US20020187955A1 - Method of inducing angiogenesis in nonischemic skeletal muscle - Google Patents
Method of inducing angiogenesis in nonischemic skeletal muscle Download PDFInfo
- Publication number
- US20020187955A1 US20020187955A1 US10/176,024 US17602402A US2002187955A1 US 20020187955 A1 US20020187955 A1 US 20020187955A1 US 17602402 A US17602402 A US 17602402A US 2002187955 A1 US2002187955 A1 US 2002187955A1
- Authority
- US
- United States
- Prior art keywords
- skeletal muscle
- nonischemic
- adcmv
- angiogenic peptide
- dna encoding
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 210000002027 skeletal muscle Anatomy 0.000 title claims abstract description 91
- 238000000034 method Methods 0.000 title claims abstract description 58
- 230000033115 angiogenesis Effects 0.000 title claims abstract description 35
- 230000001939 inductive effect Effects 0.000 title abstract description 7
- 102000008076 Angiogenic Proteins Human genes 0.000 claims abstract description 66
- 108010074415 Angiogenic Proteins Proteins 0.000 claims abstract description 66
- 230000000302 ischemic effect Effects 0.000 claims abstract description 33
- 208000002193 Pain Diseases 0.000 claims abstract description 11
- 230000036407 pain Effects 0.000 claims abstract description 10
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 10
- 230000017531 blood circulation Effects 0.000 claims abstract description 7
- 208000024891 symptom Diseases 0.000 claims abstract description 6
- 230000002238 attenuated effect Effects 0.000 claims abstract 3
- 239000013598 vector Substances 0.000 claims description 53
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims description 18
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 18
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 18
- 230000002950 deficient Effects 0.000 claims description 17
- 239000013603 viral vector Substances 0.000 claims description 17
- 108700039887 Essential Genes Proteins 0.000 claims description 9
- 239000003937 drug carrier Substances 0.000 claims description 9
- 206010022562 Intermittent claudication Diseases 0.000 claims description 4
- 208000021156 intermittent vascular claudication Diseases 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 description 56
- 210000003414 extremity Anatomy 0.000 description 31
- 241001465754 Metazoa Species 0.000 description 30
- 210000001519 tissue Anatomy 0.000 description 28
- 238000001356 surgical procedure Methods 0.000 description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 17
- 230000006698 induction Effects 0.000 description 17
- 208000028867 ischemia Diseases 0.000 description 17
- 239000008280 blood Substances 0.000 description 15
- 210000004369 blood Anatomy 0.000 description 15
- 210000003141 lower extremity Anatomy 0.000 description 15
- 230000004895 regional blood flow Effects 0.000 description 14
- 239000011780 sodium chloride Substances 0.000 description 14
- 238000011282 treatment Methods 0.000 description 14
- 238000002347 injection Methods 0.000 description 13
- 239000007924 injection Substances 0.000 description 13
- 230000000638 stimulation Effects 0.000 description 13
- 210000003205 muscle Anatomy 0.000 description 12
- 241000283973 Oryctolagus cuniculus Species 0.000 description 11
- 206010053648 Vascular occlusion Diseases 0.000 description 11
- 208000021331 vascular occlusion disease Diseases 0.000 description 11
- 230000010412 perfusion Effects 0.000 description 10
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 9
- 241000700159 Rattus Species 0.000 description 9
- 210000002565 arteriole Anatomy 0.000 description 9
- 238000012217 deletion Methods 0.000 description 9
- 230000037430 deletion Effects 0.000 description 9
- 102000058223 human VEGFA Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 210000002414 leg Anatomy 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 230000010076 replication Effects 0.000 description 8
- 238000012546 transfer Methods 0.000 description 8
- 210000000689 upper leg Anatomy 0.000 description 8
- 238000005481 NMR spectroscopy Methods 0.000 description 7
- 230000002491 angiogenic effect Effects 0.000 description 7
- 210000001367 artery Anatomy 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 210000001105 femoral artery Anatomy 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 230000036772 blood pressure Effects 0.000 description 6
- 229960003299 ketamine Drugs 0.000 description 6
- 239000002502 liposome Substances 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 241000701161 unidentified adenovirus Species 0.000 description 6
- 230000002792 vascular Effects 0.000 description 6
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 6
- 229960001600 xylazine Drugs 0.000 description 6
- 238000004679 31P NMR spectroscopy Methods 0.000 description 5
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 5
- 241000700584 Simplexvirus Species 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 210000003090 iliac artery Anatomy 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 230000035488 systolic blood pressure Effects 0.000 description 5
- 206010002091 Anaesthesia Diseases 0.000 description 4
- 244000025254 Cannabis sativa Species 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 230000037005 anaesthesia Effects 0.000 description 4
- 244000309466 calf Species 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 229960001412 pentobarbital Drugs 0.000 description 4
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000002331 radioactive microsphere Substances 0.000 description 4
- 230000003362 replicative effect Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 101710132601 Capsid protein Proteins 0.000 description 3
- 101710094648 Coat protein Proteins 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 3
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 3
- 101710125418 Major capsid protein Proteins 0.000 description 3
- 206010029113 Neovascularisation Diseases 0.000 description 3
- 101710141454 Nucleoprotein Proteins 0.000 description 3
- 208000030831 Peripheral arterial occlusive disease Diseases 0.000 description 3
- 101710083689 Probable capsid protein Proteins 0.000 description 3
- 238000002583 angiography Methods 0.000 description 3
- 230000003143 atherosclerotic effect Effects 0.000 description 3
