplasmic granules; No cell Lysis5 Wo 95/15343 2 1 7 7 8 2 3 PCT~S94/13706 Slightly Not more than 209~ of the cells are round, loosely attached, and without intra-cytoplasmic granules; occasional lysed cells are present 2 Mild Not more than 50~6 of the cells are round and devoid of intracytoplasmic granules;
extensive cell lysis and empty areas between cells 3 Moderate Not more than 70~ of the cell layerG contain rounded cells and/or are lysed 4 Severe Nearly complete destruction of the cell layerE
10 .1 2 . INTRAMUS~AR IMPT ANT~TIONS
Animals: Healthy, New Zealand White Rabbits, male and female, (Eastern Rabbit Breeding Laboratory, Taunton, MA) were used. Rabbits were individually housed u6ing suspended St 7i n~ steel cages. Upon 20 receipt, animals were placed in ~uarantine for 8 days, under the same conditions, as for the actual test.
.:Iardwood chips (Sani-chipb7'A, J. P . Murphy Fore3t Products, Montvale, ~J) were used as non-contact bedding under cages. The animal facility was 25 ~--;nt;7;n.~d at a temperature of 68 +/- 3F, with a relative humidity at 30-70~, a minimum of 10-13 complete air exchanges per hour, and a 12-hour light/dark cycle using full spectrum fluorescent lights. Animals were supplied with commercial feed 30 (Agway ProLab, Waverly, NY) under controlled conditions and municipal tap water ad 1 ihit~7m. No known ~r)nt 7m;n;7nt~ were present in the feed, bedding, or water which would be expected to interfere with the test results. Animals selected for the study were 35 chosen from a larger pool of animals. Rabbits were WO 95115343 ` 2 1 7 7 8 2 3 PCrlUS94/13706 ~

weighted to nearest l0g and individually identified by ear tattoo.
p-GlcNAc: The p-GlcNAc used was as described, above, in Section l0 . l . l .
Im~lantation Test: Two rabbits were u3ed for each implantation test. On the day of the test, the animal skin on both sides of the spinal column was clipped free of fur. Each animal was anesth~ti7~d to prevent muscular movement. ~sing 3terile hypodermic needles and stylets, four strips of the test p-GlcNAc (lmm x lmm x l0mm) were implanted into the paravertebral muscle on one side of the spine of each of two rabbits (2 . 5 to 5cm from the midline, parallel to the spinal column, and about 2.5 cm from each other) . In a similar fashion, two strips of the USP negative control plastic RS (lmm x lmm x l0mm) were implanted in the opposite muscle of each animal. Animals were maintained for a period of 7 days. At the end of the observation period, the animals were weighed and euthanized by an injectable barbituate, Euthanasia-5 (Veterinary I,aboratories, Inc., Lenexa, KS) .
Sufficient time was allowed to elapse for the tissue to be cut without bleeding. The area of the tissue surrounding the center portion of each implant strip was F~ m; n.~t~ macroscopically using a magnifying lens .
~emorrhaging, necrosis, discolorations and 1nfect jonR
were scored using the following scale: 0=Normal, l=Mild, 2=Moderate, and 3=Severe. Encapsulation, if present ~ was scored by f irst measuring the width of the capsule (~, the distance from the periphery of the implant to the periphery of the capsule) rounded to the neare6t 0 . lmm. The encapsulation was scored as f ollows:

..

WO 95/15343 2 1 7 7 8 2 3 PCr/Us~4/l37n6 Capsule Width Score None 0 up to o . 5 mm 5 0.6 - 1.0 mm 2 1.1 - 2 . 0 mm 3 Greater than 2 . 0 mm 4 The differences between the average scores for the p-GlcNAc and the positive control article were calculated. The test is considered negative if, in each rabbit, the difference between the average scores --for each category of biological reaction for the p-GlcNAc and the positive control plastic implant sites does not exceed 1.0; or, if the difference between the mean scores for all categories of biological reaction for each p-GlcNAc article and the average score for all categories for all the po~itive control plastic implant sites does not exceed 1.0, for not more than 20 one of four p-GlcNAc strips.
10 .1. 3 . INTRACUTAN~nUS INJECTIQNS
Animals: New Zealand white rabbits were used and -~;nt~;n~cl ag described, above, in Section 10.1.2.
p-GlcNAc: A solid membrane o~ p-GlcNAc which had been prepared according to the mechanical force method of purification described, above, in Section 5.3.1, was placed in an extraction flask, to which 20 ml of the a~L~,~Liate medium were added. Extraction3 were performed by heating to 70 for 24 hours. Following - this E~rocedure, extracts were cooled to room temperature. Each extraction bottle was shaken - vigorously prior to administration.
Intracut~n~ s Test: On the day of the test, animals were clipped free of fur on the dorsal side.
A volume of 0 . 2 ml of each p-GlcNAc extract was WO95/15343 2 1 7 7 8 2 3 PCr/US9.~/13706 injected intr~rut~nf~rusly at five sites on one side of each o~ two ra~bits. More than one p-GlcNAc extract was used per :rabbit. At five sites on the other side o each rabbit, 0 . 2 ml of the corresponding control 5 was in~ected. Injection sites were observed for signs of erythema, edema, and necrosis at 24, 48, and 72 hours after i~jection. Observations were scored according to the Draize Scale for the Scoring Skin Reaction (USP ~Pharmacopeia XXII, l9gO, 1497-1500; USP
10Pharmacopeia XXII, Supplement 5, 1991, 2703-2705) as shown in Table II, below:

Draize Scale for Scoring Skin RP~rt; rnR

Value Ers~thema and Eschar Formation No erythema ...................................... 0 Very slight erythema (barely perceptible) ........ 1 20Well fl~finr~ Prythema ........................... 2 Moderate to 6evere erythema ...................... 3 Severe erythema (beet redness) to slight eschar formation ~injuries in depth) .................... 4 Total possible erythema 3core = 4 25Edema Formation No edema ......................................... 0 Very slight edema (barely perceptible) ........... 1 Slight edema (edges are well defined by definite raising) ......................................... 2 Moderate edema (raised approximately lmm and ,,.rt,,nr~inr, beyond area of exposure) .......... 3 Severe edema (raised more than lmm and extending beyond area of exposure) ......................... 4 Total possible edema score = 4 Wo 95tl5343 PCTNS94/13706 All erythema and edema scores at 24, 48, and 72 hours were totaled separately and divided by 12 (i.e., 2 animals x 3 scoring periods x 2 scoring categories) to determine the overall mean score for the p-GlcNAc 5 versus the corresponding control. Animals were weighed at the end of the observation period and euthanized by injection of a barbituate, E~lthz7nz7~ia-5 (Veterinary Laboratories, Inc., Lenexa, KS). The results of the test are met if the difference between 10 the p-GlcNAc and the control means reaction scores (erythema/edema) is 1, 0 or less) .
10 .1. 4 . SYST~ IN~;~TIO~S - -Animlls: Albino Swiss mice (Mus mllR~ull7R), 15 female, (Charles River Breeding Laboratories, Wilmington, MA) were used. Groups of 5 mice were housed in polypropylene cages fitted with stz7inli~.cc steel lids. Hardwood chips (Sani-chipsl'', ~.P. Murphy Porest Products, Montvale, NJ) were used as contact --20 bedding in the cages. The animal facility was maintained as a limited access area. The animal rooms were kept at a temperature of 68 +/- 3"F, with a relative humidity of 30-709~, a minimum of 10-13 complete air exchanges per hour, and a 12 hour 25 light/dark cycle using full spectrum fluorescent lights. Mice were supplied with commercial feed and municipal tap water ad libit~ . There were no known ~-~7~tz7m7nz7ntR present in the feed, bedding, or water which would be expected to interfere with the test 30 results. Animals selected for the study were chosen from a larger pool of animals. Mice were weighed to the nearest O.lg and individually identified by ear punch .
~-GlcNAc: The samples used were as described, 35 above, in Section 10.1.1. Extracts were prepared WO95/lS343 21 778Z3 PCr/US94/13706 according to the procedures described in Section 10 .1. 3, above .
Svstemic Im ection Test: Groups of 5 mice were inj ected with either p-GlcNAc extract or a 5 corresponding control article, in the same amounts and by the same routes as set forth below:
Test Article Dosing Route Dose/Kg In~ ection or Control Rate 10 Article Extracts O . 996 Sodium Intravenous 50 ml O . l ml/sec Chloride Injection, USP
(0.9~ NaCl) 1 in 20 Intravenous 50 ml 0.1 ml/sec Alcohol in 0 . 9~ Sodium Chloride In j ection USP
( EtOH: NaCl ) Polyethylene Intraperitoneal 10 g 20 Glycol 400 (PEG 400) Cottonseed Oil Intraperitoneal 50 ml (CSO) Extracts of the p-GlcNAc prepared with PEG 400, and the corresponding control, were diluted with 0 . 99i NaCl, to obtain 200 mg of PEG 400 per ml. For the 25 Intracutaneous Test, PEG 400 was diluted with O . 9 NaCl to obtain 120 mg of PEG 400 per ml.
The animals were ob~erved imme~diately af ter injection, at 24 hours, 48 hours, and 72 hours after 30 injection. Animals were weighed at the end of the observation period and euthanized by exposure to carbon dioxide gas. The requirements of the test are met i_ none o~ the animals treated with the p-GlcNAc shows a si~n;f;c~ntly greater biological reactivity 35 than the animals treated with the control article Wo 95115343 2 1 7 7 8 2 3 PCT/USs4/13706 -- 7~ --10 . 2 RESUITS
lO . 2 . l . E~UTI~N T~.CT
The response of the cell monolayer to the p-GlcNAc test article was evaluated visually and under a 5 microscope. No cytochemical stains were used in the evaluation. No signs of cellular biological reactivity (Grade 0) were observed by 48 hours post-exposure to the negative control article or to the p-GlcNAc. Severe reactivity ~Grade 4) was noted for the positive control article, as shown below in Table III: -TABLE III
REACTIVITY GRADES
Control Articles Time p-GlcNAc Negative Positive A B A B A B
0 Xours 0 0 0 0 0 0 24 Hours 0 0 4 4 4 8 Hours 0 0 0 0 4 4 __ The p-GlcNAc o~ the invention, therefore, passes re~uirements of the elution test for biocompatibility, and, thus, is non-cytotoxic.

10.2.2 INTRAMUSCUTAR IMPr~ANTATIONS ~=
Both rabbits (A and B) tested increased in body weight and exhibited no signs of toxicity. See Table 30 IV for data. In addition, there were no overt signs of toxicity noted in either animal. Macroscopic evaluation of the test and control article implant sites showed no inflammation, encapsulation, hemorrhage, necrosis, or discoloration. See Table IV
35 for results. The test, therefore, demonstrates tha~

WO 9511~343 2 1 7 7 8 2 3 rcTlus94ll37o6 the p-GlcNAc assayed exhibits no biological reactivities, in that, in each rabbit, the dif erence between the average 3cores f or all oi the cate~ories of biological reaction for.all oi the p-GlcNAc implant 5 sites and the average score for all categorie~ ~or all the control implant ~ite~ did not exceed 1. 0 .

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All o~ the animals increased in weight. See Table V for data. There were no 5igns of erythema or edema obser~ed at any of the p-GlcNAc or control ---5 article sites. Overt si~ns of toxicity were not - observed in any animal . Because the dif f erence ~ -between the p-GlcNAc and control article mean reaction scores (erythe~a/edema) was les~ than 1. 0, the p-GlcNAc meets the requirements of the intr~ t~nPnus 10 test. See Table VI for results. Therefore, as assayed by this test, the p-GlcNAc demonstrates no biological reactivity.

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WO 95/15343 2 1 7 7 8 2 3 PCr/US~4113706 lO . 2 . 4 . S~ST~MT~ T
All of the mice treated with the p-GlcNAc extract or the control article increased in weight. See Table VII for data. In addition, there were no overt signs 5 of toxicity observed in any p-GlcNAc or control animal. See Table VI for result3. It is concluded, therefore, that none of the p-GlcNAc test animals showed a signif icantly greater biological reactivity than the animals treated with the control article.

