WO2004011498A2 - Long lasting natriuretic peptide derivatives - Google Patents
Long lasting natriuretic peptide derivatives Download PDFInfo
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- WO2004011498A2 WO2004011498A2 PCT/CA2003/001097 CA0301097W WO2004011498A2 WO 2004011498 A2 WO2004011498 A2 WO 2004011498A2 CA 0301097 W CA0301097 W CA 0301097W WO 2004011498 A2 WO2004011498 A2 WO 2004011498A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P13/00—Drugs for disorders of the urinary system
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
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- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- NP natriuretic peptide
- the natriuretic peptide family includes four structurally related polypeptide hormones: Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP), C-type Natriuretic Peptide (CNP) and, recently discovered, Dendroaspis Natriuretic Peptide (DNP), (Yandle, 1994; Wilhins et al. 1997; Stein and Levin, 1998).
- ANP Atrial Natriuretic Peptide
- BNP Brain Natriuretic Peptide
- CNP C-type Natriuretic Peptide
- DNP Dendroaspis Natriuretic Peptide
- ANP and BNP mediate natriuresis, diuresis, vasodilatation, antihypertension, renin inhibition, antimitogenesis, and lusitropic properties (increase in the heart's rate relaxation).
- CNP lacks natriuretic actions but possesses vasodilating and growth inhibiting activity (Chen and Burnett, 2000).
- the natriuretic peptide family counterbalances the effects of the renin-angiotensin-aldosterone system (Espiner 1994,Wilkins et al. 1997, Levin et al.
- ANP and BNP have been shown to be physiological antagonists of the effects of angiotensin II (Ang II) on vascular tone, aldosterone secretion, renal-tubule sodium reabsorption, and vascular cell growth (Harris et al. 1987, Itoh et al. 1990, Wilkins et al. 1997, Levin et al. 1998).
- Ang II angiotensin II
- secretion of vasopressin Obana et al. 1985
- ET-1 endothelin-1
- ANP and BNP do not cross the brain-blood barrier (BBB) but they do reach areas near the central nervous system (i.e. subfornical organ and hypothalamus).
- the actions of NPs in the brain reinforce those in the periphery.
- Natriuretic peptide receptors are present in areas adjacent to the third ventricle that are not separated from the blood by the BBB, a position that allows binding of circulating ANP as well as locally produced peptide (Langub et al., 1995 in Kelly R. and Struthers A.D., 2001).
- natriuretic peptides are mediated through the binding and the activation of cell membrane receptors leading to cyclic GMP production in target cells. These include cGMP-dependent protein kinases (PKG), cGMP-gated ion channels and cGMP- regulated phosphodiesterases (Lincoln & Cornwell 1993, de Bold et al. 1996). Three subtypes of natriuretic peptide receptors have been described: NPR-A, NPR-
- NPR-A and NPR-B are guanylyl cyclases through which the ligands induce the production of cyclic guanosine monophosphate (cGMP) (for review see Maack 1992,
- NPR-A is thought to mediate many of the effects of ANP and BNP (Maack 1992, Davidson & Struthers 1994) while CNP acts via NPR-B receptors
- NPR-C is a clearance receptor for all three natriuretic peptides, which may signal through alternative pathways (Anand-Srivastava et al.
- ANP is a 28 amino acid peptide having a 17-an ⁇ ino acid loop formed by an intramolecular disulphide linkage between two cysteine residues, an amino tail of 6 amino acids and a carboxy tail of 5 amino acids.
- Non-cardiac sites that contain ANP include the brain, anterior lobe of the pituitary gland, the lung, and the kidney (Stein and Levin, 1998).
- BNP is a 32 amino acid peptide having a 17-amino acid loop formed by an intramolecular disulphide linkage between two cysteine residues, an amino-terminal tail of 9 amino acids and a carboxy-terminal tail of 6 amino acids.
- BNP the second member of the NP family, was first detected in 1988 in extracts of porcine brain as it names suggests (Sudoh et al., 1988). However, it was subsequently shown, similarly to ANP, to be expressed primarily in the ventricular myocardium (Minamino et al., 1988; Hosoda et al., 1991) as well as in the brain and amnion (Stein and Levin, 1998).
- BNP is released into the circulation when the heart is stretched (Kinnunen et al., 1993).
- Direct studies of BNP secretion from isolated perfused heart (Ogawa et al., Circ. Res. 1991), and from in-vivo and tissue studies in humans (Mukoyama et al., J. Clin. Invest. 1991), showed that 60-80% of cardiac BNP secretion arises from the ventricle.
- ANP is shown to have several therapeutic applications such as for hypertension and pulmonary hypertension (Veale et al.), asthma, renal failure, cardiac failure and radiodiagnostic (Riboghene Inc., Press Release 1998).
- BNP is shown to have several therapeutic applications such as for hypertension, asthma and inflammatory-related diseases (Ivax Corp., 2001), hypercholesterolemia (BioNumerik Pharmaceuticals Inc, 2000), emesis (BioNumerik Pharmaceuticals Inc, 1996), erectile dysfunction (Ivax Corp., 1998), renal failure (Abraham et al., 1995), cardiac failure and diagnostic of such (Marcus et al., 1995; Miller et al., 1994), solid tumor treatment (BioNumerik Pharmaceuticals ie, 1999) and protection of common and serious toxicity with placlitaxel in metastatic breast cancer (Hausheer et al., 1998, BioNumerik Pharmaceuticals Inc, 2001).
- ANP ANP
- BNP BNP
- Three independent mechanisms are responsible for the rapid clearance of ANP and BNP: 1) binding to NPR-C with subsequent internalization and lysosomal proteolysis; 2) proteolytic cleavage by endopeptidases such as DPP IV, NEP, APA, APP and ACE; and 3) renal secretion.
- urodilatin a natriuretic peptide found to be an amino-terminal extended form of ANP, shows that the sole presence of the four additional residues at the N-terminal renders it much more resistant to enzymatic degradation (Kenny et al. 1993). Nevertheless, urodilatin has only an in vivo half-life of approximately 6 min (Carstens et al., 1998).
- native human ANP is disclosed and claimed in US patent 5,354,900.
- Peptides with longer or shorter amino-tenninal or carboxy-terminal tails of the native ANP sequence are disclosed in US patent 4,607,023, US patent 4,952,561, US patent 4,496,544 and US patent 6,013,630.
- Fragments of the native ANP comprising the carboxy-terminal tail and a part of the loop are disclosed in US patent 4,673,732.
- Dimers of ANP are proposed in US patent 4,656,158 and JP application 62,283,996.
- Different ANP constructs are proposed in JP application 04,077,499, US patent 5,248,764 and application WO 02/10195.
- ANP sequences with truncation of the amino-terminal tail, the carboxy-terminal tail or the loop, elongation of the tails, addition of alkyl group at one of the tails, amino acid substitutions in the tails or in the loop and/or substitution of the cysteine by another bridging group are proposed in US patent 4,935,492, US patent 4,757,048, US patent 4,618,600, US patent 4,764,504, US patent 5,212,286, US patent 5,258,368, US patent 5,665,704, US patent 5,846,932, EP application 0,271,041, EP application 0,341,603, application WO 90/14362, US patent 5,095,004, US patent 5,376,635, EP application 0,350,318, EP application 0,269,299, US patent 5,204,328, US patent 5,057,603, EP application 0,244,169, US patent 4,816,443, CA patent 1,267,086, EP application 0,303,243, US patent 4,861,755, US patent 5,340,920, JP application 05,
- Linear peptides having a portion thereof that has some similarities with the loop section of ANP are disclosed in US patent 5,047,397, US patent 4,804,650 and US patent 5,449,662. Also, several number of derivatives, analogs, truncations, elongations and constructs of BNP are proposed and/or patented for improving the efficiency and/or the half-life of the native form of BNP; and the related prior art references are listed herein below.
- modifications include one or more of the following modifications: truncation of the amino tail; truncation of the carboxy tail; elongation of the amino tail with the prepro sequence or a fragment thereof; addition of an alkyl group at the amino tail or the carboxy tail; and amino acid substitutions in the tails or in the loop; as disclosed in US patent 5,114,923, US patent 5 5,948,761, US patent 6,028,055, US patent 4,904,763, application JP 07,228,598 and application WO 98/45329.
- the present NP derivative comprises a NP peptide having a reactive entity coupled thereto and capable of reacting with available functionalities on a blood component, either in vivo or ex vivo, to form a stable covalent bond and provide a NP peptide- blood component conjugate.
- the NP peptide Being conjugated to a blood component, the NP peptide is prevented from undesirable cleavage by endogenous enzymes such as NEP and most likely also o prevents binding to the NPR-C receptor which is responsible for a large amount of the blood clearance, thereby extending its in vivo half-life and activity.
- the covalent bonding fonned between the NP derivative and the blood component also substantially prevents renal excretion of the NP peptide until the blood component is degraded, thereby also contributing to extend its in vivo half-life to a period of time closer to the half-life of the blood component which can 5 represent an increase of 1 000 to 10 000 times.
- the reactive entity may be on the N-terminal or the C-terminal of the NP peptide, or on any other available site along the peptidic chain.
- a lysine residue may be added or substituted at the site of the peptidic chain where the reactive entity is attached.