- 230000002715 bioenergetic effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000007491 morphometric analysis Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 239000013074 reference sample Substances 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000005166 vasculature Anatomy 0.000 description 3
- 230000007998 vessel formation Effects 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 108091029865 Exogenous DNA Proteins 0.000 description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 description 2
- 241000700157 Rattus norvegicus Species 0.000 description 2
- 210000000702 aorta abdominal Anatomy 0.000 description 2
- 230000008081 blood perfusion Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000002350 laparotomy Methods 0.000 description 2
- 238000005399 mechanical ventilation Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000001087 myotubule Anatomy 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 210000003137 popliteal artery Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000000250 revascularization Effects 0.000 description 2
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 2
- ZEYOIOAKZLALAP-UHFFFAOYSA-M sodium amidotrizoate Chemical compound [Na+].CC(=O)NC1=C(I)C(NC(C)=O)=C(I)C(C([O-])=O)=C1I ZEYOIOAKZLALAP-UHFFFAOYSA-M 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 210000002465 tibial artery Anatomy 0.000 description 2
- 230000006711 vascular endothelial growth factor production Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 239000013607 AAV vector Substances 0.000 description 1
- 102100022987 Angiogenin Human genes 0.000 description 1
- 101710190943 Angiogenin-2 Proteins 0.000 description 1
- 208000031104 Arterial Occlusive disease Diseases 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 102100028072 Fibroblast growth factor 4 Human genes 0.000 description 1
- 108090000381 Fibroblast growth factor 4 Proteins 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 229920001410 Microfiber Polymers 0.000 description 1
- 238000012565 NMR experiment Methods 0.000 description 1
- 208000005764 Peripheral Arterial Disease Diseases 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 206010039897 Sedation Diseases 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 108091008605 VEGF receptors Proteins 0.000 description 1
- 108010073923 Vascular Endothelial Growth Factor C Proteins 0.000 description 1
- 102000009520 Vascular Endothelial Growth Factor C Human genes 0.000 description 1
- 108010053096 Vascular Endothelial Growth Factor Receptor-1 Proteins 0.000 description 1
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 1
- 108010053100 Vascular Endothelial Growth Factor Receptor-3 Proteins 0.000 description 1
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 1
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 1
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 1
- 102100033179 Vascular endothelial growth factor receptor 3 Human genes 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000006427 angiogenic response Effects 0.000 description 1
- 108010072788 angiogenin Proteins 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 210000002376 aorta thoracic Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 208000021328 arterial occlusion Diseases 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- GZUXJHMPEANEGY-UHFFFAOYSA-N bromomethane Chemical compound BrC GZUXJHMPEANEGY-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- RMRJXGBAOAMLHD-IHFGGWKQSA-N buprenorphine Chemical compound C([C@]12[C@H]3OC=4C(O)=CC=C(C2=4)C[C@@H]2[C@]11CC[C@]3([C@H](C1)[C@](C)(O)C(C)(C)C)OC)CN2CC1CC1 RMRJXGBAOAMLHD-IHFGGWKQSA-N 0.000 description 1
- 229960001736 buprenorphine Drugs 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 210000005242 cardiac chamber Anatomy 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000024980 claudication Diseases 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 238000002591 computed tomography Methods 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 229940039231 contrast media Drugs 0.000 description 1
- 239000002872 contrast media Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229960003718 diatrizoate sodium Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000002815 epigastric artery Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 238000013427 histology analysis Methods 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000009851 immunogenic response Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 238000002743 insertional mutagenesis Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003601 intercostal effect Effects 0.000 description 1
- 210000001596 intra-abdominal fat Anatomy 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 210000005240 left ventricle Anatomy 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000003658 microfiber Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000037081 physical activity Effects 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000009101 premedication Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000036280 sedation Effects 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Images
Classifications
-
- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
- A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
- A61K51/1255—Granulates, agglomerates, microspheres
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
Definitions
- the present invention pertains generally to methods for inducing angiogenesis or collateral blood formation in a nonischemic skeletal muscle at risk of being affected by ischemia or a vascular occlusion, thereby maintaining or enhancing the level of perfusion of blood to the nonischemic skeletal muscle.
- Vascular atherosclerotic disease also known as peripheral arterial occlusive disease
- peripheral arterial occlusive disease is a major health problem, especially in the elderly. Its prevalence increases with age from 3% in individuals younger than 60 years old to over 20% in individuals 75 years or older. Treatment of patients suffering from peripheral arterial occlusive disease remains a considerable clinical issue despite advances in both surgical and percutaneous revascularization techniques. Many patients cannot benefit from these therapies because of the anatomic extent and distribution of arterial occlusion. In such patients, new therapeutic strategies have been sought to prevent the development of disabling symptoms related to ischemia such as claudication, resting pain and loss of tissue integrity in the distal limbs. The latter can ultimately lead to limb loss.