WO 9~d15343 2 1 7 7 8 2 3 p~US94113706 ANIM~L WEIGHT~ ANI) CI-INICAI- OBSERVATIONS
}30dy Weight Group Sex Do6e Animal Day 0 Day 3 ~eight Sigm~ of (ml) # Change Toxioity NaCl: Female 1. 03 I, , 20 ~ 6 , 22 ~ 8 2 ~ z None EtOH Female 1.06 II. 21.1 23 4 2 3 None 0 ~e6t Female 1.02 III 20 4 22 6 2.2 None soml/kg Female 1.11 IV. 22 2 24 s 2 3 ~;one Female 1 05 V 21- o 23 ~z 2.2 None Mean 21.1 23.3 SD ~/- 0.7 0 7 NaCl: Female 1 04 VI 20 7 23 2 2 s None 15EtOH Female 1.04 V}I. 20.8 23.s 2.7 None Control Female 1.02 VIII. 20.3 22 3 2.0 None 50ml/kg Female 0.91 IX. 18.2 20.6 2.4 None Female 0.94 x~ 18.7 2~ 9 2.2 None Mean 19 7 22 1 SD ~/- 1.2 1.3 20PEG Female 1.02 XI. 20.3 22 7 2.4 None Te6t Female 0.96 Xl~. 19.2 21.4 2.2 None 10ml/kg Female 0.95 XIII. 18.9 21.6 2.7 None Female 1.05 XIV. 20 9 Z2.7 1.8 Nonc Female 0.94 xv~ 18.7 21.2 2.s None Mean 19 . 6 21. 9 8D t/- 1.0 0.7 PEGFemale 1.01 XVI. 20.1 22.3 2.2 None Control Female 0.99 XVII. 19.8 22.0 2.2 None 10g/kg Female 1.10 XVIII. 22.0 24.3 2 3 None Female 1.07 XIX. 2I.4 23 6 2.2 None Female 1.03 xx~ 20.6 22.4 1.8 None Mean 20.8 22.9 SD ~/- 0.9 1.0 ~Summary of ob~3ervation~3 - o~ 4, 24, 40, and 72 h a~ter in~ ection WO 95/~5343 2 1 7 7 8 2 3 PCIIUS94/13706

11. P'XAI~PT.T~ p--Gll~NA~ ATIoN
In the Working Example presented in this Section, f ~1 p-GlcNAc membrane ( 16 . 2 mg) was dissolved in 1 ml of a dimethylA~etAm~de solution containing 5% LiCl. The S p-GlcNAc-containing solution was placed in a syringe and extruded into 50 ml of pure water to precipitate a fiber. The resulting fiber was studied with sC~nninq electron microscopy, as shown in FIG. lOA-lOB.

12 . ,T`S~AMPT.T~' rT-T T ATTAt'~TMT~'NT TO p--GlcNAc In this working example, it is 1 - L~ ~Ited that p-GlcNAc represents an ef f icient substrate f or cell at~` L and growth in culture.
15 12 .1 MATF'RTAT.. S AND MT~ oDs Cell8:The transformed mouse 3T3 fibroblast cell line was used, and was grown in DMEM supplemented with 10 fetal bovine serum (FBSl.
I~-GlcNAc ~ ` - -: p-GlcNAc was prepared 20 according to the methods described, above, in sections 5 . 3 .1 and 8 .
p-GlcNAc membranes were initially stuck to a #1 (18mm) Corning cover glass using one drop of water, and were attached by autoclaving at 121 C. for 30 25 minutes. ~fembranes prepared in this manner were then placed in culture wells of 6 well culture plates.
~ P.l 1 C~ n~6: Cell numbers were determined in media by direct counting with a hemocytometer, and on matrix by first rinsing membrane~ with ~resh medium DMEM +
30 10% FBS) followed by treatment with trypsin (1096, at 37 C. for 5 minutes) prior to counting.
,CT~M o~eratinq con~liti~nq: A Zeiss 962 il. LL, L
was utilized with an accelerating voltage of lOkv, and a working distance of 15mm. Polaroid type 55 p/n (u4) RECTIFIED SHEET (RU~E 91) WO 9SIIS343 ` 2 1 7 7 8 2 3 PCr/US94113706 ~

was utilized at various magnifications, as indicated.
Sample coat:carbon coat (lOOa) & lOOa aupd.
SPecimen PreParation: For primarv fixation, the culture growth medium was replaced with 2%
5 glutaraldehyde in Eagle' 8 DMEM without serum. Several changes were performed to ensure a complete transition from growth media to Fixative. Fixation proceeded for o . 5 hours at room temperature. Cover slips were tran3ferred to fresh vials c-~nt:~;n;n~ 2~
10 Glutaraldehyde in o . lM Na Cacodylate pX 7 . 2 with O . lM
Sucrose and ~ixed ~or a further 1. 5 hours at room temperature .
Dehydration, CPD, Mount and SPutter Coatinq:
Samples were rinsed in O . lM Na Cacodylate pX 7 . 2, 15 and cover slips were transf erred to a CPD holder .
Dehydration was performed in ethanol 6erie3 (309~, 509~, 75~, 859~, 959~ and 3 x 10096, 5 mins each), and sample3 were critical point dried. Cover slips were then mounted on Al stubs, carbon coated, using vacuum 20 Evaporator (~ looA) and Sputter Coated with loO A
AuPd .
12 . 2 RESI~LTS
p-GlcNAc membranes were tested for an abi~ity to 25 form a substrate on which cells may be grown in culture . Mouse f ibroblast cell3 were grown in wells in the presence or absence of p-GlcNAc membranes and cell counts were taken daily to assay the viability of cultures. The results of one such series of cell 30 counts in shown in FIG. 14. As in~ t~d, by day 5 a~ter plating, only the wells snnt~in;ng p-GlcNAc membranes were able to ~ nt; ml~ to sustain viable cells, demonstrating that p-GlcNAc membranes are capable of acting as efficient substrate8 for the 35 f-~n~in~ growth of cells in culture.

Further, the SEM mi- .u~L~hs depicted in FIG.
15A-15B show healthy cells strongly attached to p-GlcNAc membranes.
S 13. FlrAMPT~ P--GlcNAc/coLr~c~TN HYR~?Tr~s Presented in this Working Example is the formation and characterization of a p-GlcNAc/collagen hybrid material.

13 .1 ,M~TT~TAT~ AN~ MFTHt-n.~
MateriA 1~: Bovine Type I collagen wa6 used in preparation of the hybrids described in this study.
p-GlcNAc was prepared according to the rechAnicAl force method described, above, in Section 5.3.2.
lS Hybrid ~Le~aL~tion: Collagen (10 milligrams/ml) and p-GlcNAc (0.25 milligrams/ml) ~ ~p~n~ions were mixed, in different ratios, frozen in liquid N2 (~
80C. ), thermal soaked at -9 C. for 4 hours, and lyophilized. Material was dehydroth~rr-l ly cross-linkéd under vacuum (approximately 0. 030 Torr) at 60C.
f or 3 days .
Cell ~ ltllre: Mouse 3T3 fibroblast cells were grown on the collagenlp-GlcNAc hybrids produced.
Standard culturing ~LoceduL~as were followed, and SEM
micrographs were taken after 8 days in culture.
13 . 2 RESULTS
Collagen and p-GlcNAc suspensions were mixed in differing ratios (namely, 3: 1, 1: 1, 2: 2, and 1: 3 - 30 collagen:p-GlcNAc suspen6ion ratios), frozen, lyorhili7~d, and crosslinked. Such a pLc,ce-luLa yielded collagen/p-GlcNAc slabs. SEM mi.,~,yL..phs of the resulting materials are shown in FIGS. 16 B-E.
Fig. 16A ~ se,lLs a collagen-only control material.
S5 Note the porous ~LU~:LUL~ of the hybrid material.
RECrlFIED SHEET (RULE 91) Wo 95/15343 2 1 7 7 8 2 3 PCTIUS94/13706 ~

The collagen/p-GlcNAc hybrids of the invention provide an f~ffirj~nt three-dimensional structure for the att~rl L and ~rowth of cells, as shown in the SEM micrographs in FIGS. 17A-D.

14 . EXAMPLE: NMR CHAR~CTERIZATION OF PURE PREPARATIONS
OF D-GlcNAc Presented in this Example is an NMR (nuclear magnetic resonance) analysis of pure p-GlcNAc 10 preparations~

14 . l MATERIALS AN~ METHODS
p-GlcNAc preparations: The p-GlcNAc used in the NMR studies described here was prepared usin~ the

15 chemical purification method described, above, in Section 5 . 3 . 2, with hydro1uoric acid utilized as the chemical reagent.
NMR technicues: Solid state NMR data was obtained using a Bruker 500MH NMR spectrometer.

20 ~ r image analysis was used to transform the raw NMR spectrum data so as to ~1 im;n~tp background and to normalize h~ l in~ . An example of such transformed data are shown in FIG. 18. Transformed NMR curves such as that in Figure 18 were used to obtain areas 25 for every carbon atom type, and to then calculate the C~3 (area) to C-atom(area) ratio3 . Such values, obtained as described are provided in FIG. 20.

14 . 2 RESULTS

Solid state NMR data was obtained by measuring the C~3-NMR spectrum of a 500mg sample of p-GlcNAc. A
typical NMR spectrum is shown in FIG. l9. The individual peaks represent the contribution to the spectrum of each unir~ue carbon atom in the molecule.

The rela~ive percentage of each type of carbon atom in the r ~1 ~rlll e was determined dividing the area of the ~ WO 95115343 2 1 7 7 8 2 3 PCT/US9.1113706 peak generated by that carbon 9pecies by the total sum of the areas under all of the NMR peaks obtained in the spectrum. Thus, it was possible to calculate the ratio of each of the atoms of the molecule measured by 5 a reference atom. All p-GlcNAc molecules consist of N-acetylated gl11rrs~m;n~ re3idues having Cl, C2, C3, C4, C5 and C6 atoms, by definition. The ratio, then, of the area of the N-acetyl CH3 carbon atom peak to the areas of any of the gl-1rnP~mi n~ residue carbon 10 atom peak~, above, should be l. 0 if all of the glucosamine residues in the polymer are N-acetylated.
Data such as those in FIG. 20 were used to obtain values for the C~3 (area) ratios.
The calculated ratios in Fig. 20 are in many 15 cases eriual to or nearly equal to l.0, within experimental error, e.g. C~3/C2=1.097, CH3/C6=0.984, CH3/C5=1.007, CH3/Cl=0.886. These results are consistent with the conclusion that the p-GlcNAc material of the invention is free of rrnt~m~nAnt~ and 20 is fully acetylated (i.e. that essentially l009~ of the glllro~m; n~ residues are N-acetylated) .
15. EXAMPLE: SYNTHESIS AND BIOLOGICAL
CHARACTERIZATION OF CO~TROLLED PORE
SIZB THREE-DIMENSIONAL p-GlcNAc Described below, are methods for the production of three-dimensional p-GlcNAc ba3ed porou8 matrices having controlled average pore sizes. Such matrices have a variety of important applications, - 30 particularly, for example, as means for the encapsulation of cell~. guch cell encapsulation compositions are useful as transplantable cell-based therapeutics, and in other cell & ti3sue engineering applications such as in cartilage regeneration. The -capability to manipulate the morphology and WO 95/15343 2 ~ 7 7 8 2 3 PCT/US94113706 ~

dimensionality of p-GlcNAc materials, as demonstrated here, provides a powerful tool in ~ nl1ing the potential ap~l jr~tlnn~ of the p-GlcNAc material o~ the invention .
15.1 MATERIA~S AND MET~ODS
~ -GlcNAc startinr material: p-GlcNAc was prepared using the rl~m; rAl purification method described, above, in Section 5.3.2, with hydrofluoric 10 utilized as the chemical reagent.
Matrix ~orm~tion: Suspensions (5mls) containing 20 mg p-GlcNAc samples were made in the solvents listed below in Section 15.2, prior to lyoph;l;~t;rn.
Samples were then poured into wells of tissue culture 15 dishes and frozen at -20C. I~he frozen samples were then lyophilized to dryness, and the resulting three dimensional matrices were removed.
Sr~nn;nr electron microsco~Y techniaues: The procedures utilized here were per~ormed as described, 20 above, in Section 12.1. The images shown in FIGS.
21A-G. are 200X magnifications of the matrix material, and a scale marking of 200 microns is indicated on each of these figures.
2 5 15 . 2 RESUI TS
p-GlcNAc samples were obtained from each oE the ~ollowing solvents, as described, above, in Section 15.1:
A. Distilled water B. 109~ methanol in distilled water C 25~ methanol in distilled water D. Distilled water only E. 10~6 ethanol in distilled water F. 25~6 ethanol in distilled water G. 40~f ethanol in distilled water WO9~ 343 2 1 7 7 8 2 3 PCTIUS94113706 Samples of matrix formed using each of the solvents were subjected to scanning, electron microscopic (SEM) analysis, aa shown in FIGS. 21A-G.
These figures reveal that the average matrix pore size decreaaes as the percentage of either methanol or ethanol increases in each suspension.
Specifically, pore diameter in the two water suapensions ~FIGS. 21A and 21D) approach 200 microns on average. Pore size in the samples depicted in FIGS. 21C and 21F (25~ methanol and ethanol, respectively) are between 30 and 50 microns on =
average .
The results shown here suggest that while both ethanol and methanol may succes3fully used to control p-GlcNAc pore aize, ethanol may be more efficient than methanol in .on~hl inrJ the control o~ the p-~lcNAc matrix pore size.