- the NP peptide for derivatization according to the present invention is defined by the following formula, where it should be understood that a peptidic bond links Argis and He 1 and the line between Cys ⁇ and Cys 27 represents a direct disulfide bridge:
- Xi is Thr or absent
- X 2 is Ser, Thr, Ala or absent
- X 3 is Pro, Hpr, Val, or absent
- X is Val, He, Leu, Met, Phe, Ala, D-Ala, Nle or absent;
- X 7 is Gin, Asn, Arg, D-Arg, Asp, Lys, D-Lys or absent;
- X 8 is Gly, Pro, Ala, D-Ala, Arg, D-Arg, Asp, Lys, D-Lys, Gin, Asn or absent;
- X 9 is Ser, Thr or absent; 5 Xio is Gly, Pro, Ala, D-Ala, Ser, Thr or absent;
- X 12 is Phe, Tyr, Leu, Val, He, Ala, D-Ala, Phe with an isosteric replacement of its amide bond selected from the group consisting of N-oc-methyl, methyl amino, hydroxyl ethyl, hydrazino, ethylene, sulfonamide and N-alkyl- ⁇ -aminopropionic acid, or a Phe-replacement amino acid conferring on said analog resistance to NEP enzyme;
- o X 1 is Gly, Ala, D-Ala or Pro;
- X 14 is Arg, Lys, D-Lys, Asp, Gly, Ala, D-Ala or Pro;
- Xi 5 is Lys, D-Lys, Arg, D-Arg, Asn, Gin or Asp;
- Xi 6 is Met, Leu, He or an oxidatively stable Met-replacement amino acid;
- X 2 o is Ser, Gly, Ala, D-Ala or Pro;
- 5 X 2 ⁇ is Ser, Gly, Ala, D-Ala, Pro, Val, Leu, or He;
- X 22 is Ser, Gly, Ala, D-Ala, Pro, Gin or Asn;
- X 2 is Gly, Ala, D-Ala or Pro;
- X 26 is Gly, Ala, D-Ala or Pro;
- X 28 is Lys, D-Lys, Arg, D-Arg, Asn, Gin, His or absent;
- o X 29 is Val, He, Leu, Met, Phe, Ala, D-Ala, Nle, Ser, Thr or absent;
- X 30 is Leu, Nle, lie, Val, Met, Ala, D-Ala, Phe, Tyr or absent;
- X 31 is Arg, D-Arg, Asp, Lys, D-Lys or absent;
- X 32 is Arg, D-Arg, Asp, Lys, D-Lys, Tyr, Phe, Trp, Thr, Ser or absent;
- X 33 is His, Asn, Gin, Lys, D-Lys, Arg, D-Arg or absent;
- 5 Ri is NH 2 or a N-terrninal blocking group;
- R 2 is COOH, CONH 2 or a C-terminal blocking group.
- Preferred blood components comprise proteins such as immunoglobulins, including
- IgG and IgM serum albumin, ferritin, steroid binding proteins, transferrin, thyroxin binding protein, ⁇ -2-macroglobubn, haptoglobin etc.; serum albumin and IgG being more preferred; and serum albumin being the most preferred.
- Reactive entities are capable of forming a covalent bond with the blood component by reacting with amino groups, hydroxy groups, phenol groups or thiol groups present thereon, either in vivo or in vitro.
- the expressions "in vitro” and “ex vivo” are used in alternance in the specification and means the same in the context of the present invention since what takes place outside the body is performed in vitro, hi a preferred embodiment, the functionality on the protein will be a thiol group and the reactive entity will be a Michael acceptor, such as acrolein derivatives, ⁇ , ⁇ -unsaturated ketones, ⁇ , ⁇ -unsaturated esters, ⁇ , ⁇ -unsaturated amides, ⁇ , ⁇ - unsaturated thioesters, acrylamide, acrylic ester, vinyl benzoate, cinnamate, maleimide or maleimido-containing group such as ⁇ -maleimide-butyrylamide (GMBA) or maleimidopropionic acid (MPA), and
- a pharmaceutical composition comprising the NP derivative in combination with a pharmaceutically acceptable carrier.
- Such composition is useful for the treatment of congestive heart failure such as acute decompensated congestive heart failure of NYHA Class ⁇ , HI and IV and chronic congestive heart failure of NYHA Class HI and IV.
- the composition may also be used for the treatment of one of the following disorders or conditions: renal disorder, hypertension, asthma, hypercholesterolemia, inflammatory-related diseases, erectile dysfunction and for protection for toxicity of anti-cancer drugs.
- the present NP derivative may also be used for diagnostic or radiodiagnostic purposes.
- a conjugate comprising the present NP derivative covalently bonded to a blood component.
- a method for the treatment of congestive heart failure such as acute decompensated congestive heart failure of NYHA Class H, IH and IV and chronic congestive heart failure of NYHA Class HT and IV.
- the method comprises administering to a subject, preferably a mammal, animal or human, an effective amount of the NP derivative or the conjugate thereof, alone or in combination with a pharmaceutically acceptable carrier.
- a method for the treatment of renal disorder a method for the treatment of hypertension and a method for the treatment of asthma. These methods comprise administering to a subject, preferably a mammal, animal or human, an effective amount of the NP derivative or the conjugate thereof, alone or in
- a method for extending the in vivo half-life of a NP peptide in a subject comprising coupling to the NP peptide a reactive group which is capable of fonning a covalent bond with a blood o component, and covalently bonding the NP derivative to a blood component.
- the covalent bonding may take place in vivo or in vitro.
- the NP peptide or fragment thereof possesses natriuretic, diuretic, vasorelaxant and/or renin-angiotensin-aldosterone system modulating 5 activity. Details of the sequences of these peptides and fragments are illustrated below.
- the reactive entity is coupled to the NP peptide via a linking group, hi this case, the linking group is preferably defined as, without limitation, a straight or branched Ci-io alkyl; a straight or branched Ci-io alkyl partly or o perfluorinated; a Ci-io alkyl or fluoroalkyl wherein one or more carbon atom is replaced with O,
- the linking group can be stable or releasable so as to free the NP peptide if desired. 5
- Figure 1 shows the superposition of the LC/MS profiles of aNP peptide before and after cyclisation performed with the iodine method.
- Figure 2 shows the binding activity of commercial human ANP (hANP), o synthesized human ANP (native ANP) and four NP conjugates to guinea pig adrenal gland membranes by displacement of 125 I-rANP.
- Figure 3 shows the binding activity of synthesized human BNP (native BNP) and four NP conjugates to guinea pig adrenal gland membranes by displacement of I25 I-rANP.
- Figure 4 and 5 show the increase of cGMP production in human HELA cells being 5 incubated with in-house synthetized human ANP (native ANP), five NP conjugates and two NP peptides.
- Figure 6 shows the increase of cGMP production in human HELA cells being incubated with in-house synthetized human BNP (native BNP) and four NP conjugates.
- Figure 7 shows in vitro degradation in human plasma of hANP versus two corresponding NP conjugates.
- Figure 8 illustrates the site of cleavage of NEP enzyme along the hANP sequence.
- Figure 9 shows in vitro degradation by NEP enzyme of hANP versus a corresponding 5 NP conjugate, and capped human semm albumin as reference.
- Figure 10 shows the pharmacokinetic in rats of hANP (of commercial source and being synthetized in-house) versus two corresponding NP conjugates.
- Table 1 shows the three-letter code and one-letter code of amino acids.
- Table 2 shows the retention times of NP peptides and NP derivatives according to the present invention.
- Tables 3, 4 and 5 show three different gradients of elution of HPLC used for the analysis of NP peptide andNP derivatives of the present invention.
- Tables 6 and 7 compare the predicted and measured molecular weight of NP peptides, NP derivatives and NP conjugates.
- Table 8 shows the concentrations of 50% inhibition (EC50) and the inhibition constants (KT) calculated from the data used to draft Figure 2 i.e. binding activity of commercial human ANP (hANP), synthesized human ANP (native ANP) and four NP o conjugates to guinea pig adrenal gland membranes by displacement of I-rANP.
- Table 9 shows the concentrations of 50% inhibition (EC50) and the inhibition constants (KT) calculated from the data used to draft Figure 3 i.e. binding activity of synthetized human BNP (native BNP) and four NP conjugates to guinea pig adrenal gland membranes by displacement of 125 I-rANP.
- Table 10 lists the concentration of 50% inhibition (EC50) calculated from the data used to draft Figures 4, 5 and 6 i.e. the increase of cGMP production in human HELA cells being incubated with in-house synthetized human ANP (native ANP); in-house synthetized human BNP (native BNP); nine NP conjugates; and two NP peptides.
- Tables 11 and 12 show the gradients of elution of HPLC respectively used for the o analysis of NP peptides and NP derivatives of the present invention.
- Tables 13 and 14 show the in vivo effect of the injection of an NP derivative in SHR rats and Winstar-Kyoto rats respectively, on the increase of urine secretion and the increase of cGMP expression.
- In vivo bioconjugation is the process of covalently bonding a molecule, such as the NP derivative according to the present invention, within the body, to the targeted blood component, preferably a blood protein, in a manner that permits the substantial retention, or increase in some instances, of the biological activity of the original unmodified NP peptide in the conjugate form, while providing an extended duration of the biological activity though giving the NP peptide the biophysical parameters of the targeted blood component.
- a molecule such as the NP derivative according to the present invention
- the present NP derivative comprise a NP peptide that has 5 been chemically modified by coupling thereto a reactive entity, either directly or via a linking group which is a stable or releasable linking group.
- the reactive entity is capable of forming a covalent bond with a blood component, preferably a blood protein.
- the reactive entity must be stable in an aqueous environment.
- the covalent bond is generally formed between the reactive entity and an amino group, a hydroxyl group, or a thiol group on the blood component.
- the 0 amino group preferably forms a covalent bond with reactive entities like carboxy, phosphoryl or acyl; the hydroxyl group preferably forms a covalent bond with reactive entities like activated esters; and the thiol group preferably forms a covalent bond with reactive entities like esters or mixed anhydrides.
- the preferred blood components are mobile blood components like serum albumin, immunoglobulins, or combinations thereof, and the preferred reactive entity comprises 5 anhydrides like maleimide or maleimido-containing groups, hi a most preferred embodiment, the blood component is serum albumin and the reactive group is a maleimide-containing group.
- Protective groups maybe required during the synthesis process (which is described in detail below) to avoid interreaction between the reactive entity and the functional groups of the o NP peptide itself.
- These protective groups are conventional in the field of peptide synthesis, and can be generically described as chemical moieties capable of protecting the peptide derivative from reacting with other functional groups.
- Various protective groups are available commercially, and examples thereof can be found in US 5,493,007 which is hereby incorporated by reference. Typical examples of suitable protective groups include acetyl, 5 fluorenylniethyloxycarbonyl (FMOC), t-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ), etc.
- conjugation to a blood component defmively plays a major role in preventing the NP peptide from degradation by endogenous enzymes such as NEP and 0 preventing binding to the NPR-C receptor which the most important factor for the elimination of the natriuretic peptide from blood circulation.
- Conjugation to a blood component also overcomes renal excretion of the NP peptide as long as the blood component itself is being degraded. Therefore, the intrinsec half-life of the blood component selected for conjugation is the major determinant for the half-life of the conjugated NP peptide. 5
- the blood components are preferably mobile, which means that they do not have a fixed situs for any extended period of time, generally not exceeding 5 minutes, and more usually one minute. These blood components are not membrane-associated and are present in the blood for extended periods.