- Angiogenesis the growth of new blood vessels, is a complex process involving disruption of vascular basement membranes, migration and proliferation of endothelial cells, and subsequent blood vessel formation and maturation.
- mediators are known to elicit angiogenic responses, and administration of these mediators promotes revascularization of ischemic tissues.
- Vascular endothelial growth factor (VEGF) is one of the most specific of the known angiogenic mediators due to localization of its receptors almost exclusively on endothelial cells.
- Receptors for VEGF are upregulated under ischemic conditions, and the administration of recombinant VEGF augments development of collateral vessels and improves function in peripheral and myocardial ischemic tissue.
- aFGF acidic fibroblast growth factor
- the present invention provides a method for enhancing the level of perfusion of blood to a nonischemic skeletal muscle involving administering to a nonischemic skeletal muscle a pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) a DNA encoding an angiogenic peptide.
- the present invention further provides a method of treating, either therapeutically or prophylactically, a nonischemic skeletal muscle at risk of suffering from ischemic damage. Also provided is a method of treating a nonischemic skeletal muscle at risk of being affected by a vascular occlusion through induction of collateral blood vessel formation in the nonischemic skeletal muscle.
- a method of inducing angiogenesis is provided by the present invention.
- FIG. 1 is a graph of blood pressure ratio (BPR; ischemic/nonischemic limb) versus time after surgery (weeks) of rabbits treated with AdCMV.VEGF121 106 pfu/ml, AdCMV.VEGF121 108 pfu/ml, AdCMV.Null (control), and saline (control).
- BPR blood pressure ratio
- FIG. 2 is a graph of regional blood flow ratio measured by radioactive microspheres (RBF; ischemic/nonischemic limb) versus time after surgery (days and weeks) of the gastrocnemius muscle of rabbits treated with AdCMV.VEGF 121 10 6 pfu/ml, AdCMV.VEGF 121 10 8 pfu/ml, AdCMV.Null (control), and saline (control).
- RBF radioactive microspheres
- FIGS. 3 A- 3 C are graphs of 31P nuclear magnetic resonance (NMR) spectroscopy data showing the PCr/(PCr+Pi) ratio of the gastrocnemius muscle as a function of electrical stimulation protocol time (min).
- FIG. 3A shows data at day 1 after surgery
- FIG. 3B shows data at day 7 after surgery
- FIG. 3C show data at day 14 after surgery.
- data are shown at rest (2 min), during stimulation (4-8 min), and during recovery (10-20 min).
- FIGS. 4A and 4B are bar graphs of blood vessel length density (mm/mm3) in the adductor and gastrocnemius muscles of both injected and non-injected hindlimbs treated with AdCMV.VEGF121 and AdCMV.Null (control):
- FIG. 4A is a bar graph of arteriole length density
- FIG. 4B is a bar graph of capillary length density.
- the present invention provides a method for enhancing the level of perfusion of blood to a nonischemic skeletal muscle involving administering to a nonischemic skeletal muscle a pharmaceutical composition (e.g., a dose thereof) comprising (a) a pharmaceutically acceptable carrier and (b) a DNA encoding an angiogenic peptide.
- a pharmaceutical composition e.g., a dose thereof
- the present invention further provides a method of treating, either therapeutically or prophylactically, a nonischemic skeletal muscle at risk of suffering from ischemic damage.
- a method of inducing angiogenesis is provided by the present invention.
- inducing angiogenesis or “induction of angiogenesis,” it is meant that angiogenesis is either initiated or enhanced. Therefore, for example, when the nonischemic skeletal muscle is not already undergoing angiogenesis, the present method provides for initiation of angiogenesis in the nonischemic skeletal muscle. However, when the nonischemic skeletal muscle is already undergoing angiogenesis, the present method provides a means by which the level of angiogenesis is enhanced or heightened.
- any suitable nonischemic skeletal muscle can be subject to administration within the context of the present invention.
- the nonischemic skeletal muscle comprises receptors capable of binding the angiogenic peptide encoded by the DNA; more preferably, the nonischemic skeletal muscle comprises VEGF receptors.
- the nonischemic skeletal muscle will be part of a discrete organ, such as a limb.
- the nonischemic skeletal muscle will be at risk of suffering from ischemic damage, which results when tissue is deprived of an adequate supply of oxygenated blood.
- An interruption in the supply of oxygenated blood is often caused by a vascular occlusion.
- Vascular atherosclerotic disease, other diseases, trauma, surgical procedures, and/or other indications can cause such vascular occlusion in nonischemic skeletal muscle.
- nonischemic skeletal muscle is at risk of suffering ischemic damage from an undesirable vascular occlusion.