16. EX~MPLE: CE~I, GROWT~I ON THREE DIMENSIONAL
PCROUS p-GlrNAr ~AT~T('F;.~
Described in this Section are results demonfitrating the successful use of three dimensional p-GlcNAc porous matrices as aubstrates for the culturi~g of cells.

16 . 1 MAT~RTZ~T..C~ i~D ~qETT~on~
p-GlrN~r startinq mater; ~l: p-GlcNAc was prepared using the chemical purif ication method described, above, in Section 5.3.2, with hydro~luoric 30 acid l1ti l j 7~d as the chemical reagent.
Matr'~ forr-tion: Three-dimensional p-GlcNAc matrices were prepared by the lyo~hili7:;-ti~n of suspensions of p-GlcNAc in water, water-ethanol, or water-methanol mixtures.

WO 9~115343 2 1 7 7 8 2 3 PCTIUS94/13706 Suspensions (5 mls) f~nnti~in;n~ 20 mgs p-GlcNAc were prepared in the following solvents prior to h; 1 i 7at; nn -l. Distilled water only 2. 10~G methanol in distilled water 3. 25'6 methanol in distilled water 4. Distilled water only 5. 10% ethanol in distilled water 6. 25~ ethanol in distilled water 7. 40% ethanol in distilled water Samples were poured into circular wells of l0 plastic tigsue culture diahes and were f rozen at -20C .
The f rozen samples were then lyophilized to dryness, and the resulting three dimensional matrices were removed . Samples of each matrix were subj ected to scanning electron microscopic (SEM) analysis.
Cells: Mouse embryo BAI,BC/3T3 fibroblast cell line (clone A31), obtained from the ATCC, were used for culturing on the three dimensional porous p-GlcNAc matrices .
Cultu~inq conditions: One cm' samples o~ porous 20 matrices were placed in tis3ue culture wells and were covered with a standard tissue-r1-l turP growth medium.
~ach well was seeded and cells were cultured ~or 6 days at 3 7 C in a CO2 ; n r~l lh~t nr ( 5 % CO2 ) ~ ~rocedures: Matrix samples were fixed and 25 subjected to SEM analysis as described, above, in Section 12 . l . The matrices were prepared by h; l; 7:;n~ p-GlcNAc in distilled water. Images vary in magnification from l00X to 5000X, as indicated in figure legends (FIGS. 22A-G) .
16.2 RESUITS
SEM photographs of p-GlcXAc matrices f~nnt;~;n;n~
attached mouse f ibroblast cells attached are shown in FIGS 22A-G. These photographs show that the three 35 ~i ~inn~l p-GlcNAc matrices contain attached mouse wo gsll5343 2 t 7 7 8 2 3 PCr/US94/13706 fibroblast cells. Further, the photographs reveal that there is a close interaction and connection between the cells and the p-GlcNAc matrix material. It is also notable that the cells have a rounded three- =~
5 dimensional morphology which is different from the f lat, spread shape of the cells when cultured directly onto plastic culture dishes. Cell viabilities were --determined to be greater than 95~.

17. EXAMPLE: p-GlcNAc SUCCESSF~ Y ~ ;N18 POST su-RGI~rl ~nF~r;~IONS --The Example presented herein demonstrates the successful use of p-GlcNAc materials, specifically a p-GlcNAc membrane and gel formulation, to prevent the 15 formation of post surgical adhesions in a series of animal models for such adhesions.
17.1 MATr~lRT~T~ AND M~THODS
SYnthesiR l~-GlcN~o--lactate: p-GlcNAc membrane ~;
20 starting material was produced by the chemical method, as described, above, in Section 5.3.2, with hydrofluoric acid i,t;l i7ed as the chemical reagent.
The p-GlcNAc was converted to deacetylated p-GlcNAc by the following method. (It should be noted 25 that approximately 1. 4 g of p-GlcNAc are :lleeded to produce each 1 g of p-GlcNAc lactate, given the expected loss in mass of approximately 15~ which occurs upon deacetylation. ) Approximately 200mg of p-GlcNAc membrane material were mixed vigorously with 30 approximately 200 ml 60% NaOH. The vigorous shaking served to separate the p-GlcNAc membrane material to the extent possible. The NaOH solution used was made at least 12 hours before using. Samples were placed in an 80C water bath for 6 hrs, with periodic shaking 35 to separate and wet p-GlcNAc material. After 6 hrs, the samples were taken from water bath and the NaOH

WO 95115343 2 1 7 7 8 2 3 PCT~S94/13706 ~
-- ~6 -solution was; ~i~t~ly removed. The membrane materials were washed with ddH,O, at room temperature, until a pH of 7 wa3 reached. The membrane3 were removed rom the water and dried on a Tef lon sheet .
At this point a 2 mg sample was collected for C, H, N analysis in order to determine extent o~ ~
deacetylation. Further, solubility in 1~ acetic acid was checked, with a solubility of 1 mg/ml indicating that the p-G~cNAc material was appropriately deacetylated.
The partially deacetylated pGlcNAc was then converted to pGlcNAc-lactate using the following method: Sufficient 2-propanol (rnnt~ining 109~ water) to wet all of the partially deace~ylated pGlcNAc material and to allow for stirring was added to lg of the partially deacetylated p-GlcNAc in a 250 ml Erlenmeyer flask. (Approximately 30 mls Z-propanol necessary. ) 2-propanol must be reagent grade, and fresh prior to each synthesis. With stirring, 1.1 mL
of a 50~ agueous lactic acid solution. Lactic acid should be reagent grade, and must be analyzed to determine exact concentration of available (i . e ., non-esterif ied~ lactic acid present . This was generally ;~rrnmpl i~h~d by titration with O.lN NaOH to the phenopthalein end-point (pH 7.0) . The concentration of lactic acid used must be constant , i . e ., must be +/- 1 percent, for each p-GlcNAc synthesis. The mixture was allowed to stir for at least two hours.
It i3 possible to add low heat in order to elevate the reaction rate. Reaction time may be extended, or the amount of 50~ ar~ueous lactic acid may be increased 80 that the reaction goes to completion. After stirring, the contents of the f lask were poured through a Buchner funnel using guantitative ashless filter 35 paper. The material was washed with 15 ml of WO 95/15343 2 1 7 7 8 2 3 PCT/USg4/13706 anhydrous 2-propanol. The material was allowed to air ---dry in a f ume hood f or 2 hours and then placed in an oven at 40C overnight. For every gram of partially deacetylated p-GlcNAc starting material, a final p-5 GlcNAc-lactate weight of approximately l . 4 g, (~, an increase of 40% in mass1 should be obtained.
Form~ t; nn of ~-GlrNAr-lactate as ~ c~el: The p-GlcNAc-lactate material was formulated into a gel as follows: p-GlcNAc-lactate starting was dissolved in lO dd-deionized water to a concentration of between 0 . l-4 . 0~6 p-GlcNAc-lactate, by weight. Reagent grade propylene glycol (2-propandiol) was then added to a fi~al propylene glycol concentration of between l-lO~.
In some cases, a preservative was added to prevent 15 bacterial and/or fungal rrnt~min~tion of the product.
Typically, concentrations of p-GlcNAc-lactate of between 0 .1%-4 . 0~ were prepared. The viscosity of these preparations increases dramatically as the p-GlcNAc-lactate percentage increases, such that 20 formulations having 0 . 5% or more of the p-GlcNAc-lactate behave as gels.
An i r~ 1 models:
Sl~rar~ue-Dawlev rats: pf~ P i rn q are produced in this model by abrading or irritating the serosal 25 surface of the cecum and apposing it to an area of parietal peritoneum. The success rate for ;n~lllrin~
adhesions in control animals with this method is reported at an average 80%.
Specifically, the surgical procedure for inducing 30 post surgical adhesions in these rats involved the following. Animals were placed in dorsal recumbency and prepared and draped accordingly for aseptic surgery. Abdominal cavities were exposed through a midline in~ri~2irn. An area, approximately 0.~ cm x l.C
35 cm, of parietal peritoneum on the left abdominal wall W095115343 21 7 7 8 2 3 PCTIUS94113706 ~

was carefully excised, removing a thin layer of muscle, along with the peritoneum.
The cecum waG then elevated and isolated. An area, approximately o . 5 cm x l . 0 cm, on the lateral surface of the proximal end of the cecum was abraded by rubbing ten times with a dry gauze. The cecum was then scraped with a scalpel blade to cause minute petechial hemorrhages. The cecal abrasion and the peritoneal incision were left exposed for 15 minutes.
After 15 minutes, the test article ( e., the p-GlcNAc material) or control article was applied to the cecal wound. The cecal abrasion and the peritoneal wound were then opposed and held in contact with Allis tissue forceps for an additional 15 minutes.
The cecum was then replaced into the abdomen such that the abraded area of the cecum was adjacent to the peritoneal site. The abdominal incision was closed and the animal was allowed to recover from the anesthesia .
Fourteen days after surgery, animals were e-1thAni 7Pd and the abraded area was ~YAm~ n~ri for the formation of post surgical adhesio~s. If adhesions were present, the entire area involved in the ~lh~cinn (i.e., body wall, test or control article, and ;nt~rn:ll organs) were dissected free oi the animal.
The extent oi involvement and tenacity of adhesions was evaluated according to the following scales:
Extent of involvement ~ccOre8 0 no adhesion adhesion <= 25~ of the area 2 ~rih~.ci nn <= 5096 o the area 3 Arihl~ci nn <= 75~ of the area 4 adhesion :, 25~ of the area WO ~5/15343 2 1 7 7 8 2 3 PCI/US94/13706 _ 99 _ Te~aci~y Scores: -0 no adhesion adhesion freed with blunt dissection2 adhesion freed with aggressive dissection 3 adhesion requiring sharp dissection Additi~nAl Anir-l models: Pig and horse large animal bowel model9 were u9ed to as9e99 the prevention of peritoneal =~lh~; nnF~ .
Suxqical ~ror~ e: The animals were placed in dor8al r~rllmh~nry and prepared and draped accordingly for aseptic surgery. The Ah~lnm;n=l cavity was expo3ed through a midline incision. The small intestine was elevated and a 2 cm X 2 cm section was identified, extensively abraded (apprn~;r-t~ly 200 strikes using a scalpel), and allowed to dry for lO minutes. The test article ( e ., p-GlcNAc material ) or control article was then applied to the abraded wound, and the wounded section of the small intestine was replaced into the ahdomen. In such a large bowel type of animal model, six wounds, each separased by 4 inches of bowel ~rom the adjacent wound provide9 an environment highly prone to form adhesions. Following the last of the wounds, the Al-~ nA7 incigion ig closed and the animal is allowed to recover from the anesthesia.
AnAly9;R of periton~=l A~h~o.cinnR: Twenty one days after surgery, animals were euth=ni ~ed and the abraded area was ~ Am;n~, with adhesion formation being evaluated following a procedure similar to that of the Sprague-Dawley rat cecum model.