- Prefened mobile blood components include serum albumin, transferrin, ferritin, heptoglobin types 1-1, 2-1, 2-2 and immunoglobulins such as IgM, IgA and IgG.
- the present invention is directed to the modification of NP peptides
- NP peptide is a peptide having at least one of the l o physiologic activities of a native ANP or BNP, and particularly of human ANP and BNP. More particularly, NP peptide has natriuretic, diuretic, vasorelaxant and/or renin-angiotensin- aldosterone system modulating activity.
- Table 1 provides the three-letter code and one-letter code for natural amino acids
- the design of the NP peptide for derivatization according to the present invention is 20 based on the sequence of native human ANP and BNP. Their sequences share very high similarities. Substitution by analogous amino acids are proposed for residues that seem less involved in the pharmaceutical activity according to our structural activity analysis. Therefore, the NP peptide according to the present invention corresponds to the sequence of the following formula, where it should be understood that a peptidic bond links Argis and He ⁇ 9 and the line between Cysn and Cys 2 represents a direct disulfide bridge that forms a loop in the sequence:
- X 2 is Ser, Thr, Ala or absent
- X is Pro, Hpr, Val, or absent
- X 4 is Lys, D-Lys, Arg, D-Arg, Asn, Gin or absent;
- X 5 is Met, Leu, He, an oxidatively stable Met-replacement amino acid, Ser, Thr or absent;
- X ⁇ is Val, He, Leu, Met, Phe, Ala, D-Ala, Nle or absent;
- X 7 is Gin, Asn, Arg, D-Arg, Asp, Lys, D-Lys or absent;
- X 8 is Gly, Pro, Ala, D-Ala, Arg, D-Arg, Asp, Lys, D-Lys, Gin, Asn or absent;
- X 9 is Ser, Thr or absent
- Xio is Gly, Pro, Ala, D-Ala, Ser, Thr or absent;
- X 12 is Phe, Tyr, Leu, Val, He, Ala, D-Ala, Phe with an isosteric replacement of its amide bond selected from the group consisting of N-oc-methyl, methyl amino, hydroxyl ethyl, hydrazino, ethylene, sulfonamide and N-alkyl- ⁇ -aminopropionic acid, or a Phe-replacement amino acid conferring on said analog resistance to NEP enzyme;
- X 13 is Gly, Ala, D-Ala or Pro
- X 14 is Arg, Lys, D-Lys, Asp, Gly, Ala, D-Ala or Pro
- X1 5 is Lys, D-Lys, Arg, D-Arg, Asn, Gin or Asp;
- X ⁇ 6 is Met, Leu, He or an oxidatively stable Met-replacement amino acid
- X 20 is Ser, Gly, Ala, D-Ala or Pro
- X 2 ⁇ is Ser, Gly, Ala, D-Ala, Pro, Val, Leu, or He;
- X 22 is Ser, Gly, Ala, D-Ala, Pro, Gin or Asn;
- X 24 is Gly, Ala, D-Ala or Pro
- X 26 is Gly, Ala, D-Ala or Pro
- X 28 is Lys, D-Lys, Arg, D-Arg, Asn, Gin, His or absent;
- X 29 is Val, He, Leu, Met, Phe, Ala, D-Ala, Nle, Ser, Thr or absent;
- X 3 o is Leu, Nle, He, Val, Met, Ala, D-Ala, Phe, Tyr or absent;
- X 31 is Arg, D-Arg, Asp, Lys, D-Lys or absent;
- X 32 is Arg, D-Arg, Asp, Lys, D-Lys, Tyr, Phe, Trp, Thr, Ser or absent;
- X 33 is His, Asn, Gin, Lys, D-Lys, Arg, D-Arg or absent;
- Ri is NH 2 or a N-terminal blocking group
- R 2 is COOH, CONH 2 or a C-terminal blocking group.
- Xi is Thr or absent
- X 2 is Ala or absent
- X 3 is Pro or absent
- X 4 is Arg or absent;
- X 5 is Ser, Thr or absent;
- X ⁇ is Leu, He, Nle, Met, Val, Ala, Phe or absent;
- X 7 is Arg, D-Arg, Asp, Lys, D-Lys, Gin, Asn or absent;
- Xs is Arg, D-Arg, Asp, Lys, D-Lys, Gin, Asn or absent;
- X 9 is Ser, Thr or absent;
- Xio is Ser, Thr or absent;
- X ⁇ 2 is Phe, Tyr, Leu, Val, He, Ala, D-Ala, Phe with an isosteric replacement of its amide bond selected from the group consisting of N-oc-methyl, methyl amino, hydroxyl ethyl, hydrazino, ethylene, sulfonamide and N-alkyl- ⁇ -aminopropionic acid, or a Phe-replacement amino acid conferring on said analog resistance to NEP enzyme;
- X ⁇ 3 is Gly, Ala, D-Ala or Pro;
- X ⁇ is Gly, Ala, D-Ala or Pro;
- X 15 is Arg, Lys, D-Lys, or Asp;
- Xi 6 is Met, Leu, ⁇ e or an oxidatively stable Met-replacement amino acid
- X 2 o is Gly, Ala, D-Ala or Pro
- X 2 ⁇ is Ala, D-Ala, Val, Leu, or He;
- X 22 is Gin or Asn;
- X 2 is Gly, Ala, D-Ala or Pro;
- X 26 is Gly, Ala, D-Ala or Pro;
- X 28 is Asn, Gin, His, Lys, D-Lys, Arg, D-Arg or absent;
- X 9 is Ser, Thr or absent;
- X 3 o is Phe, Tyr, Leu, Val, He, Ala or absent;
- X 3 ⁇ is Arg, D-Arg, Asp, Lys, D-Lys or absent;
- X 32 is Tyr, Phe, Trp, Thr, Ser or absent;
- X 33 is absent;
- Ri is NH 2 or a N-terminal blocking group;
- R 2 is COOH, CONH 2 or a C-terminal blocking group
- Xi is Thr or absent
- X 2 is Ala or absent
- X 3 is Pro or absent
- X 4 is Arg or absent;
- X 5 is Ser or absent;
- X ⁇ is Leu or absent
- X 7 is Arg, Asp or absent
- X 8 is Arg, Asp or absent
- X 12 is Phe or Phe with an isosteric replacement of its amide bond selected from the group consisting of N-oc-methyl, methyl amino, hydroxyl ethyl, hydrazino, ethylene, sulfonamide and N-alkyl- ⁇ -aminopropionic acid;
- X 15 is Arg or Asp
- X 20 is Gly
- X 2 ⁇ is Ala; X 22 is Gin;
- X 24 is Gly
- X 26 is Gly
- X 28 is Asn or absent
- X 29 is Ser or absent;
- X 3 o is Phe or absent;
- X 3 ⁇ is Arg, Asp or absent
- X 32 is Tyr or absent
- X 33 is absent
- Ri is NH 2 or a N-terminal blocking group
- R 2 is COOH, CONH 2 or a C-terminal blocking group.
- Native human ANP is among the NP peptides in accordance with first embodiment of the present invention.
- Further preferred NP peptides i accordance with the first embodiment of the present invention are SEQ ID NO: 1, SEQ ID NO: 8, SEQ ED NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19.
- Preferred NP derivatives comprising NP peptides according to the first embodiment of the present invention, are SEQ LD NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ TD NO:l 1, SEQ ID NO: 14, SEQ DD NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20.
- SEQ LD NO: 2 SEQ ID NO: 3
- SEQ ID NO: 4 SEQ ID NO: 5
- SEQ ID NO: 6 SEQ ID NO: 7
- SEQ ID NO: 9 amino acid sequence
- Xi is absent
- X 2 is Ser, Thr or absent; X is Pro, Hpr, Val or absent;
- X 4 is Lys, D-Lys, Arg, D-Arg, Asn, Gin or absent;
- X 5 is Met, Leu, He, an oxidatively stable Met-replacement amino acid or absent;
- X ⁇ is Val, He, Leu, Met, Phe, Ala, D-Ala, Nle or absent;
- X 7 is Gin, Asn or absent
- X 8 is Gly, Pro, Ala, D-Ala or absent
- X 9 is Ser, Thr or absent
- Xio is Gly, Pro, Ala, D-Ala or absent;
- X ⁇ 2 is Phe, Tyr, Leu, Val, He, Ala, D-Ala, Phe with an isosteric replacement of its amide bond selected from the group consisting of N-oc-methyl, methyl amino, hydroxyl ethyl, hydrazino, ethylene, sulfonamide and N-alkyl- ⁇ -aminopropionic acid, or a Phe-replacement amino acid conferring on said analog resistance to NEP enzyme;
- X ⁇ is Gly, Ala, D-Ala or Pro
- X ⁇ is Arg, Lys, D-Lys, or Asp
- X 15 is Lys, D-Lys, Arg, D-Arg, Asn or Gin;
- X 16 is Met, Leu, He or an oxidatively stable Met-replacement amino acid;
- X 2 o is Ser, Gly, Ala, D-Ala or Pro;
- X 2 ⁇ is Ser, Gly, Ala, D-Ala or Pro;
- X 22 is Ser, Gly, Ala, D-Ala or Pro
- X 2 is Gly, Ala, D-Ala or Pro
- X 26 is Gly, Ala, D-Ala or Pro
- X 2 s is Lys, D-Lys, Arg, D-Arg, Asn, Gin or absent;
- X 29 is Val, ⁇ e, Leu, Met, Phe, Ala, D-Ala, Nle or absent;
- X 3 o is Leu, Nle, He, Val, Met, Ala, D-Ala, Phe or absent;
- X 3 ⁇ is Arg, D-Arg, Asp, Lys, D-Lys or absent;
- X 32 is Arg, D-Arg, Asp, Lys, D-Lys or absent;
- X 33 is His, Asn, Gin, Lys, D-Lys, Arg, D-Arg or absent;
- Ri is NH 2 or a N-terminal blocking group
- R 2 is COOH, CONH 2 or a C-terminal blocking group.