- Such methods are well known to physicians who treat such conditions, and include clinical evaluation (history and physical examination), Doppler, treadmill test to evaluate time to development of symptoms (e.g., pain), CT scan, NMR angiography, 31P NMR spectroscopy, and contrast angiograms.
- Induction of angiogenesis in nonischemic skeletal muscle at risk of being affected by a vascular occlusion is an effective means of preventing and/or attenuating any resulting ischemia.
- the target nonischemic skeletal muscle preferably is one that is at risk of being affected by a vascular occlusion.
- any DNA encoding an angiogenic peptide operably linked to suitable expression signals can be used within the context of the present invention.
- the angiogenic peptide is a VEGF protein, and more preferably, the angiogenic peptide is VEGF121, VEGF145, VEGF165, VEGF189, or a mammalian counterpart, which are variously described in U.S. Pat. Nos. 5,332,671 (Ferrara et al.), 5,240,848 (Keck et al.); and 5,219,739 (Tischer et al.).
- the angiogenic peptide is VEGF121 or VEGF165, particularly VEGF121.
- VEGF121 does not bind to heparin with a high degree of affinity, as does VEGF165.
- VEGF moieties are advantageous over other angiogenic peptides because VEGF proteins do not induce the growth of tissues not involved in the production of new vasculature.
- angiogenic peptides include VEGF II, VEGF-C, fibroblast growth factors (FGFs) (e.g., aFGF, bFGF, and FGF-4), angiopoiteins, angiogenin, angiogenin-2, and P1GF, which are variously described in U.S. Pat. Nos.
- FGFs fibroblast growth factors
- Promiscuous induction of angiogenesis can cause blindness, increase the aggressiveness of tumor cells, and lead to a multitude of other negative side-effects.
- the present invention involves the administration of a DNA encoding the angiogenic peptide in a localized manner to nonischemic skeletal muscle. While any suitable means of administering the DNA encoding the angiogenic peptide to the nonischemic skeletal muscle can be used within the context of the present invention, preferably, such a localized administration to the nonischemic skeletal muscle is accomplished by directly injecting the DNA encoding the angiogenic peptide into the nonischemic skeletal muscle or by topically applying the DNA encoding the angiogenic peptide to the nonischemic skeletal muscle. By the term “injecting,” it is meant that the DNA encoding the angiogenic peptide is forcefully introduced into the nonischemic skeletal muscle. Any suitable injection device can be used within the context of the present invention. However, it is desirable that whatever means of administering the DNA encoding the angiogenic peptide is chosen, the induction of angiogenesis in non-targeted tissue is minimized.
- the DNA encoding the angiogenic peptide can be delivered, for example, by intramuscular injection or a catheter inserted into the proximal portion of the femoral artery or arteries.
- the DNA encoding the angiogenic peptide can be delivered by a catheter or like device inserted sufficiently deeply into the proximal portion of the organ- or tissue-feeding artery or arteries so that gene transfer is effected substantially only into the cells of the target organ or tissue.
- a number of different delivery methods are available for administering a DNA encoding an angiogenic peptide, including plasmid DNA, plasmid-liposome complexes, and viral vectors.
- Any suitable viral vector can be used in the context of the present inventive method to administer the DNA encoding an angiogenic peptide.
- suitable viral vectors are adenoviral vectors, herpes simplex viral vectors, and adeno-associated viral vectors.
- Plasmids genetically engineered circular double-stranded DNA molecules, can be designed to contain an expression cassette for delivery of a specific DNA.
- plasmids were the first method described for gene transfer of DNA encoding an angiogenic peptide, their level of efficiency is poor, compared with other techniques.
- liposomes used for plasmid-mediated gene transfer strategies have various compositions, they are typically synthetic cationic lipids.
- the positively charged liposome forms a complex with a negatively charged plasmid.
- These plasmid-liposome complexes enter target cells by fusing with the plasma membrane.
- Advantages of plasmid-liposome complexes include their ability to transfer large pieces of DNA encoding an angiogenic peptide and their relatively low potential to evoke immunogenic responses in the host.
- the adenovirus is a 36 kb double-stranded DNA virus that efficiently transfers DNA in vivo to a variety of different target cell types, including skeletal muscle.
- the virus is made suitable by deleting some of the genes required for viral replication; the expendable E3 region is also frequently deleted to provide additional room for a larger DNA insert.
- the resulting replication deficient adenoviral vectors can accommodate up to 7.5 kb of exogenous DNA and are capable of being produced in high titers and efficiently transferring DNA to replicating and non-replicating cells.
- Of particular importance for transfer of DNA to the skeletal muscle, in which the host cell is a terminally differentiated cell is the ability of adenoviral vectors to efficiently transfer DNA to non-replicating cells.
- the newly transferred genetic information remains epi-chromosomal, thus eliminating the risks of random insertional mutagenesis and permanent alteration of the genotype of the target cell.
- the herpes simplex virus is another viral vector that can be used to administer a DNA encoding an angiogenic peptide.
- the mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb.