17 . 2 ~I~
When injury occurs, the body sets in motion a complex set of responses designed to restore the injured area. In the final stages of healing, 35 rnnn~rtive tissue forms at the wound site to WO 95/ls343 2 1 7 7 8 2 3 Pcr/us9~1l37o6 regenerate the body structure and protect the affected area from further damage. In some instances this cascade of events does not work properly and can lead to life threatening conditions.
For example, as a visceral organ heals following surgery, a fibrin clot generated during the surgical procedure may invade the surface of adjoining organs forming a link which allows for fibrobla5t migration.
This migration leads to collagen deposition and tissue growth, which in turn causes the organs involved to adhere to one another.
Such adhesions, referred to as post surgical adhesions, may produce pain, obstruction and malfunctlon by distorting the organ or organs involved. Immobilized jolnts, intestinal obstruction and infertility are often linked to the formation of post-aurgical adhesions. ~urthermore, post surgical adhesion will complicate and extend the length of future surgical procedures in the surrounding region.
2 0 This last issue is of particular relevance to open heart surgeries and cesarean section obstetrical procedures where additional surgeries may be reguired.
The formation of adhesions is very common following ~h~ l, cardiovascular and orthopedic surgical procedures.
When adhesions become pathological and seriously interfere with organ function, surgical adhesiolysis (sharp or blunt dissection of the adhesion in conjunction with meticulous surgical techniques) is the treatment that is currently used to eliminate adhesions. In 1991, approximately 500, 000 adhesiolysis procedures were performed. This procedure is, however, notoriously ineffective, with the frequency of recurrence of adhesion formation 35 reported to be as high as 9096. Further, no other -Wo 95/15343 2 1 7 7 8 2 3 PCT/USg.l/13706 technique or composition has proven effective in the prevention of such post surgical adhesions.
The results pre~eneed herein, there~ore, are sign;f;~n~ in that they demonstrate the effectiveness 5 of the p-GlcNAc materials of the invention for the prevention of post surgical adhesions. Specifically, the results presented here demonstrate the e~ficacy of p-GlcNAc baæed solid and liquid formulations as barriers to the fQrmation of Ahtl~ ;n;~ post surgical lO adhesions in accepted rat and pig animal model 13y8 tems .
One of the accepted animal models used to ~tudy adhesion formation employs visceral-parietal peritoneal adhesions in ~prague-Dawley rats. Both l5 partially deacetylated p-GlcNAc membranes and p-GlcNAc-lactate gel formulations prevented and/or considerably reduced the ; nr; ri~n~-e of adhesion formation as compared with either non-treated controls treated with InterCE~D1M (Johnson ~ Johnson), the only 2 0 commercially available product :Eor this indication .
Specifically, a total of l~ rat~ were u~ed to test p-GlcNAc-lactate gel formulations. 12 animals were used a~ controls, with 6 receiving no treatment and 6 receiving InterCeed'M. 6 animal~ received 0.259f 25 p-GlcNAc-lactate gel, lO~ propylene glycol, water.
Animals recelving the p-GlcNAc-lactate gel treatment showed a significantly reduced inr~ nre Of postoperative adhesion formation, compared to either of the controls, as shown, below, in Table VIII.

WO 95/1~343 2 1 7 7 8 2 3 p~" s9~"3706 Extent of Tenacity Involvement Control ~No treatment) l +/- 2 .1 1 +/ - 1. 5 InterCEED'M 1 +/- 1. 8 1 +/- 1. 5 p-GlcNAc-lactate gel 0 +/- 0 . 8 1 +/- 1. 2 Partially aeacetylated p-GlcNAc membranes were also tested ~or their ability to prevent to occurrence o~ post surgical adhesions ln the rat animal model. A
A total of 22 rats were used in the study. 12 animal3 15 were used a8 controls, with 6 receiving no treatment and 6 receiving InterCEED'M Ten animalb each received a lcm x lcm membrane oi~ an approximately 60%
deacetylated p-glcNAc formulation. The animals which received the partially deacetylated p-GlcNAc membrane 20 showed a 8iSn~ nt reduction in the int~ n-e of f ormation o E po~stoperative adhesions, as ~ d with the non-treated controlb and InterCEED'U, as shown, ~elow, in Table IX.
TABLE IX
Extent o~ Tenacity Involvement 30 Control (No t~.=~tr^nt ) 3 +/- 1. 8 1 +/- 0 . 6 InterCEED~ 3 +/- 1.6 1 +/- 0.4 p-GlcNAc-membrane 1 +/- 0 . 8 1 +/- 0 . 3 WO 95115343 2 1 7 7 8 2 3 PCT/US9~/13706 Large animal bowel models i or the prevention of peritoneal adhesions were also used to test p-GlcNAc compositions. Specifically, six pigs and one horse were used to study both the partially deacetylated p-5 GlcNAc membrane and the p-GlcNAc-lactate gel The partially deacetylated p-GlcNAc membrane consisted of a 2 cm X 2 cm piece of 60~ deacetylated p-GlcNAc membrane, while the p-GlcNAc-lactate gel consisted of O . 25% p-GlcNAc lactate formulated with lO~ propylene 10 glycol and water Control animals received no treatment to the wounded site.
The results of these large animal studies revealed that, while the control sites formed multiple adhesions and scare tissue in the surrounding site, 15 both the p-GlcNAc membrane and gel formulations ef E~ectively prevented the formation of adhesions Samples ~rom control and treated sites were additionally f'Y~mi n~d using SEM, which showed an increased amount of fibrosis in the control sites as 20 compared to the treated tissues.

18. EXAMPLE: BIODEGRADABILITY OF p-GlcNAc ~AT~R T AT..S
The Example presented in this Section a5 demonstrates that p-GlcNAc materials o~ the invention may be prepared which exhibit controllable in vitro and in vivo biodegradability and rates of resorption.
18.1 MA~RTl~r~ ~ND l~ T~r)DS
p-GlcNA~ r-teri~ : Prototype I was made by the method described, above, in Section 5 . 3 . 2, via the chemical method, with hydro~luoric acid being utilized as the r~ l reagent Prototype I represented 100 acetylated p-GlcNAc.
The p-GlcNAc starting material of prototype 3A
was made by the method described, above, in Section WO 95/15343 2 1 7 7 8 2 3 PCT/US9~113706 5.3.2, via the chemical method, with hydrofluoric acid being utilized as the chemical reagent. The p-GlcNAc material was then deacetylated by the me~hod described, above, in Section 5.4 . SFor~ Al ly, the 5 p-GlcNAc material was treated with a 40~6 NaOH solution at 60C. for 10~ minutes . The resulting prototype 3A
was determined to be 30~ deacetylated.
The p-Glc~Ac starting material of prototype 4 was made by the method described, above, in Section 5.3.2, 10 via the chemical method, with hydrofluoric acid being utilized as the chemical rea~ent. The p-GlcNAc material was then deacetylated by treatment with a g0 NaOH solution at 60C. for 3C minutes. Next, the fibers were suspended in distilled water, frozen at -15 20C., and lyophilized to dryness. Prototype 4 wasalso determined to be 30~ deacetylated.
Ah~ in:~l im~lantation model: Sprague Dawley albino rats were utilized for the ~1 ~ ; niql implantation model studies. Animals were anesthetized 20 and prepared for surgery, and an incision was made in the skin and ;~hrlt~minzll muscles. The cecum was located and lifted out. A 1 cm x 1 cm membrane of p-GlcNAc materia~ was placed onto the cecum, and the incision was closed with nylon. Control animals were those in 25 which no material was placed onto the cecum.
Animals were opened at 14 and Z1 days post implantation. Photographs were taken during the implant and explant procedures (FIGS. 23A-E). Samples of cecum were prepared for histopathology after the 30 explant procedure.
D-GlcNAc in vitro deqradation lYsozYme-chitinase assaY: The assay i8 a r~lor;r^tric assay for N-acetyl glucosamine, and was performed as follows: 150/11 of a reaction sample was pipetted into 13xlOOmm glass 35 ~ poc;~hle tegt tubes, in duplicate. 251zl of 0.25M

Wo 9S/~s343 PCT/US9l/13706 potassium phosphate buffer (pH 7.1) was added to each test tube, followed by the addition of 35~L1 of 0 . 8M
potassium borate solution ~pH 9 . 8) . Tubes were immediately immersed into an ice-bath for a minimum of 2 minutes. Samples were then removed from the ice-bath, lml of freshly prepared DMA~3 reagent was added, and the samples were vortexed. DMA3 ~Dimethyl aminobenzaldehyde) reagent was made by adding 70mls of glacial acetic acid and lOmls of 11. 6N (concentrated) HCl to 8 grams of p-dimethyl ~nint~h~n7~ldehyde Samples were then incubated at 37C for 20 minutes.
To prepare a standard curve, the following procedure was utilized. A GlcNAc stock solution was diluted to 0 . lmg/ml with 0 . OlOM sodium acetate buffer (pH 4 5), and 01l1, 20~L1, 30~L1, 90~L1 or 120lL1 of the diluted GlcNAc solution was added to a set of test tubes. This wa6 followed by the addition of 150~L1, 130111, 60111 or 30f~1, respectively, of 0. OlOM sodium acetate buffer (pH 4.5) to the test tubes. Next, 25~1 of 0 25M potassium phosphate buffer (pH 7.1) and 35~L1 of 0 . 8M potassium borate buf f er (pH 9 . 8 ) were added to each test tube. A duplicate set of test tubes is prepared by the same procedure.
The test tubes are capped and boiled at 100C. for ~~
for exactly 3 minutes. The tubes are then immersed in an ice bath. The tubes are removed from the ice bath and lml of DMA~3 reagent, fres~ly prepared according to the method described above in the Section, is added to each tube. The tubes are incubated at 37C for 20 - 30 minutes. The absorbance of the contents of each tube is read at 585nM. Absorbance should be read as quickly as possible. The standard curve is plotted on graph paper and used to determine the concentration o~
N-acetyl glucosamine in the reaction samples. A
typical standard curve is shown in FIG. 23.

WO 95/15343 2 1 7 7 g 2 3 PCTrUS9~/13706 18 . 2 RESULTS
The in-vitro biodegradability of p-Glc~Ac materials was studied in experiments which assayed the relative susceptibility of p-GlcNAc membrane materials 5 to degradation, ~y lysozyme . p-GlcNAc membranes were exposed to an excesæ o~ lysozyme in a lOmM acetate buffer, and the subsequent release oi N acetyl glucosamine was determined using the assay described, above, in SectLon 18.1 The results of these experiments; n~1; r jZItf~l that partially deacetylated membranes are more~ susceptible to digestion by lysozyme (see FIG. 24) and, further, that the rate of lysozyme degradation is directly related to the extent of deacetylation (see FIG. 25, which compares the degradation rates of a 50% to a 251 deacetylated p-Glc~c membrane).
Additionally, experiments were performed which addressed the in-vivo biodegradability of p-GlcNAc materials . Such experiments utilized an ~h~ ; n;
implantation model Th~e~ p-GlcNAc materials, as listed below, were tested.
~-GlcNAc materials tested-1) p-GlcNAc, fully acetylated tdesignated prototype 1);
2 ) partially deacetylated p-GlcNAc membran~
(designated prototype 3A); and 3) lyophilized and partially deacetylated p-GlcNAc I ' ,r;3n~ (designated prototype 4~ .
The fully acetylated p-GlcNAc (prototype 1 ) was resorbed within 21 days, as shown in FIGS. 26A-26C. The partially deacetylated p-GlcNAc membrane (prototype 3A) was completely resorbed within 14 days, as shown in FIGS . 26D-26E~. ~ The lyophilized and PclluS94/l37o6 partially deacetylated p-GlcNAc membrane (prototype 4 ) had not yet been completely resorbed af ter 21 days post-implantation .
Histopathology analyses showed that once the 5 p-GlcNAc material has been resorbed there were no histological differences detectable between tissue samples obtained from the treated and from the control animals .