- Xi is absent;
- X 2 is Ser or absent;
- X is Pro or absent;
- X 4 is Lys or absent;
- X 5 is Met, He or absent
- Xg is Val or absent
- X 7 is Gin or absent; Xs is Gly or absent;
- X 9 is Ser or absent
- Xio is Gly or absent
- X1 2 is Phe or Phe with an isosteric replacement of its amide bond selected from the group consisting of N-oc-methyl, methyl amino, hydroxyl ethyl, hydrazino, ethylene, sulfonamide and N-alkyl- ⁇ -aminopropionic acid;
- X 14 is Arg or Asp
- X 15 is Lys or Arg; X 2 o is Ser;
- X 22 is Ser
- X 24 is Gly
- X 26 is Gly;
- X 28 is Lys, Arg or absent;
- X 29 is Val or absent
- X 3 o is Leu or absent
- X 31 is Arg, Asp or absent
- X 32 is Arg, Asp or absent; X 33 is His or absent.
- Native human BNP is among the NP peptides in accordance with second embodiment of the present invention.
- Further preferred NP peptides in accordance with the second embodiment of the present invention are SEQ HD NO: 21, SEQ BD NO: 22, SEQ HD NO: 23, SEQ HD NO: 25, SEQ HD NO: 28, SEQ HD NO: 31 , SEQ HD NO: 34, SEQ DD NO: 37, SEQ HD NO: 39, SEQ HD NO: 42, SEQ HD NO: 45, SEQ ID NO: 48 and SEQ HD NO: 51.
- Preferred NP derivatives comprising NP peptides according to the second embodiment of the present invention, are SEQ HD NO: 24, SEQ DD NO: 26, SEQ HD NO: 27, SEQ DD NO: 29, SEQ HD NO: 30, SEQ HD NO: 32, SEQ HD NO: 33, SEQ DD NO: 35, SEQ HD NO: 36, SEQ HD NO: 38, SEQ DD NO: 40, SEQ DD NO: 41, SEQ HD NO: 43, SEQ HD NO: 44, SEQ DD NO: 46, SEQ DD NO: 47, SEQ DD NO: 49, SEQ DD NO: 50, SEQ DD NO: 52, SEQ DD NO: 53, SEQ DD NO: 54, SEQ DD NO: 55, SEQ DD NO: 56 and SEQ DD NO: 57.
- the amino acids of the sequences of the NP peptides given in the present application may be D-amino acids or L-amino acids or combinations thereof, unless otherwise specified. L- amino acids
- the functionality on the protein will be a thiol group and the reactive entity will be a maleimide or maleimido-containing group such as ⁇ -maleimide-butyrylamide (GMBA) and maleimidopropionic acid (MPA).
- the reactive entity can be linked to the NP peptide via a stable or releasable linking group.
- the linking group corresponds is represented by formula V-W where V is a functional group reacting with the NP peptide and W is a chain moiety attached to the reactive entity.
- V is an ether, a thioether, a secondary or tertiary amine, a secondary or tertiary amide, an ester, a thioester, an imine, an hydrazone, a semicarbazone, an acetal, an hemi-acetal, a ketal, an hemi-ketal, an aminal, an hemi-aminal, an sulfonate, a sulphate, a sulfonamide, a sulfonamidate, a phosphate, a phophoramide, a phosphonate or a phosphonamidate, and preferably a primary amide.
- the linking group is preferably selected in the group consisting of hydroxyethyl motifs such as (2-amino) ethoxy acetic acid (AEA), ethylenediamine (EDA), 2-[2-(2- amino)ethoxy)] ethoxy acetic acid (AEEA); one or more alkyl chains (C1-C10) motifs such as glycine, 3-aminopropionic acid (APA), 8-aminooctanoic acid (AOA), 4-aminobenzoic acid (APhA).
- hydroxyethyl motifs such as (2-amino) ethoxy acetic acid (AEA), ethylenediamine (EDA), 2-[2-(2- amino)ethoxy)] ethoxy acetic acid (AEEA); one or more alkyl chains (C1-C10) motifs such as glycine, 3-aminopropionic acid (APA), 8-aminooctanoic acid (AOA), 4-amino
- linking groups are (2-amino) ethoxy acetic acid (AEA), ethylenediamine (EDA), and 2-[2-(2-amino)ethoxy)] ethoxy acetic acid (AEEA).
- Examples of combinations of linking group and reactive entity include, without limitations, (AEEA-EDA)-MPA; (AEEA-AEEA)-MPA, (AEA-AEEA)-MPA and the like.
- one or more additional amino acids may be added or substituted to the peptide at the site of coupling the reactive entity, via a linking group or not, prior to performing such coupling on the added or substituted amino acid, in order to facilitate the coupling procedure.
- the addition or substitution of amino acid(s) may be made at the N- terminal, the C-terminal, or therebetween. It is preferred to substitute an amino acid of the sequence of the NP peptide with Lys, D-Lys, Orn, D-Orn or 2,4-diaminobutanoic acid (DABA) and couple the reactive group on it, optionally via a linking group. To do so, lysine is the most preferred.
- Male nide groups are most selective for sulfhydryl groups on peptides when the pH of the reaction mixture is kept between 6.5 and 7.4. At pH 7.0, the rate of reaction of maleimido groups with sulfhydryls is 1000-fold faster than with amines. When a stable thioether linkage between the maleimido group and the sulfliydryl is formed, it cannot be cleaved under physiological conditions.
- the NP derivatives of the invention can provide specific labeling of blood components.
- the specific labeling particularly with a maleimide, offers several advantages. Free thiol groups are less abundant in vivo than amino groups, and as a result, maleimide derivatives covalently bond to fewer proteins. For example, in serum albumin, there is only one free thiol group per molecule. Thus, a NP peptide - maleimide - albumin conjugate will tend to comprise a 1:1 molar ratio of peptide : albumin, i addition to albumin, IgG molecules (class D) also have free thiols.
- IgG molecules and serum albumin make up the majority of soluble proteins in the blood, i.e., about 80-85%, they also make up the majority of the free thiol groups available to covalently bond to a NP derivative having a maleimido-containing group.
- Cys 34 of albumin is predominantly in the anionic form, which dramatically increases its reactivity, hi addition to the low pK value of Cys 34 , another factor which enhances the reactivity of Cys 34 is its location, which is in a hydrophobic pocket close to the surface of one loop of region V of albumin. This location makes Cys 34 accessible to ligands of all kinds, and is an important factor in Cys 34 's biological role as free radical trap and free thiol scavenger. As a result, the reaction rate acceleration can be as much as 1000-fold relative to rates of reaction of peptide-maleimides with other free-thiol containing proteins and with free thiols containing low molecular weight molecules.
- peptide-maleimide-albumin conjugates Another advantage of peptide-maleimide-albumin conjugates is the reproducibility associated with the 1:1 loading of peptide to albumin specifically at Cys 34 .
- Conventional activation techniques such as with glutaraldehyde, DCC, EDC and other chemical activators of, for example, free amines, lack this selectivity.
- human albumm contains 59 lysine residues, 25-30 of which are located on the surface of albumin and accessible for conjugation. Activating these lysine residues, or alternatively modifying a peptide to couple through these lysine residues, results in a heterogeneous population of conjugates.
- NP derivatives being capable of selectively covalently bonding with one functionality on a targeted blood component whith a degree of selectivity of 80% or more.
- the degree of selectivity is 90% or more, and more preferably, 95% or more.
- the desired conjugates of NP derivatives to blood components may be prepared in vivo by administration of the derivatives directly to the subject, which may be an animal or a human.
- the administration may be done in the form of a bolus, or introduced slowly over time by infusion using metered flow or the like.
- the conjugate may also be prepared ex vivo or in vitro by combining blood samples or purified blood components with the NP derivatives, allowing covalent bonding of the NP derivatives to the functionalities on blood components, and the resulting blood solution or the resulting purified blood component conjugates may be administered to the subject, animal or human.
- the purified blood components can be of commercial source, prepared by recombinant techniques or purified from blood samples. The blood may be treated to prevent coagulation during handling ex vivo.
- the invention is also directed to the therapeutic uses and other related uses of NP derivatives and fragments thereof having an extended half-life in vivo, and one or more of the following ANP-associated properties and BNP-associated properties:
- NPR-A natriuretic peptide receptors
- the NP derivatives or NP conjugates can be administered to patients that would benefit from inducing natriuresis, diuresis and vasodilatation.
- the NP derivatives and conjugates of the present invention are particularly useful to treat cardiac failure such as congestive heart failure (CHF) and more particularly acute decompensated CHF of NYHA Class D, DI and IV and chronic CHF of NYHA Class ID and IV.
- CHF congestive heart failure
- NP derivatives or NP conjugates can be administered in a single dose in acute CHF or following a long term medication for chronic CHF.
- NP derivatives or NP conjugates can be administered alone or in combination with one or more of the following types of compounds: ACE inhibitors, beta blockers, diuretics, spironolactone, digoxin, anticoagulation and antiplatelet agents, and angiotensin receptor blockers.
- NP derivatives and NP conjugates of the present invention include renal disorders and diseases, asthma, hypertension and pulmonary hypertension. More particularly for the NP derivatives and conjugates based on formula D, the following diseases and conditions can also be treated: inflammatory-related diseases, erectile dysfunction and hypercholesterolemia; and also be used as protectant for toxicity of anti-cancer drugs.
- Two or more NP derivatives or conjugates of the present invention may be used in combination to optimize their therapeutic effects. They can be administered in a physiologically acceptable medium, e.g. deionized water, phosphate buffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma, proteinaceous solutions, mannitol, aqueous glucose, alcohol, vegetable oil, or the like.
- PBS phosphate buffered saline
- Other additives which may be included include buffers, where the media are generally buffered at a pH in the range of about 5 to 10, where the buffer will generally range in concentration from about 50 to 250 mM, salt, where the concentration of salt will generally range from about 5 to 500 mM, physiologically acceptable stabilizers, and the like.
- the compositions may be lyophilized for convenient storage and transport.
- the NP derivatives and conjugates of the present invention may be administered orally, pulmonary, parenterally, such as intiavascularly (TV), intraarterially (IA), intramuscularly (TM), subcutaneously (SC), or the like.
- Administration by transfusion may be appropriate in some situations, hi some cases, administration may be oral, nasal, rectal, transdermal or by aerosol. It can be suitable to employ a single dose or multiple daily doses so as to build the desired systemic dosage, hi the case of chronic use, the inverval of administration are established in relation with subject's needs.
- the NP derivative or conjugate may be administered by any convenient means, including syringe, trocar, catheter, or the like. The particular manner of administration will vary depending upon the amount to be administered, whether a single bolus or continuous administration, or the like.