- Most replication-deficient HSV vectors contain a deletion to remove one or more intermediate-early genes to prevent replication.
- Advantages of the herpes vector are its ability to enter a latent stage that could potentially result in long-term DNA expression, and its large viral DNA genome that can accommodate exogenous DNA up to 25 kb.
- Adeno-associated virus (AAV) vectors represent other viral vectors that can be used to administer a DNA encoding an angiogenic peptide.
- AAV is a DNA virus, which is not known to cause human disease and which requires coinfection by a helper virus (i.e., an adenovirus or a herpes virus) for efficient replication.
- helper virus i.e., an adenovirus or a herpes virus
- AAV vectors used for administration of a DNA encoding an angiogenic peptide have approximately 96% of the parental genome deleted such that only the terminal repeats remain, which contain recognition signals for DNA replication and packaging. This eliminates immunologic or toxic side effects due to expression of viral genes.
- adenoviral vector is preferably deficient in at least one gene function required for viral replication.
- the adenoviral vector is deficient in at least one essential gene function of the E1 region of the adenoviral genome (e.g., the E1a and/or E1b region), particularly the E1a region. More preferably, the vector is deficient in at least one essential gene function of the E1 region and part of the E3 region (e.g., an XbaI deletion of the E3 region).
- the vector is deficient in at least one essential gene function of the E1 region and at least one essential gene function of the E4 region.
- adenoviral vectors deficient in at least one essential gene function of the E2a region and adenoviral vectors deficient in all of the E3 region also are contemplated here and are well known in the art.
- Adenoviral vectors deleted of the entire E4 region can elicit lower host immune responses.
- Suitable replication deficient adenoviral vectors are disclosed in International Patent Applications WO 95/34671 and WO 97/21826.
- suitable replication deficient adenoviral vectors include those with a partial deletion of the E1a region, a partial deletion of the E1b region, a partial deletion of the E2a region, and a partial deletion of the E3 region.
- the replication deficient adenoviral vector can have a deletion of the E1 region, a partial deletion of the E3 region, and a partial deletion of the E4 region.
- the adenoviral vector's coat protein can be modified so as to incorporate a specific protein binding sequence, as described in U.S. Pat. No. 5,770,442 (Wickham et al.), or the adenoviral vector's coat protein can be modified so as to decrease the adenoviral vector's ability or inability to be recognized by a neutralizing antibody directed against the wild-type coat protein, as described in International Patent Application WO 98/40509.
- Other suitable modifications to the adenoviral vector are described in U.S. Pat. Nos.
- the adenoviral vector also can include a DNA encoding another peptide, for example, an angiogenic peptide receptor or another angiogenic peptide.
- Suitable angiogenic peptide receptors include, for example, FLT-1, FLK-1, and FLT-4.
- the DNA operably linked to expression signals and encoding the angiogenic peptide, can be inserted into any suitable region of the adenoviral vector as an expression cassette.
- the DNA segment is inserted into the E1 region of the adenoviral vector.
- the DNA segment can be inserted as an expression cassette in any suitable orientation in any suitable region of the adenoviral vector, preferably, the orientation of the DNA segment is from right to left.
- the expression cassette having an orientation from right to left it is meant that the direction of transcription of the expression cassette is opposite that of the region of the adenoviral vector into which the expression cassette is inserted.
- An adenoviral vector illustrative of the present inventive vector is deficient in the E1a region, part of the E1b region, and part of the E3 region of the adenoviral genome and contains the DNA encoding human VEGF121 or human VEGF165 under the control of the CMV immediate early promoter in the E1 region of the adenoviral genome.
- Such a vector supports in vivo expression of VEGF that is maximized at one day following administration and is not detectable above baseline levels as little as one week after administration. This is ideal inasmuch as it is sufficient to provide substantial growth of new vasculature while minimizing adverse neovascularization at distal sites.
- the angiogenic peptide desirably is administered to the nonischemic skeletal muscle in a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and the DNA encoding the angiogenic peptide.
- any suitable pharmaceutically acceptable carrier can be used within the context of the present invention, and such carriers are well known in the art.
- the choice of carrier will be determined, in part, by the particular site to which the composition is to be administered and the particular method used to administer the composition.
- Formulations suitable for injection include aqueous and non-aqueous solutions, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
- the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use.
- sterile liquid carrier for example, water
- Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
- the pharmaceutically acceptable carrier is a buffered saline solution.
- the pharmaceutical carrier also can contain peptides, for example, an angiogenic peptide receptor, an angiogenic peptide, or a factor necessary for the development of blood vessels.
- additional peptides can be encoded by a DNA, which can be a plasmid or contained within a viral vector (e.g., HSV, adenovirus, or AAV). It should be appreciated that a plasmid or viral vector comprising the DNA encoding an angiogenic peptide can be the same or different than the plasmid or viral vector that comprises the DNA encoding these additional peptides.
- the DNA encoding the angiogenic peptide is administered in small volumes of carrier.