19. EXAMPLE: ~-GlcNAc HEMOSTASIS STUDIES =_ The experiments described herein study the efficacy of the p-GlcNAc materials of the invention for the control of bleeding. The success of the p-Glc~Ac materials in controlli~g bleeding is, further, ~=-15 compared against commercially available hemostatic products .
19.1 MATERIALS AND METHODS
~-GlcNAc and control materials: partially

20 deacetylated (approximately 709~) p-GlcNAc membranes were made using the chemical separation technique described, above, in Section 5.3.2, with hydrofluoric acid being utilized as the chemical reagent, and the techniques described, above, in Section 5.4. 2 cm x 1 25 cm pieces were used. p-GlcNAc-lactate gel (4% p-GlcNAc-lactate, formulated in propylene glycol and water) was produced following the methods described, above, in Section 17.1. The control material utilized for the study of bleeding in the spleen and liver was 30 Gelfoam~ (Upjohn Company) . Gelfoam'M and Avitene~
(Medchem Products, Inc. ) were the control materials used in the study of small blood vessel bleeding.
Test animals: New Zealand White rabbits were used. 3 animals received two wounds in the spleen and 35 one wound in the liver. 4 animals received five Wo 95/15343 2 1 7 7 8 2 3 PCT/US9~/13706 surgical wounds to blood vessels of similar size in the caudal mesenteric artery system.
'iurqical Pre~aration: The animals were anesthetized with k~t~ ICl and Xylazine. The 5 animals were placed in dorsal recumbency, and all the hair f rom the abdomen was removed . The abdomen was then scrubbed with povidone-iodine and 70~s isopropyl alcohol and draped ~or aseptic surgery.
Idver/s~leen surclical orocedures: A midline 10 incision was made and either the spleen or liver was exteriorized and packed with moist lap sponges. A 3-4 mm diameter cork bore was used to make a circular wound of about 2 mm depth at one end of the organ.
Once the splenic tissue was removed, a pre-weighed 15 4 ~ X 4 " gauze sponge was used to absorb all the blood lost from the splenic wound for a period of one minute. The sponge was re-weighed to ~uantify the amount of blood lost f rom that particular wound . The test animal was then treated by application to the 20 wound of one of the treatment materials. The time until hemostasis and the amount of blood lost prior to hemostasis was recorded After hemostasis in the fir6t wound was achieved, a second wound in the spleen and one wound in the 25 liver were made following the same procedure.
Small blood vessel surqical ~rocedure. A midline incision was made and the small bowel was exteriorized exposing the ,c,audal mesenteric artery system. The bowel was packed with moist lap sponges and f ive blood 30 vessels of about the same size were identified. A
scalpel was used to make a wound of about 1 mm depth at one of the vessels . A pre-weighed 4 " x 4 " gauze sponge was used to absorb all the blood lost from the vessel wound i~or a period o~ one minute. The sponge 35 was re-weighed to sluantify the amount~ of blood lost WO 9Y15343 2 1 7 7 8 2 3 PCT/USg4/13706 f rom that particular wound . The animal was then treated by application to the wound of one of the treatment materials The time until hemostasi~ and ~~
the amount of blood lost prior to hemostasis was 5 recorded.
After hemostasis in the first wound was achieved, four more wounds were made following the same procedure .
19 . 2 RES~l; S ~ -p-GlcNAc materials were tested for their ability to control bleeding in the spleen and liver o~ rat animal models. The p-GlcNAc materials tested were:
1) partially deacetylated (approximately 70~) p-15 GlcNAc; and 2) p-GlcNAc-lactate gel (49,~ p-GlcNAc-lactate, formulated in propylene glycol and water).
The effectiveness of these p-GlcNAc materials was compared to Gelf oam'M (Upj ohn Company) .
Each material was tested three times (twice in 20 the spleen and once in the liver). Both of the p-GlcNAc based materials exhibited an effectiveness in controlling bleeding within the f irst minute af ter application which was comparable to that of GelfoamTV.
The p-GlcNAc based materials have additional 25 advantages~ Specifically, the p-GlcNAc materials do not need to be held in place during the procedure, may be left in the body, where they will be resorbed within two to three weeks (Gelfoam~ is not indicated for this purpose), are compatible with both general 30 and m;n;m:qlly invasive surgical procedures.
Next, the efficacy of p-GlcNAc based materials in - the control of blee~ing in small blood ves~els was studied, and compared against commercially available hemostatic products.

WO 95/1~343 2 1 7 7 8 2 3 PCT/US9VI3706 - llo Each material was tested five times (twice in one of the animals and once in the other three animals) .
The p-GlcNAc membrane and gel formulations were easily applied to the site and co~trolled the bleeding within 5 2 minutes. Gelfoam'M, which had to be held in place in order to perform its function achieved hemostasis within the same 2 minute range as the p-GlcNAc materials . Avitene`V, a f ibrous material made of collagen, was difficult to handle and required more 10 than f ive minutes to control the bleeding .
Thus, the results described herein demonstrate that the p-GlcNAc materials tested here represent effective, convenient hemostatic agents.
20. EXAMP~E: o-GlcNAc DRUG DELIVER~ SYSTEMS
Described herein are studies demonstrating the successful use of p-GlcNAc materials to deliver anti-tumor drugs to the site of malignant skin cancer and colon cancer tumors such that the delivered anti tumor 2 0 drugs exhibit a therapeutic impact upon the tumors .
20.1 MATERIALS AND METHC)DS ~_ p-GlcNAc-lactate druq deliverY com~ositions:
Mixtures of 5'-fluorouracil (5'-FU) and p-GlcNAc-25 lactate were formulated as follows; O . 5mL of 5 ' -FU
(50mg/ml,) was mixed with 0.5mL of propylene glycol, and 2 . OmL of 4~ p-GlcNAc-lactate was added and mixed .
The p-GlcNAc-lactate was produced using the techniques described, above, in Section ---. Even after 30 extensive mixing, the 5'FU did not completely dissolve into the p-GlcNAc-lactate gel. Assuming complete mixing, the final c~n~ntr;stion of 5'-FU would be 6 . 25mg/mL .
Mixtures of mitomycin ~Mito) and p-GlcNAc-lactate 35 were formulatea as follows; 0.5mg of Mito (lyophili~ed

21 77823 9S/15343 PC r/US9 J/13706 powder) were dissolved in 5ml of propylene glycol, and 0 . 5ml of the Mito solution was mixed with 0 . 5mL of MPT' 8 4'6 p-GlcN-lactate preparation to give a final Mito rnnr~ntration of 0 2mg/ml and a final p-GlcNAc-5 lactate rnnrPntration of 2~. The materials were compatible, with the Mito dissolving easily into the p-GlcNAc-lactate gel.
p-GlcNAc ~ ~ ~ 5 ' FU de~ iverv CQml:)ositi(~nF::
Samples of 5'-fluorouracil (5'-FU) were immobilized 10 into discs of pure p-GlcNAc membrane material produced using the chemical separation method described, above, in Section 5.3.2, with hydrofluoric acid being llti 1 i 7~'i as the chemical reagent . Each disc described here had a diameter of 1 5cm, as described here.
For the preparation of high dose (HD) discs, 0 . 64mL of a 50mg/mL solution of 5 ~ -FU was mixed with suspensions containing approximately 8 mg of pure p-GlcNAc. The mixtures were allowed to stand for several hours to promote the absorption of 5 ' -FU into the p-GlcNAc, and were then dried at 55C for 3 5 hours. The resulting HD discs contained a total of 32mg 5 ' -FU, which is equivalent to approximately twice the normal total 14 day dose of 5 ' -FU typically given to a cancer patient, Low dose (LD) 5 ' -FU containing p-GlcNAc discs were prepared in the same manner, except that the LD
discs rnnt~3inf~d 17mg o 5'-FU, an amount er1uivalent to er~ual the normal total human dose for a 1~ day period, normalized to the weight of the experimental mice - 30 bal3ed on the typical dose of 5'-FU per Kg body weight f or humans .
- Te~t AniTn~ : For the 5'FU study, SCID (severe combined immunodeficiency) mice ~ere inoculated with 8l~hr~t~n~0ug flank injections of HT-29 colon cancer cells; (ATCC; lXlOs cells per inoculum) obtained by Wo 9511~343 2 1 7 7 8 2 3 PCrlUS9~/13706 standard tissue culture methods, in order to produce HT-29 colon cancer tumors. These injections led to palpable tumors which were harvested in 14-21 days.
Tumors were dissected and necrotic tissue was cut away. The HT-29 colon cancer tumors were sliced into 3x3x3mm pieces.
The experimental SCID mice were anesthetized via intra-peritoneal injections with a standard dose of avetin, and a slice of HT-29 colon caner tumor was implanted onto the cecum of each mouse. Specifically, each mouse was surgically opened to expose its abdomen and the cecum was located, which was nicked with a scalpel to make a small incision. A 3x3x3mm tumor slice was 6utured over the incision onto the cecum using 5 . o silk sutures. The abdomen was then closed using Clay Adams staples.
All mice were caged 6ingly and fed for two weeks.
All mice were healthy and none had obstructed colons at the end of the two week period.
On day 14, each mouse was anesthetized, and its abdomen was reopened. The growing tumors were measured ~length and horizontal dimensions). Tumors were then treated with the p-GlcNAc/anti-tumor drug or were used as controls.
Six mice were used for the p-GlcNAc-lactate 5 ' FU
study, and 15 mice were used for the p-GlcNAc membrane 5 ~ FU study .
For the mitomycin study, nine SCID mice were inoculated with sub-cutaneous injections of A431 squamous cell skin cancer cells (ATCC; lX10s cell~ per inoculum) . Tumors resulted in all mice within 14 days.
Treatments: For the p-GlcNAc-lactate 5'FU study, animals were treated once daily by "painting" the 5 ' -3 5 f 1 uo rourac i 1 ( 5 ~ - FU ) - cont a i n i ng p - GlcNAc ge 1 mixture onto the skin area over the tumor mass. Measurements of the tumor size were obtained daily. Control animals included animals treated with p-GlcNAc alone, without 5 ' -FU, and animals which received no 5 treatment.
For the p-GlcNAc membrane 5 ' FU study, the HT29 colon tumors in the SCID mice were treated by surgically implanting discs of the drug-r~nt~;nlng p-GlcNAc membrane material directly onto their surf ace, lO after having allowed the tumor to grow on the colon for 14 days. Mice were sacrificed 14 days following the implant procedure. Mea~u, ~ tF~ of tumor volumes were made immediately prior to implanting the drug-rr)nt~;n;nr p-GlcNAc membranes on day 0 and at the ---lS termination of the experiment on day 14. Control animals ; nrl ~ ones treated with the p-GlcNAc membrane without 5 ' -FU, and controls which received no treatment. Additionally, two animals received daily systemic injections of 5'-FU in doses equivalent to 2 0 the HD and LD regimen .
For the p-GlcNAc-lactate Mito study, animals were treated daily as in the p-GlcNAc-lactate 5 ' -FU study, with 3 animals being treated with the Mito containing mixture. In addition, 3 animals were treated with p-GlcNAc minus Mito, 2 animal3 received no treatment, and l animal received propylene glycol.
2 0 . 2 RESYI.TS
20 . 2 . l ~-GlcNAr-LAcTATE 5 ' FU -Experiments designed to study the effect of p-GlcNAc-lactate 5 ' FU drug delivery systems on tumor size were conducted, as described, above, in Section 20 .l.
The largest length and width dimension were measured for each tumor and the cross-sectional area WO 9511~343 2 1 7 7 8 2 3 PCT/US9~/13706 ~

using these dimensi~ns was calculated. The cross-sectional area values are shown in Table X, below.

~ WO 95/15343 2 1 7 7 8 2 3 PCrNS9~113706 Table X
Aninal # Treatment Tumor ~ize (cm') Day 0 Day 4 Day 11 Day 15 CL + 5FU 63 90 168 156 2 CL + 5FU 48 56 70 88 Control - -4 CL 58 110 150 195,30 Control 15 5 Nothing 40 64 132 234 6 Nothing 28 42 100 132 9~ Tn~-reaSe in Size Day 0 Day 4 Day 11 Day 15 CL + 5FU 0 43 167 147 2 CL + 5FU 0 17 47 84 Control Control 5Nothing 0 61 232 488 - 306 Nothing 0 48 253 366 The data comparing p-GlcNAc-lactate 5 ' FU
treated animals with controls are shown in FIG~. 27-28. The data summarized in Table X and FIGS. 27-28 35 clearly suggest that the E~T-29 subc~ An~r~ tumors in the rats treated with the 5'-FU contA;nln~ p-GlcNAc-Wo 95/15343 2 1 7 7 8 2 3 PCT/US9VI3706 ~1 lactate gels have a signif icantly retarded rate of growth compared to controls Their growth has been slowed 2.5-fold in comparison to ~he p-GlcNAc-lactate gel controls and 4 - f old compared to the no treatment 5 controls.
20.2.2 ~-GlcNAc-l~ACTATE MIT0 Experiments designed to study the effect of p-10 GlcNAc-lactate 5'FU drug delivery~systems on tumor~
size were also cond~cted, as described, above, in Section 20.1~ -The largest length and width dimensions weremeasured for each tumor and the cross sectional area 15 using these dimensions was calculated. The cross-SF~rt inn~ 1 area values were as shown in Table XI, below .