- the blood of the mammalian host may be monitored for the activity of NP peptides and/or presence of the NP derivatives or conjugates.
- NP peptides and/or presence of the NP derivatives or conjugates By taking a blood sample from the host at different times, one may determine whether the NP peptide has become bonded to the long- lived blood components in sufficient amount to be therapeutically active and, thereafter, detennine the level of NP peptide in the blood. If desired, one may also determine to which of the blood components the NP peptide is covalently bonded. Monitoring may also take place by using assays of peptide activity, HPLC-MS, antibodies directed to peptides, or fluorescent- labeled or radiolabeled derivatives.
- Another aspect of this invention relates to methods for detennining the concentration of the NP peptide or its conjugate in biological samples (such as blood) using antibodies specific to the NP peptide and to the use of such antibodies as a treatment for toxicity potentially associated with such NP peptide or conjugate.
- This is advantageous because the increased stability and life of the NP peptide in the patient might lead to novel problems during treatment, including increased possibility for toxicity.
- the use of anti-NP antibodies, either monoclonal or polyclonal, having specificity for NP, can assist in mediating any such problem.
- the antibody may be generated or derived from a host immunized with the particular NP derivative, or with an immunogenic fragment of the NP peptide, or a synthesized immunogen corresponding to an antigenic determinant of the NP peptide.
- Preferred antibodies will have high specificity and affinity any of the NP peptide, the derivatized form thereof and the conjugated form thereof.
- Such antibodies can also be labeled with enzymes, fluorochromes, or radiolabels.
- Antibodies specific for a particular NP derivative may be produced by using purified ; NP peptides for the induction of derivatized NP-specific antibodies. By induction of antibodies, it is intended not only the stimulation of an immune response by injection into animals, but analogous steps in the production of synthetic antibodies or other specific binding molecules such as screening of recombinant immunoglobulin libraries. Both monoclonal and polyclonal antibodies can be produced by procedures well known in the art. 0
- the antibodies may also be used to monitor the presence of the NP peptide in the blood stream.
- Blood and/or serum samples may be analyzed by SDS-PAGE and western blotting. Such techniques allow determination of the level of conjugation of the NPderivative.
- the anti-NP antibodies may also be used to treat toxicity induced by administration of the NP derivative, and may be used ex vivo or in vivo. Ex vivo methods would include immuno-dialysis treatment for toxicity employing anti-therapeutic agent antibodies fixed to solid supports. In vivo methods include administration of anti-NP antibodies in amounts effective to induce clearance of antibody-agent complexes. 0
- the antibodies may be used to remove the NP derivatives and conjugates thereof, from a patient's blood ex vivo by contacting the .blood with the antibodies under sterile conditions.
- the antibodies can be fixed or otherwise immobilized on a column matrix and the patient's blood can be removed from the patient and passed over the matrix.
- the 5 NP derivatives will bind to the antibodies and the blood containing a low concentration of NP, then may be returned to the patient's circulatory system.
- the amount of NP derivative removed can be controlled by adjusting the pressure and flow rate.
- Preferential removal of the NP derivative from the serum component of a patient's blood can be effected, for example, by the use of a semipermeable membrane, or by otherwise first separating the serum component from o the cellular component by ways known in the art prior to passing the serum component over a matrix containing the anti-therapeutic antibodies.
- the preferential removal of NP- conjugated blood cells, including red blood cells can be effected by collecting and concentrating the blood cells in the patient's blood and contacting those cells with fixed anti-NP antibodies to the exclusion of the serum component of the patient's blood. 5
- the anti-NP antibodies can be administered in vivo, parenterally, to a patient that has received the NP derivative or conjugates for treatment.
- the antibodies will bind the NP derivative and conjugates. Once bound, the NP activity will be hindered if not completely blocked thereby reducing the biologically effective concentration of NP derivatives in the patient's bloodstream and minimizing harmful side effects if any. hi addition, the bound antibody-NP complex will facilitate clearance of the NP derivative and conjugates from the patient's blood stream.
- the reactive entity via a linking group or not), such as MPA, is activated as a succinate ester for example (one skilled in the art can use haloacyl or p-nitrophenyl or others) and reacted with an amino group of NP peptide or derivative thereof produced by Solid Phase Synthesis or by recombinants means (see Example 2).
- the amino group is selected from the group consisting of the amino group of the C-terminal residue, the amino group of the N-terminal residue, or the amino group of the lateral chain of an amino acid such as Lys, D-Lys, Om, D-Orn and DABA.
- Peptide derivative synthesis NP peptides may be synthesized by standard methods of solid phase peptide chemistry well known to any one of ordinary skill in the art.
- the peptide may be synthesized by solid phase chemistry techniques following the procedures described by Steward et al. in Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, HI., (1984) using a Rainin PTI SymphonyTM synthesizer.
- peptides fragments may be synthesized and subsequently combined or linked together to form a larger peptide (segment condensation). These synthetic peptide fragments can also be made with amino acid substitutions and/or deletion at specific locations.
- the protected and/or derivatized amino acid is then either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected and under conditions suitable for forming the amide linkage.
- the protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added, and so forth.
- any remaining protecting groups are cleaved sequentially or concurrently to afford the final peptide.
- this general procedure it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide (segment condensation).
- the particularly preferred method of preparing the present NP derivatives of the present invention is solid phase peptide synthesis where the amino acid ⁇ -N-tenninal is protected by an acid or base sensitive group.
- Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein. Examples of N-protecting groups and carboxy-protecting groups are disclosed in Greene, "Protective Groups In Organic Synthesis," (John Wiley & Sons, New York pp. 152-186 (1981)), which is hereby incorporated by reference.
- N-protecting groups comprise, without limitation, loweralkanoyl groups such as formyl, acetyl ("Ac"), propionyl, pivaloyl, t-butylacetyl and the like; other acyl groups include 2-chloroacetyl, 2- bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, -chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl, o-mtrophenylsulfonyl, 2,2,5,7,8-pentamethylchroman-6- sulfonyl (pmc), and the like; carbamate forming groups such as t-amyl,
- Preferred ⁇ -N-protecting group are o-nitrophenylsulfenyl; 9-fluorenylmethyloxycarbonyl; t-butyloxycarbonyl (boc), isobomyloxycarbonyl; 3,5-dimethoxybenzyloxycarbonyl; t- amyloxycarbonyl; 2-cyano-t-butyloxycarbonyl, and the like, 9-fluorenyl-methyloxycarbonyl (Fmoc) being more preferred, while preferred side chain N-protecting groups comprise 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene- sulfonyl, Cbz, Boc, and adamantyloxycarbonyl for side chain amino groups like lysine and arginine; benzyl, o-bromobenzyloxycarbonyl, 2,6-dich
- a carboxy-protecting group conventionally refers to a carboxylic acid protecting ester or amide group.
- Such carboxy protecting groups are well known to those skilled in the art, having been extensively used in the protection of carboxyl groups in the penicillin and cephalosporin fields as described in US 3,840,556 and 3,719,667, the disclosures of which are hereby incorporated herein by reference.
- carboxy protecting groups comprise, without limitation, C ⁇ -C 8 loweralkyl; arylalkyl such as phenethyl or benzyl and substituted derivatives thereof such as alkoxybenzyl or nitrobenzyl groups; arylalkenyl such as phenylethenyl; aryl and substituted derivatives thereof such as 5-indanyl; dialkylaminoalkyl such as dimethylaminoethyl; alkanoyloxyalkyl groups such as acetoxymethyl, butyryloxymethyl, valeryloxymethyl, isobutyryloxymethyl, isovaleryloxymethyl, 1- (propionyloxy)-l -ethyl, l-(pivaloyloxyl)-l -ethyl, 1 -methyl- l-(propionyloxy)-l -ethyl, pivaloyloxymethyl, propionyloxymethyl; cycloalkanoyloxyalkyl groups such as cycl
- amide carboxy protecting groups comprise, without limitation, aminocarbonyl and loweralkylaminocarbonyl groups.
- loweralkyl, cycloalkyl or arylalkyl ester for example, methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, sec-butyl ester, isobutyl ester, amyl ester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl ester and the like or an alkanoyloxyalkyl, cycloalkanoyloxyalkyl, aroyloxyalkyl or an arylalkylcarbonyloxyalkyl ester are preferred.
- Preferred amide carboxy protecting groups are loweralkylaminocarbonyl groups.
- the ⁇ -C-terminal amino acid is attached 5 to a suitable solid support or resin.
- suitable solid supports useful for the above synthesis are those materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the media used.
- the preferred solid support for synthesis of ⁇ -C-terminal carboxy peptides is a Ramage Amide LinkerTM Resin (R. Ramage et al., THL, 34, p. 6599 (1993)).
- the preferred solid support for ⁇ -C-terminal o amide peptides Fmoc-protected Ramage Amide LinkerTM Resin.
- the Fmoc group is cleaved with a secondary amine, preferably piperidine, prior to coupling with the ⁇ -C-terminal amino acid as described above.
- the preferred method 5 for coupling to the deprotected 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy- acetamidoethyl resin is O-benzotriazol-l-yl-N j HN'jN'-tetramethyluroniumhexafluoro- phosphate (HBTU, 5 equiv.), diisopropylethylamine (DIEA, 5 equiv.), and optionally 1- hydroxybenzotriazole (HOBT, 5 equiv.), in DMF.
- the coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer in a conventional manner as is o well known in the art.
- the removal of the Fmoc protecting group from the ⁇ -N-terminal side of the growing peptide is accomplished conventionally, for example, by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in about 6-fold molar 5 excess, and the coupling is preferably carried out in DMF.
- the coupling agent is normally O- benzotriazol-l-yl-N,N,N',N'-tetramethyluroniumhexafluoro-phosphate (HBTU, 5 equiv.), diisopropylethylamine (DIEA, 5 equiv.), and optionally 1-hydroxybenzotriazole (HOBT, 5 equiv.).
- the peptide is removed from the resin and deprotected, either in successive operations or in a single operation. Removal of the polypeptide and deprotection can be accomplished conventionally in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising thioanisole, triisopropylsilane, phenol, and trifluoroacetic acid, hi cases wherein the ⁇ -C-terminal of the 5 polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide may be removed by transesterification, e.g. with methanol, followed by aminolysis or by direct transamidation.