- Administration of small volumes is such that the tissue to be vascularized (i.e., the nonischemic skeletal muscle) is perfused with the DNA encoding the angiogenic peptide and very little or no DNA encoding the angiogenic peptide is carried by the blood, lymphatic drainage, or physical mechanisms (e.g., gravitational flow or osmotic flow) to tissues not targeted.
- the proper dosage is such that the level of perfusion is enhanced to the nonischemic skeletal muscle.
- the dosage is sufficient to have a therapeutic and/or prophylactic effect on nonischemic skeletal muscle that is at risk of being affected by ischemia or a vascular occlusion.
- the dosage should be such that induction of angiogenesis in non-targeted tissue is minimized.
- the dosage also will vary depending upon the angiogenic peptide. Specifically, the dosage will vary depending upon the particular method of administration, including the nature of the vector and DNA encoding and controlling the expression of the angiogenic peptide.
- a dose typically will be at least about 1 ⁇ 106 pfu (e.g., 1 ⁇ 106-1 ⁇ 1013 pfu) to the nonischemic skeletal muscle, for example, a human hindlimb.
- the dose preferably is at least about 1 ⁇ 107 pfu (e.g., about 1 ⁇ 107-1 ⁇ 1013 pfu), more preferably at least about 1 ⁇ 108 pfu (e.g., about 1 ⁇ 108-1 ⁇ 1011 pfu), and most preferably at least about 1 ⁇ 109 pfu (e.g., about 1 ⁇ 109-1 ⁇ 1010 pfu).
- the dose typically is for a volume of targeted tissue of about 100 cm3, more typically about 150 cm3.
- adenoviral vector deleted of the E1a region, part of the E1b region, and part of the E3 region of the adenoviral genome wherein the vector carries human VEGF121 or VEGF165 under the control of a standard CMV immediate early promoter
- about 107-1013 pfu, preferably about 109-1011 pfu are administered to a targeted tissue (e.g., to a discrete organ containing the targeted nonischemic skeletal muscle) with an estimated volume of about 150 cm3.
- a substantial level of VEGF production is achieved in the nonischemic skeletal muscle without producing detectable levels of VEGF production in distal tissues.
- a total of 112 6-month-old male New Zealand White rabbits (HRP Inc. Rabbitry, Denver, Pa.), mean weight 4.0 ⁇ 0.2 kg, and 36 10-month-old male Wistar rats (Wistar Rats Colony, Gerontology Research Center, NIA, NIH, Baltimore, Md.), mean weight 550 ⁇ 50 g, were used in the experimental protocols.
- AdCMV.VEGF121 is an E1a-, partial E1b-, partial E3-adenovirus vector that carries an expression cassette in the E1 position containing the CMV immediate early promoter/enhancer driving the cDNA for the 121-residue form of human VEGF.
- AdCMV.Null used as a control vector in this study, is similar to AdCMV.VEGF121 but with no gene in the expression cassette.
- IM intramuscularly
- Rabbits were pre-anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg), intubated using a laryngoscope and an uncuffed 3.5-neonatal orotracheal tube, and placed under mechanical ventilation. Stable anesthesia was achieved using a mixture of 1.5% isoflurane and oxygen. The surgical procedure to induce unilateral hindlimb ischemia in rats was performed under intraperitoneal anesthesia with ketamine (60 mg/kg) and xylazine (10 mg/kg). Both species underwent a similar surgical procedure as described below.
- a longitudinal incision was performed in the thigh, extending distally from the inguinal ligament to a point just above the knee.
- the femoral artery was dissected free along its entire length, as were all its major branches including the inferior epigastric, deep femoral, lateral circumflex, and superficial epigastric arteries.
- the external iliac artery was ligated with 5-0 silk (Ethicon, Inc., Somerville, N.J.).
- the femoral artery was completely excised from its proximal origin as a branch of the external iliac artery to the point distally where it bifurcates into the saphenous and popliteal arteries. Rabbits received 0.9% normal saline (50 ml IV) during surgery. Rats and rabbits were given post-operatory analgesia (buprenorphine 0.04 mg/kg) twice daily for the first two days after the procedure.
- This example demonstrates angiogenesis in a nonischemic skeletal muscle by administration of an adenoviral vector comprising a DNA encoding an angiogenic peptide. Further demonstrated by this example is that perfusion of blood is maintained in the skeletal muscle upon induction of ischemia. Angiogenesis in the hindlimb of rabbits was measured physiologically by calf blood pressure and blood flow measurements with radioactive microspheres and anatomically by post-mortem contrast angiography after administration of the adenoviral vector comprising VEGF121 to the nonischemic hindlimb and subsequent induction of ischemia.
- calf blood pressure was measured weekly in both hindlimbs of thirty-three rabbits using a Doppler flowmeter (Vascular Mini-Lab III, Parks Medical Electronics, Aloha, Oreg.).
- ketamine 50 mg/kg
- xylazine 5 mg/kg
- the hindlimbs were shaved and cleaned
- the pulse of the posterior tibial artery was identified using a Doppler probe
- the systolic blood pressure in both limbs was determined according to standard techniques. Briefly, a 2.5 cm wide cuff was applied over the thigh, and the Doppler probe was placed over the posterior tibial artery.