~ WO 95/15343 2 t 7 7 8 2 3 PCTiUSg4/13706 Table XI
Animal # Treatment Tumor Size (cm2) Day 0 Day 3 Day 5 Day 8 5 1 pGlcN-L + 23 23 42 49 Mi to 2 pGlcN-L + 23 16 54 63 Mi to lO3 pGlcN-L + 72 99 Term Term Mito 4pGlcN-L 27 54 140 203 control 155 pGlcN-L 30 54 96 140 control 6pGlcN-L 30 58 200 221 control 207 Nothing 48 75 126 300 8Nothing 44 80 207 Dead 9Propylene ~9 86 180 216 glycol ~ rncrf~A~e in Size -~-Day 0 Day 3 Day 5 Day 8 pGlcN-L + 0 0 83 135 Mi to 30 2 pGlcN-L + 0 -30 135 174 Mito 3 pGlcN-L + 0 38 Term Term Mi to Wo 95/153~3 2 1 7 7 8 2 3 Pcr/13SsJ/13706 e 4 pGlcN-~ 0 1oo 419 652 control 5 pGlcN-~ 0 80 220 367 control 6 pGlc~-~ 0 93 567 637 control 7 Nothing o 56 16} 525 10 8 Nothing 0 82 370 Dead 9 Propylene 0 76 267 341 glycol ~rhe data comparing p-GlcNAc-lactate Mito treated animals with controls are shown in FIGS. 29-30. The data 51 r; z~l in Table XI and FIGS . 29-30 clearly suggest that the tumors growing in the rats treated with the Mitomycin-containing p-GlcNAc-lactate gels animals have a $ignificantly retarded rate of growth.
Their growth was been ælowed by 4-fold in comparison to the p-GlcNAc-lactate gel controls and 4-fold compared to the no treatment controls.
20 . 2 . 3 ~-ÇlcNAc MEMBRANE 5 ' FU
Next, experiments deæigned to study the effect of p-GlcNAc membrane 5 ' FU drug delivery systems on skin cancer tumor size were conducted, as described, abo~re, in Section 20.1.
The tumor volume data obtained during the study, including percent change in volume cau$ed by the dif f erent tr~i~ tr- o n t c, iS summarized in Table XI I, below. Tumors were assumed to be cylindrical in shape. Their Yolumes were determined by measuring their width and length, and using the following ~ WO9SIIS343 21 7 7 8 2 3 PCT~US94JI3706 equation: V=7rr~1, where the radius r is 0 . 5 times the width and l is the length WO95J15343 21 77823 PCTIUS9J1~3706 ~
- 120 ~
~ t~
~ ~ o ~ o ~ ~ ~ 3 .~ .
o ~ ~ . o ~ X ~ . o .
m X
O ~ ~ ~ t--r . . c O
a Q a a a a ~ ~ a ~ o l x E ' o~ E
3 ~

", ~ ~ r~
o o ~ o o o o ~ o l_ o ~ o o U
o ~ ~ ~t ,, o 2 8 - a a a b ~ ~

WO 95/15343 2 1 7 7 8 2 3 PCT/US9~113706 FIG 31 summarizes a portion of the data presented, above, in Table XII. as shown in FIG. 31, the data strongly suggest that tumors treated with the >
high dose (HD) 5' -FU-~ rntAln;ng p-GlcNAc membranes 5 have stopped growing and have, i~ all cases, actually gotten significantly smaller. The low dose (~D) polymer materials resulted in disease stability and slight decrease in tumor size. In contrast, the tumors in the control animals conti~ued to rapidly l0 increase in size. It is interesting to note that two of the three control animals which were treated via IV
died during the study, i nrl; (-At i ng that systemic delivery of the equivalent amount of ~'-FU is lethal, whereas site-specif ic delivery via the p-GlcNAc 15 polymer is efficacious in ridding the animal o the disease .
20 . 3 CC~NCLUSION ~ ~
The data presented in this Section strongly 20 suggest that the site-specific delivery of anti-tumor drugs has a positive ef ect in retarding and reversing tumor growth. Successful results were obtained using p-GlcNAc drug delivery compositions produced having two different ormulations, namely p-GlcNAc-lactate 25 and p-GlcNAc membrane ormulations. Further, successful results were ~l1htA;n~l using two different anti-tumor drugs, ~'-FU and Mito. Thus, the p-GlcNAc drug delivery systems of the invention exhibit anti-tumor activity, useful, for example, in the delivery 30 of drugs specifically to the site of the tumor cells of interest.
It is apparent that many modif ications and variations of this invention as set forth here may be 35 made without departing from the ~ spirit and scope ~ Wo 95/15343 2 1 7 7 8 2 3 PCT/US9~113706 thereof, The specific embodiments described above are given by way of example only, and the invention is llmited only by the terms of the appended claim~.
-

Claims (165)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for isolating poly-.beta.-1.fwdarw.4-N-acetylglucosamine comprising about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons comprising:
a) culturing a microalgae comprising a cell body and a poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber in a sterile culture solution having a neutral pH;
b) agitating the culture in step (a) about every 8 to 12 hours;
c) subjecting the microalgae to a mechanical force for a time sufficient to separate the cell body from the poly-.beta.-1.fwdarw. 4-N-acetylglucosamine fiber;
d) segregating the poly-.beta.-1-.fwdarw.4-N-acetylglucosamine fiber from the cell body; and e) treating the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber with an organic solvent or a detergent, so that all protein, substantially all other organic contaminants, and substantially all inorganic contaminants are removed from the segregated poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber, and the poly-.beta.-1-.fwdarw.4-N-acetylglucosamine is isolated.

2. The method of claim 1 wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine isolated comprises about 4,000 to about 15,000 N-acetylglucosamine monosaccharides and has a molecular weight of about 800,000 daltons to about 3 million daltons.

3. The method of claim 1 wherein the mechanical force is a shear force or a cutting force.

4. The method of claim 1 wherein the microalgae is a diatom.

5. The method of claim 4 wherein the diatom is of the genus Thalassiosira.

6. The method of claim 5 wherein the diatom of the genus Thalassiosira is Thalassiosira fluviatilis or Thalassiosira weissflogii.

7. A method of isolating poly-.beta.-1 .fwdarw.4-N-acetylglucosamine comprising about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons comprising:
a) treating a microalgae comprising a cell body and a poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber with a chemical capable of weakening the microalgae cell wall at a concentration that does not disrupt the cell body for a sufficient time so that the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber is released from the intact cell body;
b) segregating the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber from the cell body; and c) removing all protein, substantially all other organic contaminants, and substantially all inorganic contaminants from the segregated poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber;
so that the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is isolated.

8. The method of claim 7 wherein the poly-.beta.-1.fwdarw.4-acetylglucosamine isolated comprises about 4,000 to about 15,000 N-acetylglucosamine monosaccharides and has a molecular weight of about 800,000 daltons to about 3 million daltons.

9. The method of claim 7 wherein the chemical is hydrofluoric acid.

10. The method of claim 7 further comprising neutralizing the segregated poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber prior to step (c).

11. The method of claim 7 wherein the microalgae is a diatom.

12. The method of claim 11 wherein the diatom is of the genus Thalassiosira.

13. The method of claim 12 wherein the diatom of the genus Thalassiosira is Thalassiosira fluviatilis or Thalassiosira weissflogii.

14. The method of claim 1 wherein the pH of the sterile culture solution is about 7.0 to about 7.4.

15. The method of claim 14 wherein the pH of the sterile culture solution is maintained by carbon dioxide dissolved in the sterile culture solution.

16. The method of claim 14 wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber is segregated from the cell body by fixed angle centrifugation.

17. The method of claim 14 wherein the organic solvent is ethanol.

18. The method of claim 14 wherein the detergent is sodium dodecyl sulfate.

19. A poly-.beta.-1.fwdarw.4-N-acetylglucosamine comprising about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons.

20. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 having about 4,000 to about 15,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, and having a molecular weight of about 800,000 daltons to about 3 million daltons.

21. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 or 20 wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a cell culture substrate.

22. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 or 20 wherein the poly-.beta.-1.fwdarw.4-acetylglucosamine is a mat, string, rope, microsphere, microbead, membrane, fiber, powder, or sponge.

23. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 or 20 wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a three dimensional matrix formulation.

24. A poly-.beta.-1.fwdarw.4-N-acetylglucosamine derivative comprising the poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 or 20 wherein at least one N-acetylglucosamine monosaccharide has been deacetylated.

25. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine derivative of claim 24 wherein at least about 25% to about 75% of the N-acetylglucosamine monosaccharides have been deacetylated.

26. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine derivative of claim 25 wherein at least about 70% of the N-acetylglucosamine monosaccharides have been deacetylated.

27. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine derivative of claim 24 wherein the derivative is a mat, string, rope, microsphere, microbead, membrane, fiber, powder, or sponge.

28. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine derivative of claim 24 wherein the derivative is a three dimensional matrix formulation.

29. A poly-.beta.-1.fwdarw.4-N-acetylglucosamine derivative comprising the poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 or 20 wherein at least one monosaccharide contains a sulfate group, a sulfonyl group, an O-aryl group, an N-aryl group, an O-alkyl group, an N-alkyl group, an N-alkylidene group, or an N-arylidene group.

30. A poly-.beta.-1.fwdarw.4-N-acetylglucosamine derivative comprising the poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 or 20 wherein at least one monosaccharide is a phosphorylated derivative, a nitrated derivative, an alkali derivative, or a deoxyhalogen derivative.

31. A poly-.beta.-1.fwdarw.4-N-acetylglucosamine derivative comprising the poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 or 20 wherein at least one monosaccharide forms a salt or a metal chelate.

32. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 or 20 wherein the poly-.beta.-1.fwdarw.4-acetylglucosamine is isolated from a microalgae source.

33. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 32 wherein the microalgae source is a diatom source.

34. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 33 wherein the diatom is of the genus Thalassiosira.

35. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 34 wherein the diatom of the genus Thalassiosira is Thalassiosira fluviatilis or Thalassiosira weissflogii.

36. A poly-.beta.-1.fwdarw.4-glucosamine comprising about 4,000 to about 150,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 640,000 daltons to about 24 million daltons.

37. The poly-.beta.-1.fwdarw.4-glucosamine of claim 36 having about 4,000 to about 15,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, and having a molecular weight of about 640,000 daltons to about 2.4 million daltons.

38. A poly-.beta.-1.fwdarw.4-glucosamine comprising about 4, 000 to about 150,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, wherein at least one glucosamine monosaccharide has been acetylated.

39. The poly-.beta.-1.fwdarw.4-glucosamine of claim 38 wherein at least about 25% to about 75% of the glucosamine monosaccharides have been acetylated.

40. The poly-.beta.-1.fwdarw.4-glucosamine of claim 39 wherein at least about 30% of the glucosamine monosaccharides have been acetylated.

41. The poly-.beta.-1.fwdarw.4-glucosamine of claim 36 or 38 wherein the poly-.beta.-1.fwdarw.4-glucosamine is a mat, string, rope, microsphere, microbead, membrane, fiber, powder, or sponge.

42. The poly-.beta.-1.fwdarw.4-glucosamine of claim 36 or 38 wherein the poly-.beta.-1.fwdarw.4-glucosamine is a three dimensional matrix formulation.

43. A poly-.beta.-1.fwdarw.4-glucosamine derivative comprising the poly-.beta.-1.fwdarw.4-glucosamine of claim 36 or 38 wherein at least one monosaccharide contains a sulfate group, a sulfonyl group, an O-acyl group, an N-acyl group, an O-alkyl group, an N-alkyl group, an N-alkylidene group, or an N-arylidene group.