- a cleavage reagent comprising thioanisole, triisopropylsilane, phenol, and trifluoroacetic acid
- the protected peptide may be purified at this point or taken to the next step directly.
- the removal of the side chain protecting groups is accomplished using the cleavage mixture described above.
- the fully deprotected peptide can be purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (such as Amberlite XADTM); silica gel adsorption chromatography;
- NP peptides and derivatives are cyclic.
- the thiol groups of the peptide can be reduced by a tallium, iodine or by the sulphoxide method.
- the iodine method is exemplified herein below in Example 1 and the sulphoxide method is exemplified herein below
- the cyclisation is preferably made with the sulphoxide method.
- a final purification is performed on the cyclised product.
- the preferred method of purification is by HPLC.
- the synthesis process for the production of the NP derivatives of the present 25 invention will vary widely, depending upon the nature of the various elements, i.e., the sequence of the NP peptide, the linking group and the reactive entity, comprised in the NP derivative.
- the synthetic procedures are selected to ensure simplicity, high yields and repetitivity, as well as to allow for a highly purified product.
- the chemically reactive entity will be coupled at the last stage of the synthesis, for example, with a carboxyl group, 3 o esterification to form an active ester. Specific methods for the production of the embodiment of NP derivatives of the present invention are described below.
- the chemically reactive entity be placed at a site to allow the peptide to covalently bond to the blood component while retaining a substantial proportion, if 35 not all, activity and/or beneficial effects of the corresponding NP peptide.
- the reactive group at a site along the peptidic sequence of the NP peptide selected so as to not interfere with the binding activity and the pharmacologic activity of the NP peptide.
- hi vitro assays may be used to select the best site to attach the reactive group.
- HBTU 0-benzotriazol-l-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate
- the Fmoc protective group was removed using 20% piperidine/DMF.
- a Boc-protected amino acid was used at the N-terminus in order to generate the free N ⁇ -terminus after the peptide was cleaved from the resin. All amino acids used during the synthesis possessed the L-stereochemistry unless otherwise stated. Glass reaction vessels were SigmacotedTM and used during the synthesis.
- the NP peptides and NP derivatives prepared in Examples 1 to 20 comprise NP peptides in accordance with the first preferred embodiment of the present invention
- ones prepared in Examples 21 to 57 comprise NP peptides in accordance with the second preferred embodiment of the present invention.
- a peptidic bond links the last amino acid on the first line and the first amino acid on the second line for each sequence given in the examples.
- the line between the two cysteines in each sequence illustrated in the present application represents a direct disulfide bridge that forms a loop in the sequence.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale.
- Boc-Ser(tBu)-OH Tliey were dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using 0-benzotriazol-l-yl-N, N, N', N'-tetramethyl-uromum hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was achieved using a solution of 20% (V/V) piperidine in N,N- dimethylformamide (DMF) for 20 minutes.
- DMF diisopropylethylamine
- Step 2 The peptide was cleaved from the resin using 85% TFA 5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold (0-4°C) Et 2 0. The crude peptide was collected on a polypropylene sintered funnel, dried, redissolved in a 20% mixture of acetonitrile in water (0.1% TFA) and lyophilized to generate the corresponding crude material used in the purification process. 5 Step 3: The resulting peptide fully deprotected and was purified according to the standard purification procedure detailed herein below. The desired fractions were collected pooled together and lyophilised.
- Step 4 The lyophilate of step 3 was placed in 2.5 mL AcOH/H 2 0 (1 : 1). Then iodine (I 2 ) (6 eq.) was added and followed by mass spectrometry (LC/MS) to monitor the reaction. The solution 0 was stirred at room temperature for 12 hours. After the elapsed time, a solution of vitamine C
- Step 5 The lyophilate of Step 4 was purified using standard purification procedure (detailed herein below).
- Step 1 Native Atrial Natriuretic peptide (provided by Phoenix Pharmaceuticals Inc., Belmont, CA, USA, catalog number 005-06) was placed in DMF. To the solution was added MPA- o AEEA-COO(Su) and N-Methyl Morpholine. The solution was stirred for 6 hours and then the solution was diluted (1:1) with water and it was purified according to the standard methodology.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale.
- N,N- dimethylformamide DMF
- HBTU O-benzotriazol-1-yl- N, N, N, N'-tetiamethyl-uronium hexafluorophosphate
- DIEA diisopropylethylamine
- Step 2 The peptide was cleaved from the resin using 85% TFA 5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold (0-4°C) Et 2 0.
- the crude peptide was collected on a polypropylene sintered funnel, dried, redissolved in a 20% mixture of acetonitrile in water (0.1%) TFA) and lyophilized to generate the corresponding crude material used in the purification process.
- Step 3 The resulting peptide fully deprotected, except for the Acm groups which remained attached to the thiol portion of the cysteine, and was purified according to the standard purification procedure detailed herein below. The desired fractions were collected pooled together and lyophilised.
- Step 4 The lyophilate of step 3 was placed in neat TFA (trifluoroacetic acid) (lmg/mL). Then anisole (100 eq.) was added followed by methyltrichlorosilane (lOeq.) and finally by diphenylsulphoxide (100 eq.). The solution was stirred at room temperature for 18 hours. After the elapsed time, the solution was placed in a separatory funnel with 2N Acetic acid (ImL/mg of peptide) and cold ether (5mL/mL of TFA). After multiple extractions, the desired cyclised peptides, present in the aqueous solution, were collected, combined together and lyophilised.
- Step 5 The lyophilate of Step 4 was purified using standard purification procedure (detailed herein below).
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly- OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)- H, Fmoc-Tyr(tBu)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, F oc- Asp(tBu)-OH, Fmo
- N,N- dimethylformamide DMF
- HBTU O-benzotriazol-1-yl- N, N, N', N'-tetramethyl-uronium hexafluorophosphate
- DIEA diisopropylethylamine
- Step 2 The selective deprotection of the Lys (Aloe) group was performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh 3 ) 4 dissolved in 5 mL of C 6 H 6 :CHC1 3 (1:1) : 2.5% NMM (v:v): 5% AcOH (v:v) for 2 h. The resin is then washed with CHC1 3 (6 x 5 mL), 20% AcOH in DCM (6 x 5 mL), DCM (6 5 mL), and DMF (6 5 mL). Step 3: The synthesis was then re-automated for the addition of the Fmoc-AEEA-OH.
- Step 5 The resulting peptide fully deprotected, except for the Acm groups which remained attached to the thiol portion of the cysteine, was purified according to the standard purification procedure. The desired fractions were collected pooled together and lyopliilised.
- Step 6 The lyophilate of step 3 was placed in neat TFA (trifluoroacetic acid) (lmg/mL). Then anisole (100 eq.) was added followed by methyltrichlorosilane (lOeq.) and finally by diphenylsulphoxide (lOOeq.). The solution was stirred at room temperature for 18 hours. After the elapsed time, the solution was placed in a separatory funnel with 2N Acetic acid (ImL/mg of peptide) and cold ether (5mL/mL of TFA). After multiple extractions the aqueous solution were collected, combined toghether and lyophilised.
- TFA trifluoroacetic acid
- Step 7 The lyophilate of Step 4 was purified using standard purification methodology.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc- Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc- Lys(Aloc)-OH, Fmoc-Gly-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmo
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale.
- Step 1 Same as Step 1 in example 2 using urodilatin as starting material.
- Urodilatin is provided by Bachem, Torance, CA, USA, catalog number H-3046.1000. 5
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)- 5 OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc- Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala- OH, Fmoc-Gly-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-
- Step 2-5 The steps were performed in the same manner as Example 3.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Asp(tBu)- OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc- Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Frnoc-Tyr(tBu)-OH, Fmoc-Asp(tBu)- OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc- Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala- OH, Fmoc-Gly-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-As
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly- OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, F
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly- OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-He-OH, Fmoc
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly- OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH,
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly- OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-
- Step 2-5 The steps were performed in the same manner as Example 3.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly- OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Met
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly- OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Met
- N,N-dimethylformamide N,N-dimethylformamide
- HBTU O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
- DIEA diisopropylethylamine
- Step 2-5 The steps were performed in the same manner as Example 3.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)- o OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- 0 Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, F
- Step 2 The peptide was cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% o phenol, followed by precipitation by dry-ice cold (0-4°C) Et 2 0. The crude peptide was collected on a polypropylene sintered funnel, dried, redissolved in a 40% mixture of acetonitrile in water (0.1% TFA) and lyophilized to generate the corresponding crude material used in the purification process.
- DMF diisopropylethylamine
- Step 3 The resulting peptide fully deprotected, except for the Acm groups which remained 5 attached to the thiol portion of the cysteine, and was purified according to the standard purification procedure detailed herein below. The desired fractions were collected pooled together and lyophilised.
- Step 4 Hie lyophilate of step 3 was placed in neat TFA (trifluoroacetic acid) (lmg/mL). Then anisole (100 eq.) was added followed by methyltrichlorosilane (lOeq.) and finally by 0 diphenylsulphoxide (100 eq.). The solution was stirred at room temperature for 18 hours. After the elapsed time, the solution was placed in a separatory funnel with 2N Acetic acid (ImL/mg of peptide) and cold ether (5mL/mL of TFA). After multiple extractions the aqueous solution were collected, combined together and lyophilised.
- TFA trifluoroacetic acid
- Step 5 The lyophilate of Step 4 was purified using standard purification procedure (detailed 5 herein below).
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- 5 OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fm
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following o protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, F oc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, F
- OH, MPA-OH were dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetiamethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was achieved using a solution of 20% (V/V) piperidine in N,N- o dimethylformamide (DMF) for 20 minutes.
- DMF N,N-dimethylformamide
- Step 2-5 The steps were performed in the same manner as Example 21.
- Step 1 Solid phase peptide synthesis was carried out on a lOO ⁇ mole scale.
- the following protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc- His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He- OH, Fmoc-Arg(Pbf)-OH
- N,N-dimethylformamide N,N-dimethylformamide
- HBTU O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
- DIEA diisopropylethylamine
- Step 2 The selective deprotection of the Lys (Aloe) group was performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh 3 ) 4 dissolved in 5 mL of C 6 H 6 :CHC1 3 (1:1) : 2.5% NMM (v:v): 5% AcOH (v:v) for 2 h. The resin is then washed with CHC1 3 (6 5 mL), 20% AcOH in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). Step 3: The synthesis was then re-automated for the addition of the Fmoc-AEEA-OH.