- the cuff was rapidly inflated to approximately 30 mm Hg above the anticipated systolic pressure and then slowly deflated. The pressure at which the Doppler flow signal reappeared was recorded as the systolic pressure. A single observer, blinded to the treatment regimen, performed all measurements.
- the calf blood pressure ratio (BPR) was then defined as a ratio of systolic pressure of the ischemic limb to systolic pressure of the normal limb. Thus, the lower the ratio, the more impaired the arterial perfusion of the ischemic limb.
- FIG. 1 is a graph of BPR vs. time after surgery (weeks), shows that the animals reached their final recovery ratio of approximately 0.50-0.60 after treatment with AdCMV.VEGF121 at 108 pfu/ml after 4 weeks, AdCMV.VEGF121 at 106 pfu/ml after 10 weeks, and the control groups (AdCMV.Null and saline) after 9-12 weeks. Further analysis revealed a faster rate of recovery between weeks 1 and 4 for AdCMV.VEGF121 than both controls (P ⁇ 0.0001) and animals treated with AdCMV.VEGF121 at 106 pfu/ml (P ⁇ 0.001).
- the catheter was connected to a withdrawal syringe pump (Model SP210iw, World Precision Instruments, Sarasota, Fla.) for blood collection and to a single channel blood pressure monitor (Model 50110, Stoelting, Wood Dale, Ill.).
- a withdrawal syringe pump Model SP210iw, World Precision Instruments, Sarasota, Fla.
- a single channel blood pressure monitor Model 50110, Stoelting, Wood Dale, Ill.
- the chest was opened at the left fourth intercostal space level, the left heart chambers were exposed, and 3.3 ⁇ 106 radioactive microspheres (15.5 ⁇ m diameter) labeled with 51Cr (NEN Life Science Products, Boston, Mass.) were injected directly into the left ventricle within a 20-second period.
- the vial containing the microspheres was placed in warm water (40° C.) for thirty minutes and then, immediately before injection, vigorously shaken (Daigger Vortex, Model Genie 2, Scientific Industries, Inc., Bohemia, N.Y.) for one minute to assure proper mixing of the beads in the solution.
- An arterial blood reference sample was withdrawn at a constant rate of 2 ml/min starting thirty seconds before, and continued for ninety seconds after, the injection was completed. Animals then were killed with a sodium pentobarbital overdose, and the entire gastrocnemius muscles of both limbs were removed.
- Each muscle was cut in three approximately equal parts (proximal, middle and distal), weighed, and put in 50 ml conical polypropylene tubes (Coming Labware & Equipment, Coming, N.Y.). Twenty ml of 2 M KOH and 10 ml of 2% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) were added to each vial for tissue digestion. After 24 hours at 50° C. in a constant temperature shaking water bath, the tissue samples were fully dissolved. All samples then were filtered using glass microfiber filters with 1.6 ⁇ m diameter pores (Whatman Filters, Whatman International Ltd., England).
- the filters containing the microspheres were placed into liquid scintillation vials with 10 ml of liquid scintillation cocktail (CytoScint ES, ICN Biomedical Research Products, Costa Mesa, Calif.). To prevent the occurrence of chemiluminescence in the samples, 1 ml of acetic acid was added to each vial.
- the level of radioactivity in each sample was determined using a liquid scintillation counter (Model LS5801, Beckman Coulter, Inc., Fullerton, Calif.).
- FIG. 2 which is a graph of the calculated ratio between RBF in the ischemic and nonischemic gastrocnemius muscles, shows that animals reached their final recovery ratios of approximately 1.0 and 0.9, for AdCMV.VEGF121 and the controls, respectively, after treatment with AdCMV.VEGF121 (at 106 or 108 pfu/ml) after 1 week, and the controls (AdCMV.Null and saline) after 4 weeks. Significant differences were found between treatment groups and between time points for RBF in the ischemic limb (P ⁇ 0.0001 for both effects of time and treatment).
- a total of 5,000 units of heparin were given to prevent clot formation.
- the animal was killed with an overdose of sodium pentobarbital and immediately placed under the fluoroscope (Digimax MP4000 Series III Workstation, Acomma Medical Imaging Inc., Wheeling, Ill.).
- a total of 5 ml contrast media (Hypaque sodium 50%, diatrizoate sodium, Nycomed Inc., Princeton, N.J.) was injected into the right common iliac artery using an infusion syringe pump (Model 848, Edco Scientific Inc., Chapel Hill, N.C.) at a constant rate of 20 ml/min.
- Serial images of the ischemic hindlimb were recorded and printed out for further analysis.
- This example therefore demonstrates the induction of angiogenesis or collateral blood formation in a nonischemic skeletal muscle at risk of being affected by, and subsequently affected by, ischemia or a vascular occlusion after treatment with a pharmaceutical composition comprising a DNA encoding an angiogenic peptide.