44. A poly-.beta.-1.fwdarw.4-glucosamine derivative comprising the poly-.beta.-1.fwdarw.4-glucosamine of claim 36 or 38 wherein at least one monosaccharide is a phosphorylated derivative, a nitrated derivative, an alkali derivative, or a deoxyhalogen derivative.

45. A poly-.beta.-1.fwdarw.4-glucosamine derivative comprising the poly-.beta.-1.fwdarw.4-glucosamine of claim 36 or 38 wherein at least one monosaccharide forms a salt or metal chelate.

46. A poly-.beta.-1.fwdarw.4-N-acetylglucosamine derivative comprising the poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 wherein at least one monoaaccharide contains lactate.

47. A poly-.beta.-1.fwdarw.4-glucosamine derivative comprising the poly-.beta.-1.fwdarw.4-glucosamine of claim 36 or 38 wherein at least one monosaccharide contains lactate.

48. A poly-.beta.-1.fwdarw.4-acetylglucosamine comprising about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons isolated by a process comprising:
a) culturing a microalgae comprising a cell body and a poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber in a sterile culture solution having a neutral pH;
b) agitating the culture in step (a) every 8 to 12 hours;
c) subjecting the microalgae to a mechanical force for a time sufficient to separate the cell body from the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber;
d) segregating the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber from the cell body; and e) treating the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber with an organic solvent or a detergent;

so that all protein, substantially all other organic contaminants, and substantially all inorganic contaminants are removed from the segregated poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber, and the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is isolated.

49. A poly-.beta.-1.fwdarw.4-N-acetylglucosamine comprising about 4,000 to about 15,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, and having a molecular weight of about 800,000 daltons to about 3 million daltons isolated by a process comprising:
a) culturing a microalgae comprising a cell body and a poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber in a sterile culture solution having a neutral pH;
b) agitating the culture in step (a) every 8 to 12 hours;
c) subjecting the microalgae to a mechanical force for a time sufficient to separate the cell body from the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber;
d) segregating the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber from the cell body; and e) treating the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber with an organic solvent or a detergent;
so that all protein, substantially all other organic contaminants, and substantially all inorganic contaminants are removed from the segregated poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber, and the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is isolated.

50. A poly-.beta.-1.fwdarw.4-N-acetylglucosamine comprising about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons isolated by a process comprising:
a) treating a microalgae comprising a cell body and a poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber with a chemical capable of weakening the attachment between the cell body and the fiber at a concentration that does not disrupt the cell body for a sufficient time so that the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber is released from the intact cell body;
b) segregating the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber from the cell body; and c) removing all protein, substantially all other organic contaminants, and substantially all inorganic contaminants from the segregated poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber;
so that the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is isolated.

51. A poly-.beta.-1.fwdarw.4-N-acetylglucosamine comprising about 4,000 to about 15,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, and having a molecular weight of about 800,000 daltons to about 3 million daltons isolated by a process comprising:
a) treating a microalgae comprising a cell body and a poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber with a chemical capable of weakening the attachment between the cell body and the fiber at a concentration that does not disrupt the cell body for a sufficient time so that the poly-.fwdarw.-1.fwdarw.4-N-acetylglucosamine fiber is released from the intact cell body;
b) segregating the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber from the cell body; and c) removing all protein, substantially all other organic contaminants, and substantially all inorganic contaminants from the segregated poly-.beta.-1.fwdarw.4-acetylglucosamine fiber;
so that the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is isolated.

52. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 48, 49, 50 or 51, wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine exhibits the infrared spectrum shown in FIG. 4A or FIG. 4D.

53. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 48, 49, 50 or 51 wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine exhibits an infrared spectrum which does not contain a peak at 1740 cm-1

54. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 48, 49, 50 or 51 wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine yields an elemental analysis of approximately 47.13-47.42 weight percent carbon, 6.26-6.53 weight percent hydrogen, 6.76-7.15 weight percent nitrogen and 39.19-39.91 weight percent oxygen.

55. A drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition comprising poly-.beta.-1.fwdarw.4-N-acetylglucosamine comprising about 4,000 to 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons within which a drug is encapsulated.

56. The drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 55 wherein at least one N-acetylglucosamine monosaccharide has been deacetylated.

57. The drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 55 or 56 wherein the drug encapsulated is an antibiotic, an anti-inflammatory, an antifungal, an antoprotozoal or a spermicidal drug.

58. A drug/poly-.beta.-1.fwdarw.4-glucosamine composition comprising poly-.beta.-1.fwdarw.4-glucosamine comprising about 4,000 to about 150,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 640,000 daltons to about 24 million daltons within which a drug is encapsulated.

59. The drug/poly-.beta.-1.fwdarw.4-glucosamine composition of claim 58 wherein the drug encapsulated is an antibiotic, an anti-inflammatory, an antifungal, an anti-protozoal or a spermicidal drug.

60. A drug/poly-.beta.1.fwdarw.4-N-acetylglucosamine composition comprising poly-.beta.-1.fwdarw.4-N-acetylglucosamine of about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons wherein at least one N-acetylglucosamine monosaccharide has been deacetylated, and where at least one peptide is functionally attached to the deacetylated monosaccharide of the poly-.beta.-1.fwdarw.4-N-acetylglucosamine.

61. The drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 60 wherein the peptide is covalently attached to a deacetylated monosaccharide.

62. The drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 60 wherein the peptide is non-covalently attached to a deacetylated monosaccharide.

63. The drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 60 wherein the peptide is a growth factor, hormone, peptide recognition sequence, laminin, integrin, or cell adhesion molecule.

64. A drug/poly-.beta.-1.fwdarw.4-glucosamine composition comprising poly-.beta.-1.fwdarw.4-glucosamine of about 4,000 to about 150,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 640,000 daltons to about 24 million daltons wherein at least one glucosamine monosaccharide of the poly-.beta.-1.fwdarw.4-glucosamine contains a peptide functionally attached thereto.

65. The drug/poly-.beta.-1.fwdarw.4-glucosamine composition of claim 64 wherein the peptide is covalently attached to a glucosamine monosaccharide.

66. The drug/poly-.beta.-1.fwdarw.4-glucosamine composition of claim 64 wherein the peptide is non-covalently attached to a glucosamine monosaccharide.

67. The drug/poly-.beta.-1.fwdarw.4-glucosamine composition of claim 64 wherein the peptide is a growth factor, hormone, peptide recognition sequence, laminin, integrin, or cell adhesion molecule.

68. Use of the drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 55 or 56 for delivering the drug into the system of a patient as the poly-.beta.-1.fwdarw.4-N-acetylglucosamine degrades.

69. Use of the drug/poly-.beta.-1.fwdarw.4-glucosamine composition of claim 58 or 59 for delivering the drug into the system of a patient as the poly-.beta.-1.fwdarw.4-glucosamine degrades.

70. Use of the drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 60, of or 62 for controlled delivery of the attached peptide into the system of a patient as the poly-.beta.-1.fwdarw.4-N-acetylglucosamine degrades.

71. Use of the drug/poly-.beta.-1.fwdarw.4-glucosamine composition of claim 64, 65 or 66 for controlled delivery of the attached peptide into the system of a patient as the poly-.beta.-1.fwdarw.4-glucosamine degrades.

72. Use of the drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 57 for delivering the drug into the system of a patient as the poly-.beta.-1.fwdarw.4-N-acetylglucosamine degrades.

73. A biodegradable barrier-forming material comprising poly-.beta.-1.fwdarw.4-N-acetylglucosamine comprising about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons.

74. The biodegradable barrier-forming material of claim 73 wherein at least one N-acetylglucosamine monosaccharide is deacetylated.

75. The biodegradable barrier-forming material of claim 74 wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine has at least one lactate moiety functionally attached to the deacetylated monosaccharide.

76. A biodegradable barrier-forming material comprising an isolated poly-.beta.-1.fwdarw.4-glucosamine comprising about 4,000 to about 150,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 640,000 daltons to about 24 million daltons.

77. The biodegradable barrier-forming material of claim 76 wherein the poly-.beta.-1.fwdarw.4-glucosamine has at least one lactate moiety functionally attached to the glucosamine monosaccharide.

78. Use of the biodegradable barrier-forming material of claim 73, 74 or 75 for reducing post-surgical adhesions at a surgical site prior to performing a surgical procedure, so that lubrication is provided and surgical trauma to tissue is reduced.

79. Use of the biodegradable barrier-forming material of claim 76 or 77 for reducing post-surgical adhesions at a surgical site prior to performing a surgical procedure, so that lubrication is provided and surgical trauma to tissue is reduced.

80. Use of the biodegradable barrier-forming material of claim 73, 74 or 75 for reducing post-surgical adhesions at a surgical site after the completion of a surgical procedure, so that a physical barrier between traumatized tissue and non-traumatized tissue is produced.

81. Use of the biodegradable barrier-forming material of claim 76 or 77 for reducing post-surgical adhesions at a surgical site at the completion of a surgical procedure, so that a physical barrier between traumatized tissue and non-traumatized tissue is produced.

82. Use of the biodegradable barrier-forming material of claim 73, 74 or 75 to reduce the amount of traumatized tissue, fibrosis and scar tissue in wound healing.

83. Use of the biodegradable barrier-foaming material of claim 76 or 77 to reduce the amount of traumatized tissue, fibrosis and scar tissue in wound healing.

84. Use of the biodegradable barrier-forming material of claim 73, 74 or 75 to reduce blood loss in hemostasis.

85. Use of the biodegradable barrier-forming material of claim 76 or 77 to reduce blood loss in hemostasis.

86. The biodegradable barrier-forming material of claim 73, 74, 75, 76 or 77 wherein the material is a gel, sponge, film or membrane.

87. A hybrid composition, comprising poly-.beta.-1.fwdarw.4-N-acetylglucosamine comprising about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons, crosslinked to collagen.

88. The hybrid composition of claim 87 wherein the poly-.beta.1.fwdarw.4-N-acetylglucosamine has about 4,000 to about 15,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, and has a molecular weight of about 800,000 daltons to about 3 million daltons.

89. The hybrid composition of claim 87 in which the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a derivative wherein at least one N-acetylglucosamine monosaccharide has been deacetylated.

90. The hybrid composition of claim 88 in which the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a derivative wherein at least one N-acetylglucosamine monosaccharide has been deacetylated.

91. The hybrid composition of claim 87 in which the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a derivative wherein at least one monosaccharide contains a sulfate group, a sulfonyl group, an O-acyl group, an N-acyl group, an O-alkyl group, an N-alkylidene group, or an N-arylidene group.

92. The hybrid composition of claim 88 in which the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a derivative wherein at least one monosaccharide contains a sulfate group, a sulfonyl group, an O-acyl group, an N-acyl group, an O-alkyl group, an N-alkylidene group, or an N-arylidene group.

93. The hybrid composition of claim 87 in which the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a derivative wherein at least one monosaccharide is a phosphorylated derivative, a nitrated derivative, an alkali derivative or a deoxyhalogen derivative.

94. The hybrid composition of claim 88 in which the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a derivative wherein at least one monosaccharide is a phosphorylated derivative, a nitrated derivative, an alkali derivative or a deoxyhalogen derivative.

95. The hybrid composition of claim 87 in which the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a derivative wherein at least one monosaccharide forms a salt or a metal chelate.

96. The hybrid composition of claim 88 in which the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a derivative wherein at least one monosaccharide forms a salt or a metal chelate.

97. A hybrid composition, comprising poly-.beta.-1.fwdarw.4-glucosamine comprising about 4,000 to about 150,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 640,000 daltons to about 24 million daltons, crosslinked to collagen.

98. The hybrid composition of claim 97 wherein the poly-.beta.-1.fwdarw.4-glucosamine has about 4,000 to 15,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, having a molecular weight of about 640,000 daltons to about 2.4 million daltons.

99. The hybrid composition of claim 97 in which the poly-.beta.-1.fwdarw.4-glucosamine is a derivative wherein at least one glucosamine monosaccharide has been acetylated.

100. The hybrid composition of claim 98 in which the poly-.beta.-1.fwdarw.4-glucosamine is a derivative wherein at least one glucosamine monosaccharide has been acetylated.

101. The hybrid composition of claim 97 in which the poly-.beta.-1.fwdarw.4-glucosamine is a derivative wherein at least one monosaccharide contains a sulfate group, a sulfonyl group, an O-acyl group, an N-acyl group, an O-alkyl group, an N-alkylidene group, or an N-arylidene group.