- Step 4 The peptide was cleaved from the resin using 85% TFA/5% TIS/5%> thioanisole and 5% phenol, followed by precipitation by dry-ice cold (0-4°C) Et 2 0.
- the crude peptide was collected on a polypropylene sintered funnel, dried, redissolved in a 40%) mixture of acetonitrile in water (0.1% TFA) and lyophilized to generate the corresponding crude material used in the purification process.
- Step 5 The resulting peptide fully deprotected, except for the Acm groups which remained attached to the thiol portion of the cysteine, was purified according to the standard purification procedure. The desired fractions were collected pooled together and lyophilised.
- Step 6 The lyophilate of step 3 was placed in neat TFA (trifluoroacetic acid) (lmg/mL). Then anisole (100 eq.) was added followed by methyltrichlorosilane (lOeq.) and finally by diphenylsulphoxide (100eq.). The solution was stirred at room temperature for 18 hours. After the elapsed time, the solution was placed in a separatory funnel with 2 ⁇ Acetic acid (ImL/mg of peptide) and cold ether (5mL/mL of TFA). After multiple extractions the aqueous solution were collected, combined toghether and lyophilised. Step 7: The lyophilate of Step 4 was purified using standard purification methodology.
- TFA trifluoroacetic acid
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- 5 OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fm
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following o protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, F
- OH, MPA-OH were dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was achieved using a solution of 20% (V/V) piperidine in N,N- o dimethylformamide (DMF) for 20 minutes.
- DMF N,N-dimethylformamide
- Step 2-5 The steps were performed in the same manner as Example 21.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc- His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile- OH, Fmoc-Arg(Pbf)-OH, F
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmo
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fm
- DMF diisopropylethylamine
- Step 2-5 The steps were performed in the same manner as Example 21.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-De-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmo
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc- His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fnioc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He- OH, Fmoc-Arg(Pbf)-OH, F
- N,N-dimethylformamide N,N-dimethylformamide
- DIEA diisopropylethylamine
- Step 2-7 The steps were performed in the same manner as Example 24.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmo
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fm
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale.
- Step 2-7 The steps were performed in the same manner as Example 24.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-De-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmo
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc- His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile- OH, Fmoc-Arg(Pbf)-OH, F
- Fmoc-AEEA-OH, MPA-OH were dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using 0-benzotriazol-l-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes. Step 2 to 7: The steps were performed in the same manner as Example 24.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Cys(Acm)-OHa, Fmoc-Gly- OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc-Cys(Acm)-OH,.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-C
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale.
- the following protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-De-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly- OH, Fmoc-Phe-OH, Boc-Cy
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc-Cys(Acm)- OH.
- N,N-dimethylformamide DMF
- O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
- HBTU HBTU
- DIEA diisopropylethylamine
- Step 2-5 The steps were performed in the same manner as Example 21.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-C
- N,N-dimethylformamide N,N-dimethylformamide
- HBTU O-benzotriazol-1-yl-N, N, N', N'-tetramethyl- uronium hexafluorophosphate
- DIEA diisopropylethylamine
- Step 2-5 The steps were performed in the same manner as Example 21.
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly- OH, Fmoc-Phe-OH, Boc-Cys(
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-As ⁇ (tBu)-OH, Fm
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmo
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc- His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-De- OH, Fmoc-Arg(Pbf)-OH, Fm
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, * Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, F oc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmo
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- i o Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, F
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following
- N,N- dimethylformamide DMF
- HBTU O-benzotriazol-1- yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Asp(tBu)- OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH,
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Asp(tBu)- OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH,
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH Fmoc- His(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-He- OH, Fmoc-Arg(Pbf)-OH, F
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-De-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmo
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc- His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-De- OH, Fmoc-Arg(Pbf)-OH, Fm
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-
- DMF N,N-dimethylformamide
- HBTU O-benzotriazol-1 -yl-N, N, N', N'-tetramethyl- uronium hexafluorophosphate
- DIEA diisopropylethylamine
- Step 1 Solid phase peptide synthesis was carried out on a 100 ⁇ mole scale. The following protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc- l o Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)
- Table 6 shows the predicted molecular weight (Predicted) and measured molecular weight (Measured) of compounds that are NP peptides and derivatives according to the present invention. All the molecular weights are expressed in g/mol. Molecular weight has been measured by Quadrupole Electro Spray mass spectroscopy. The predicted molecular weight has been established by addition of the theoretical mass of each atom. The differences between the predicted molecular weight and the measured molecular weight are negligible and indicate that the compounds synthesized are the desired compounds.
- Cyclisation is obtained by reduction of the thiol group of both cysteine residues of the 5 peptide so as to form an intiamolecular disulphide bridge and details of the process are in the specification and are exemplified in Step 4 of Example 1 and in Step 4 of Example 3.
- an Ellman test was performed on the final cyclised material as taught in G.L. Ellman, Arch. Biochem. Biophys., 82 (70) 1959 and G.L. Ellman, Biochem. Pharmacol., 7 (68) 1961.
- the Ellman test allows determination of thiol o groups that would not form disulphide bridges. The absence of free thiol groups indicates that the cyclisation was successful.
- FIG. 1 shows in supe ⁇ osition the LC/MS spectrums of the intermediates of synthesis of the compound of Example 1 before cyclisation illustrated in dotted line ( — ) and the corresponding final products after cyclisation illustrated in continuous line ( — ), wherein the cyclisation was performed with iodine as exemplified in Step 4 of Example 1.
- the intermediates have a molecular ion fragment of 771.2 (M+4) that corresponds to a mass of o 3080.8 and the final products have a molecular ion fragment of 770.5 (M+4) that corresponds to a mass of 3078.0.
- M+4 molecular ion fragment of 771.2
- M+4 molecular ion fragment of 770.5
- the reduction of the mass of 2.8 results from the loss of two hydrogens during the formation of the disulphide bridge.
- the sha ⁇ ness of the peaks of the linear intermediates and the cyclic final products indicate that all the intermediates were cyclised.
- the derivative is conjugated to a blood component.
- the blood component is human serum albumin (HSA).
- HSA is provided by Cortex-BiochemTM, San Leandro, CA, USA.
- Example 58 Preparation of 1 mM of the compound of Example 3:HSA conjugates.
- HSA 25% g/lOOml
- the HSA solution is vortexed.
- 50 ⁇ L of the compound of Example 3 at a concentration of lOmM in nanopure water, is added.
- the resulting solution is incubated at 37°C for 4 hours, and stored at 20°C.
- Example 59 Preparation of 1 mM of the compound of Example 4:HSA conjugates. Conjugation to HSA is performed in the same manner as Example 58.
- Example 60 Preparation of 1 mM of the compound of Example 5:HSA conjugates. Conjugation to HSA is performed in the same manner as Example 58.
- Example 61 Preparation of 1 mM of the compound of Example 6:HSA conjugates. Conjugation to HSA is performed in the same manner as Example 58.
- Example 62 Preparation of 1 M of the compound of Example 7:HSA conjugates. Conjugation to HSA is performed in the same manner as Example 58.
- Example 63 Preparation of 1 mM of the compound of Example 14:HSA conjugates. Conjugation to HSA is performed in the same manner as Example 58.
- Example 64 Preparation of 1 mM of the compound of Example 18:HSA conjugates.
- Example 65 Prepare 1 mM of the compound of Example 54:HSA conjugates. Conjugation to HSA is performed in the same manner as Example 58.
- Example 66 Preparation of 1 mM of the compound of Example 55:HSA conjugates. Conjugation to HSA is performed in the same manner as Example 58.
- Example 67 Preparation of 1 mM of the compound of Example 56:HSA conjugates. Conjugation to HSA is performed in the same manner as Example 58.
- Example 68 Preparation of 1 mM of the compound of Example 57:HSA conjugates. Conjugation to HSA is performed in the same manner as Example 58.
- LC/MS liquid chromatography/mass spectrometry
- Example 58 Conjugates of compound of Example 3 with HSA 2.2%
- Example 59 Conjugates of compound of Example 4 with HSA: 4.4%
- Example 60 Conjugates of compound of Example 5 with HSA 3.6%
- Example 61 Conjugates of compound of Example 6 with HSA: ⁇ 1%
- Example 62 Conjugates of compound of Example 7 with HSA ⁇ 1%
- Example 63 Conjugates of compound of Example 14 with HSA: 1.2%
- Example 64 Conjugates of compound of Example 18 with HSA: 1.3%
- Example 65 Conjugates of compound of Example 54 with HSA 1.4%
- Example 66 Conjugates of compound of Example 55 with HSA: 2.4%
- Example 67 Conjugates of compound of Example 56 with HSA 0.8%
- Example 68 Conjugates of compound of Example 57 with HSA: 2.1%
- Table 7 shows the predicted molecular weight (Predicted) and measured molecular weight (Measured) of conjugates of NP derivatives according to the present invention. All the molecular weights are expressed in g/mol. Molecular weight has been measured by Quadrupole Electro Spray mass spectroscopy. The predicted molecular weight has been established by addition of the theoretical mass of each atom. The differences between the predicted molecular weight and the measured molecular weight are negligible and indicate that the compounds synthesized are the desired compounds.
- NP derivatives The potency of NP derivatives is evaluated as their ability to bind NPR receptors in guinea pig adrenal glands and to elevate cGMP levels in a rat primary lung fibroblasts assay.
- cell lines can be used to perform these in vitro assays such as aortic smooth muscle cells, glomeruli mesangial cells and adrenal cells.
- Human, rat, and ginea pig cell lines or other 5 species cell lines can be used with a preference for human cell lines.
- Membranes for binding studies are prepared as follow.
- Adrenal glands were collected from anesthetized normal Duncan Hartley Guinea Pig and homogenized using a o polytron in 50 mM Tris-HCl buffer containing 150 mM NaCl, 5mM MgCl 2 , 5mM MnCl 2 ; pH 7.4 at 25°C.
- the homogenate was centrifuged for 10 minutes at 39,000 x g (4°C). The pellet was resuspended and washed.
- the membranes were resuspended in the same buffer supplemented with 1 mM Na 2 EDTA+0.2% BSA. Protein concentration is measured using the BCA protein assay kit (Pierce).