102. The hybrid composition of claim 98 in which the poly-.beta.-1.fwdarw.4-glucosamine is, a derivative wherein at least one monosaccharide contains a sulfate group, a sulfonyl group, an O-acyl group, an N-acyl group, an O-alkyl group, an N-alkylidene group, or an N-arylidene group.

103. The hybrid composition of claim 97 in which the poly-.beta.-1.fwdarw.4-glucosamine is a derivative wherein at least one monosaccharide is a phosphorylated derivative, a nitrated derivative, an alkali derivative or a deoxyhalogen derivative.

104. The hybrid composition of claim 98 in which the poly-.beta.-1.fwdarw.4-glucosamine is a derivative wherein at least one monosaccharide is a phosphorylated derivative, a nitrated derivative, an alkali derivative or a deoxyhalogen derivative.

105. The hybrid composition of claim 97 in which the poly-.beta.-1.fwdarw.4-glucosamine is a derivative wherein at least one monosaccharide forms a salt or a metal chelate.

106. The hybrid composition of claim 98 in which the poly-.beta.-1.fwdarw.4-glucosamine is a derivative wherein at least one monosaccharide forms a salt or a metal chelate.

107. An isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species comprising a cell encapsulated by a poly-.beta.-1.fwdarw.4-N-acetylglucosamine species, wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine species comprises about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, is free of protein, substantially free of other organic contaminants, and substantially free of inorganic contaminants, and has a molecular weight of about 800,000 daltons to about 30 million daltons.

108. The cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species of claim 107 wherein at least one acetylglucosamine monosaccharide of the poly-.beta.-1.fwdarw.4-N-acetylglucosamine species has been deacetylated.

109. The isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species of claim 107 or 108 which is contained within a thermoplastic capsule.

110. The isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species of claim 109 wherein the thermoplastic capsule contains hydroxyethyl methylacrylate-methylmethacrylate copolymer.

111. The isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species of claim 107 or 108 wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine species is a membrane, three-dimensional porous matrix or a gel.

112. The isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species of claim 107 or 108 wherein the cell is a recombinantly engineered cell.

113. The isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species of claim 107 or 108 wherein the cell is a pancreatic islet cell.

114. The isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species of claim 107 or 108 wherein the cell is a adrenal chromaffin cell.

115. The isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species of claim 107 or 108 wherein the cell is derived from liver, pancreas, parathyroid, skin, cartilage, nerve tissue, bone, tendon, ligaments or blood vessels.

116. The isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species of claim 107 or 108 wherein the cell is derived from skin, cartilage, nerve tissue, bone, tendon, ligaments or blood vessels.

117. An isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species comprising a cell encapsulated by a poly-.fwdarw.-1.fwdarw.4-glucosamine species, wherein the poly-.beta.-1.fwdarw.4-glucosamine species comprises about 4,000 to about 150,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, is free of protein, substantially free of other organic contaminants, and substantially free of inorganic contaminants and has a molecular weight of about 640,000 daltons to about 24 million daltons.

118. The isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species of claim 117 which is contained within a thermoplastic capsule.

119. The isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species of claim 118 wherein the thermoplastic capsule contains hydroxyethyl methylacrylate-methylmethacrylate copolymer.

120. The isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species of claim 117 wherein the poly-.beta.-1.fwdarw.4-glucosamine species is a membrane, three-dimensional porous matrix or a gel.

121. The isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species of claim 117 wherein the cell is a recombinantly engineered cell.

122. The isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species of claim 117 wherein the cell is a pancreatic islet cell.

123. The isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species of claim 117 wherein the cell is a adrenal chromaffin cell.

124. The isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species of claim 117 wherein the cell is derived from liver, pancreas, parathyroid, skin, cartilage, nerve tissue, bone, tendon, ligaments or blood vessels.

125. The isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species of claim 117 wherein the cell is derived from skin, cartilage, nerve tissue, bone, tendon, ligaments or blood vessels.

126. An isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species according to claim 107 or 108 for therapeutic use, wherein cellular producers secreted by the encapsulated cells are released during said use.

127. An isolated cell/poly-.beta.-1.fwdarw.-glucosamine encapsulation species according to claim 117 for therapeutic use, wherein cellular products secreted by the encapsulated cells are released during said use.

128. An isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species according to claim 107 or 108 for therapeutic use against reduced or lost organ or tissue function to augment said reduced or lost function of the specific organ or tissue.

129. An isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species according to claim 117 for therapeutic use against reduced or lost organ or tissue function to augment said reduced or lost function of the specific organ or tissue.

130. An isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species according to claim 107 or 108 for therapeutic use for seed tissue regeneration at the site of an injury to promote tissue regeneration at the site of said injury.

131. An isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species according to claim 117 for therapeutic use for seed tissue regeneration at the site of an injury to promote tissue regeneration at the site of said injury.

132. The isolated cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine encapsulation species according to claim 126 wherein the cellular product secreted by the cell is insulin, nerve growth factor, a blood clotting factor, dopamine, an enkephalin, a dystrophin or human growth hormone.

133. The isolated cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation species according to claim 127 wherein the cellular product secreted by the cell is insulin, nerve growth factor, a blood clotting factor, dopamine, an enkephalin, a dystrophin or human growth hormone.

134. A method for immunoisolation of a cell comprising:
coating the cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine species of claim 107 or 108 with a coating having a polyelectrolyte charge opposite to the charge of the cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine species, so that the cell within the cell/poly-.beta.-1.fwdarw.4-N-acetylglucosamine species is immunoisolated.

135. An anti-tumor drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition comprising poly-.beta.-1.fwdarw.4-N-acetylglucosamine comprising about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons within which an anti-tumor drug is encapsulated.

136. The anti-tumor drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 135 wherein at least one N-acetylglucosamine monosaccharide has been deacetylated.

137. The anti-tumor drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 135 or 136 wherein the anti-tumor drug is 5'-fluorouracil, mitomycin, cis-platin, taxol, adriamycin, actinomycin, a bleomycin, a daunomycin or a methamycin anti-tumor drug.

138. An anti-tumor drug/poly-.beta.-1.fwdarw.4-glucosamine composition comprising poly-.beta.-1.fwdarw.4-glucosamine comprising about 4,000 to about 150,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants and having a molecular weight of about 640,000 daltons to about 24 million daltons within which an anti-tumor drug is encapsulated.

139. The anti-tumor drug/poly-.beta.-1.fwdarw.4-glucosamine composition of claim 138 wherein the anti-tumor drug is 5'-fluorouracil, mitomycin, cis-platin, taxol, adriamycin, actinomycin, a bleomycin, a daunomycin or a methamycin anti-tumor drug.

140. An anti-tumor drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition comprising poly-.beta.-1.fwdarw.4-N-acetylglucosamine of about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons wherein at least one N-acetylglucosamine monosaccharide has been deacetylated, and where at least one anti-tumor drug is functionally attached to the deacetylated monosaccharide of the poly-.beta.-1.fwdarw.4-N-acetylglucosamine.

141. The anti-tumor drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 140 wherein the drug is a peptide covalently attached to the deacetylated monosaccharide.

142. The anti-tumor drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 140 wherein the drug is a peptide non-covalently attached to a deacetylated monosaccharide.

143. An anti-tumor drug/poly-.beta.-1.fwdarw.4-glucosamine composition comprising about 4,000 to about 150,000 glucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants and having a molecular weight of about 640,000 daltons to about 24 million daltons wherein at least one glucosamine monosaccharide of the poly-.beta.-1.fwdarw.4-glucosamine contains an anti-tumor drug functionally attached thereto.

144. The anti-tumor drug/poly-.beta.-1.fwdarw.4-glucosamine composition of claim 143 wherein the drug is a peptide covalently attached to the glucosamine monosaccharide.

145. The anti-tumor drug/poly-.beta.-1.fwdarw.4-glucosamine composition of claim 143 wherein the drug is a peptide non-covalently attached to the glucosamine monosaccharide.

146. Use of the anti-tumor drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 135, 136 or 137 for delivering the anti-tumor drug into the system of a patient as the poly-.beta.-1.fwdarw.4-N-acetylglucosamine degrades.

147. Use of the anti-tumor drug/poly-.beta.-1.fwdarw.4-glucosamine of claim 138 or 139 for delivering the anti-tumor drug into the system of a patient as the poly-.beta.-1.fwdarw.4-glucosamine degrades.

148. Use of the anti-tumor drug/poly-.beta.-1.fwdarw.4-N-acetylglucosamine composition of claim 140, 141 or 142 for delivering the attached anti-tumor drug into the system of a patient as the poly-.beta.-1.fwdarw.4-N-acetylglucosamine degrades.

149. Use of the anti-tumor drug/poly-.beta.-1.fwdarw.4-glucosamine composition of claim 143, 144 or 145 for delivering the attached anti-tumor drug moiety into the system of a patient as the poly-.beta.-1.fwdarw.4-glucosamine degrades.

150. The method of claim 10 further comprising, after step (c):
d) placing the isolated poly-.beta.-1.fwdarw.4-N-acetylglucosamine in a basic pH environment.

151. The method of claim 150 further comprising:
e) adding a drug to the isolated poly-.beta.-1.fwdarw.4-N-acetylglucosamine so that the drug and the isolated poly-.beta.-1.fwdarw.4-N-acetylglucosamine are in the basic pH environment; and f) lowering the pH of the basic pH environment, so that the drug is encapsulated within the poly-.beta.-1.fwdarw.4-N-acetylglucosamine.

152. A method for isolating poly-.beta.-1.fwdarw.4-N-acetylglucosamine comprising about 4,000 to about 150,000 N-acetylglucosamine monosaccharides covalently attached in a .beta.-1.fwdarw.4 conformation, free of protein, substantially free of other organic contaminants, substantially free of inorganic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons comprising:
a) treating a microalgae comprising a cell body and a poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber with a biological agent capable of inhibiting poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber synthesis for a sufficient time so that the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber is released from the cell body;
b) segregating the poly-.beta.-1.fwdarw.4-N-acetylglucosamine fiber from the cell body; and c) removing all protein, substantially all other organic contaminants, and substantially all inorganic contaminants from the segregated poly-.beta.-1.fwdarw.4-N-acetylglucosamine fibers so that poly-.beta.-1.fwdarw.4-N-acetylglucosamine is isolated.

153. The method f claim 152 wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine isolated comprises about 4,000 to about 15,000 N-acetylglucosamine monosaccharides and has a molecular weight of about 800,000 daltons to about 3 million daltons.

154. The method of claim 152 wherein the biological agent is polyoxin-D.

155. The method of claim 152 wherein the microalgae is a diatom.

156. The method of claim 155 wherein the diatom is of the genus Thallasiosira.

157. The method of claim 156 wherein the diatom of the genus Thallasiosira is Thallasiosira fluviatilis or Thallasiosira weissflogii.

158. Poly-.beta.-1.fwdarw.4-N-acetylglucosamine isolated by the method of claim 152 or 153.

159. The poly-.beta.-1.fwdarw.4-glucosamine of claim 38 wherein at least one peptide is functionally attached to a glucosamine monosaccharide.

160. The poly-.beta.-1.fwdarw.4-glucosamine of claim 159 wherein the peptide is covalently attached to a glucosamine monosaccharide.

161. The poly-.beta.-1.fwdarw.4-glucosamine of claim 159 wherein the peptide is non-covalently attached to a glucosamine monosaccharide.

162. The poly-.beta.-1.fwdarw.4-glucosamine of claim 159 wherein the peptide is a growth factor, hormone, peptide recognition sequence, laminin, integrin, or cell adhesion molecule.

163. A cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation comprising a cell encapsulated by the poly-.beta.-1.fwdarw.4-glucosamine of claim 38.

164. The cell/poly-.beta.-1.fwdarw.4-glucosamine encapsulation of claim 163 wherein the encapsulation is contained within a thermoplastic capsule.

165. The poly-.beta.-1.fwdarw.4-N-acetylglucosamine of claim 19 wherein the poly-.beta.-1.fwdarw.4-N-acetylglucosamine is a poly-.beta.-1.fwdarw.4-N-acetylglucosamine-lactate.