- the binding assay is done by incubation of membranes with 5 0.016 nM 125 I-rANF and increasing concentrations of either NP peptides or NP derivatives (10 "5 -10 “ ⁇ M) for 60 minutes at 4°C. All assays were done in duplicate. Separation of bound and free radioactive rANF was achieved by rapid filtration through polyethylenimine-treated Whatman GF/C filters soaked in assay buffer. Filters were washed, dried and counted for radioactivity in a gamma-counter. 0 Binding assays results of the NP derivatives comprising NP peptides of formula I are presented on Figure 2 and the binding assays results of the NP derivatives comprising NP peptides based on formula D are presented on Figure 3.
- “Native ANP” is the peptide having the human ANP sequence that has been synthesized in our laboratories (see Example 1) and "hANP” is the commercial peptide provided by Phoenix Pharmaceuticals Inc., Belmont, CA, USA, and catalogue number 005- 06.
- native ANP and commercial hANP both inhibited the binding of 125 I-ANF to the receptor in a concentration-dependent manner with apparent o inhibition constants (Ki values) of 3.4 x 10 "10 M and 6.0 x 10 "10 M, respectively.
- Conjugates of NP derivatives of Examples 3 and 5 also inhibited the binding of 125 I-ANF to the receptor of adrenal glands in a concentration-dependent manner with apparent Ki values of 2.4 x 10 "9 M and 2.9 x 10 "9 M respectively.
- Conjugates of NP derivatives of Examples 6 and 7 had a lower binding affinity and avidity for the NPR receptors.
- the derivatives of Examples 6 and 7 are 5 modified in the loop in comparison with the derivatives of Examples 3 and 5, which are modified at the N-terminus and C-terminus respectively.
- Table 8 shows the concentrations at 50% of inhibition (EC50) and the inhibition constants (KI) that were calculated with the data from which originates the graph in Figure 2. 0
- “Native BNP” is the peptide having the human BNP sequence that has been synthesized in our laboratories (see Example 21). As it can be seen on Figure 3, native BNP inhibited the binding of I-ANF to the receptor in a concentration-dependent manner with an apparent inhibition constant (Ki value) of 4.8 x 10 "9 M. Conjugates of NP derivatives of Examples 54 and 55 also inhibited the binding of 125 I-ANF to the receptor of adrenal o glands in a concentiation-dependent manner with apparent Ki values of 1.5 x 10 "8 M and 5.5 x 10 " M respectively. Conjugates of NP derivatives of Examples 56 and 57 had a lower binding affinity and avidity for the NPR receptors. The derivatives of Examples 56 and 57 are modified in the loop in comparison with the derivatives of Examples 54 and 55, which are modified at the N-terminus and C-terminus respectively.
- Table 9 shows the concentrations at 50% of inhibition (EC50) and the inhibition constants (KT) that were calculated with the data from which originates the graph in Figure 3.
- a human cervix epithelial adenocarcinoma cell line was used for in vitro activity studies. Hela cells express high levels of natriuretic peptide receptors with guanylate cyclase activity.
- the lyophilised enzymes contained in a vial of NEP enzyme are solubilized with 100 ⁇ L of 0.1 M Tris-HCl buffer pH 8.0. It was vortexed and sonnicated to ensure a complete dissolution of the enzymes.
- One vial contains between 800 and 950 U of enzymes.
- a solution of conjugates is prepared at 250 ⁇ M with 0.1 M Tris-HCl buffer pH 8.0.
- the site of hydrolysis of NEP on the sequence of ANP is the Cys-Phe peptidic bond at the beginning of the loop, as illustrated in Figure 8.
- the BNP sequence is also cleaved by NEP at the same site, i.e. at the Cys-Phe peptidic bond at the beginning of the loop.
- radioimmunoassay For detection of the non-hydrolysed NP peptide, radioimmunoassay (RIA) is performed using a commercial polyclonal antibody raised against human native ANP (Product # RGG-8798, Peninsula Laboratories Inc. Division of Bachem, San Carlos, CA, USA).
- radioimmunoassay 50 ⁇ L of either NP conjugate calibration standards, quality control samples, or diluted test samples in assay buffer (0.05M phosphate buffer, pH 7.5, 0.08% sodium azide, 0.025M EDTA, and 0.1% gelatin) is added to the appropriately labeled 12 x 75 mm borosilicate glass test tubes. 50 ⁇ L of assay buffer is added to the NSB (Non Specific Binding) and zero-standard (Reference) tubes. Then, 300 ⁇ L of assay buffer is added to each NSB tube and 200 ⁇ L of this same buffer is added to each of the other 12 x 75 mm borosilicate glass test tubes.
- assay buffer 0.05M phosphate buffer, pH 7.5, 0.08% sodium azide, 0.025M EDTA, and 0.1% gelatin
- HSA is albumin with a cysteine residue bonded to it.
- Pharmacokinetic studies of the derivatives are carried out in male Sprague-Dawley rats by subcutaneous (250 nmol/kg) or intravenous (50 nmol/kg) injection. Serial blood 5 samples were taken at pre-dose and 5 min, 30 min, 1 hr, 2 hrs, 4 hrs, 8 hrs, 24 hrs, 48 hrs, 72 hrs and 96 hrs post-agent administration. Blood samples were collected into tubes containing K2- EDTA and aprotinin, then centrifuged to obtain plasma and kept frozen until analysis by radioimmunoassay (RIA). A commercial polyclonal antibody raised against human native ANP (Product # RGG-8798, Peninsula Laboratories Inc.
- the assay sensitivity is 300 to 10 000 pM.
- Specific monoclonal antibodies need to be prepared and used for detecting each NP derivative that contains a NP peptide significatively different from the ANP and BNP.
- synthetic antibodies are available for derivatives of ANP and BNP.
- the commercially available antibodies may successflly be used in 5 the RIA.
- the bioavailability of free NP peptides is compared with the bioavailability of conjugated NP peptides. It can be seen that the conjugated ANP (conjugates of NP peptide of Example 3) administrered by intravenous injection (A) or by subcutaneous o injection (•) are still bioavailable after 96 hrs whereas free ANP (NP peptide of Example 3) administered by intravenous injection (o) or by subcutaneous injection (O) are not present in the blood stream within 5 min.
- the half-life of the conjugated ANP (conjugates of Example 58) 5 administrered by intravenous injection (A) or by subcutaneous injection (•) is 17,5 ⁇ 1,5 hours and 14,8 ⁇ 0,6 hours respectively.
- the half-life of free ANP (NP peptide of Example 3) administered by subcutaneous injection (o) is 0,2 ⁇ 0,06 hour and the one for ANP administered by intravenous injection (D) could not be calculated since it was too short.
- SHR rats are genetically hypertensive rats, which develop significantly elevated systolic blood pressure (BP) by 4 weeks of age. As a consequence of sustained elevated blood pressure throughout their lifetimes, these rats develop congestive heart failure by around 1 year of age. hi addition to high blood pressure, this model is also characterized by left ventricular hypertrophy and left ventricular fibrosis. SHR rats have been used previously in 5 studies of the in vivo effects of atrial natriuretic peptide. Single doses of ANP analogues produced a temporary drop in BP, while continuous infusions were required to sustain a decrease in systolic BP (De May et. al. J Pharm Exper Therap, 1987).
- the pacing model in dogs involves the implantation of programmable cardiac o pacemakers. After a surgical recovery period, the heart rate is increased incrementally from
- This model allows for the study of different stages of heart failure, evolving from the normal heart, to asymptomatic left ventricular dysfunction, to overt congestive heart failure (Luchner et. al. Eur J Heart Failure, 2000). Characteristics of this model include increases of heart rate, increased cardiac filling pressure, 5 low cardiac output, edema formation and activation of the sympathetic nervous system and other vasoconstrictor hormones (Arnolda et al, Austr. NZ J Med., 1999). The pacing model has been used previously in studies of the effects of both ANP and BNP on heart failure (Luchner et al, 2000; and Yamamoto et al, Am J Physiol, 1997).
- Tables 13 and 14 show in vivo results in SHR rats of 7 week old and in Winstar- Kyoto rats of 7 week old respectively.
- the increase of urine secretion and the increase of cGMP expression have been measured 24 and 48 hours after injection of compound of Example 3. Concentrations of 1, 2 and 4 mg of compounds per kg of rats have been tested in 5 comparison with saline solution. Contiol values have been taken before injection (pre-dose).
- the urine secretion ( Vol.) is expressed in mL/day of urine exceeding the value at pre-dose.
- the cGMP expression (cGMP) is reported in ⁇ mol/day and was measured by RIA method.
Abstract
Description
Claims
Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2003246500A AU2003246500A1 (en) | 2002-07-31 | 2003-07-29 | Long lasting natriuretic peptide derivatives |
EP03771007A EP1530588A2 (en) | 2002-07-31 | 2003-07-29 | Long lasting natriuretic peptide derivatives |
JP2004523679A JP2006514607A (en) | 2002-07-31 | 2003-07-29 | Long-lasting sodium excretion increasing peptide derivative |
US10/471,348 US20040266673A1 (en) | 2002-07-31 | 2003-07-29 | Long lasting natriuretic peptide derivatives |
CA002488348A CA2488348A1 (en) | 2002-07-31 | 2003-07-29 | Long lasting natriuretic peptide derivatives |
US11/040,810 US20050176641A1 (en) | 2000-05-17 | 2005-01-21 | Long lasting natriuretic peptide derivatives |
US11/067,556 US7601691B2 (en) | 1999-05-17 | 2005-02-25 | Anti-obesity agents |
US11/923,222 US7906482B2 (en) | 1999-05-17 | 2007-10-24 | Anti-obesity agents |
US11/926,843 US20080199532A1 (en) | 1999-05-17 | 2007-10-29 | Long Lasting Natriuretic Peptide Derivatives |
US12/253,856 US20090275506A1 (en) | 2000-05-17 | 2008-10-17 | Long lasting natriuretic peptide derivatives |
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JP (2) | JP2006514607A (en) |
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Also Published As
Publication number | Publication date |
---|---|
JP2006514607A (en) | 2006-05-11 |
WO2004011498A3 (en) | 2004-06-24 |
JP2010047614A (en) | 2010-03-04 |
CA2488348A1 (en) | 2004-02-05 |
AU2003246500A1 (en) | 2004-02-16 |
EP1530588A2 (en) | 2005-05-18 |
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