- This example demonstrates perfusion of blood in nonischemic skeletal muscle.
- the bioenergetic profile of the gastrocnemius muscle was used as an indirect indicator of gastrocnemius muscle perfusion. Histology was also performed to determine the capillary and arteriole length densities in the skeletal muscles of the hindlimbs, another indirect indicator of the level of blood perfusion in the gastrocnemius muscle.
- 31P-NMR spectroscopy was used to determine the bioenergetic profile of the gastrocnemius muscles of both hindlimbs at rest and during exercise induced by electrical stimulation of these muscles. NMR tests were conducted in twenty-two rats on days 1, 7, and 14 after surgery. All data were acquired on a 1.9-T/3 1-cm NMR spectrometer (Biospec, Bruker Medizintechnik GmbH, Ettlingen, Germany).
- the foot of the stimulated leg was tied to a strain gauge force transducer (Grass Instruments Manufacturing, Braintree, Mass.) using a 3-0 silk suture.
- the force transducer was connected to a strain gauge conditioner, preamplifier, and chart recorder (Gould Instrument Systems, Inc., Cleveland, Ohio), allowing continuous monitoring of the muscle contraction force during the electrical stimulation.
- the electrical stimulation was applied as a train of pairs of pulses of 100 ⁇ s length separated by a 200 ms interval and repeated once every two seconds. The voltage of these pulses was incremented over about thirty seconds until the observed contraction force no longer increased, thereby determining the stimulation voltage for that leg.
- the animal was positioned in the NMR magnet, and the surface coil tuning was adjusted for exact resonance. Radio-frequency pulses were applied every two seconds with adiabatic frequency and amplitude shaping to compensate for the surface coil's RF inhomogeneity.
- the proton NMR signal from the coil was detected and used as a guide to magnetic field shipping for 31 P-spectroscopy.
- the exact proton resonance frequency of the water peak was used to calculate the expected frequency for 31 P, based on the gyromagnetic ratios of the two nuclei.
- the RF transmitter was set to the calculated 31 P frequency, and a preliminary 31 P spectrum was recorded with a one-minute acquisition time.
- FIGS. 3 A-C which collectively are graphs of NMR data (PCr/(PCr+Pi)) obtained in rats as a function of time after surgery, show: At day 1, AdCMV.VEGF121 pre-treated ischemic limbs recovered approximately 0.8 of the original PCr/(PCr+Pi), and AdCMV.Null pre-treated ischemic limbs only recovered approximately 0.3 of the original PCr/(PCr+Pi) (see FIG. 3A).
- AdCMV.VEGF 121 pre-treated ischemic limbs recovered approximately 0.8 of the original PCr/(PCr+Pi), and AdCMV.Null pre-treated ischemic limbs recovered approximately 0.6 of the original PCr/(PCr+Pi) (see FIG. 3B).
- AdCMV.VEGF 121 and AdCMV.Null pre-treated ischemic limbs recovered approximately 0.8 of the original PCr/(PCr+Pi) (see FIG. 3C).
- Animals pre-treated with AdCMV.VEGF 121 showed an improved bioenergetic profile of the gastrocnemius muscle after femoral artery removal when compared to controls.
- AdCMV.VEGF121 was injected either with AdCMV.VEGF121 (2 ⁇ 109 pfu/ml) or AdCMV.Null (2 ⁇ 109 pfu/ml), as previously described. Fifteen days after injection of the viral vector, animals were anesthetized as usual, and a median laparotomy was performed. Both legs were then perfused via the abdominal aorta with 10% buffered formalin at 100 mm Hg for fifteen minutes. Subsequently, the adductor and gastrocnemius muscles were immersion-fixed in formalin for 48 hours.
- Sections were deparafinized, rinsed in phosphate buffered saline (PBS), incubated at 37° C. for sixty minutes with mouse monoclonal anti- ⁇ -smooth muscle actin (clone 1A4, Sigma Chemical Co., St. Louis, Mo.) diluted 1:30 in PBS, and subsequently incubated at 37° C. for sixty minutes with anti-mouse IgG tetramethyrhodamine B isothiocyyanate (TRITC) labeled antibody, diluted 1:60 in PBS. Finally, sections were rinsed in PBS and embedded in Vectashield (Vector Laboratories, Burlingame, Calif.) mounting medium.
- VBS phosphate buffered saline
- the total area of the muscle present in each section was examined at ⁇ 200 magnification.
- measurements of the profiles of any artery and arteriole included the length of its major and minor luminal diameter and wall thickness along the minor axis.
- the morphometric analysis allows the estimate of the length density of vessels arranged in any variety of orientations. This methodology is based on the evaluation of each vascular profile individually as it is encountered. Specifically, for n profiles counted in an area A, the length density Ld is equal to the sum of the ratio of the major or long axis to the minor or wide axis of each profile.
- FIG. 4A which is a graph of arteriole length densities in the adductor and gastrocnemius muscles of both hindlimbs, shows that the length density in the adductor muscle was approximately 11 mm/mm3 for the limb injected with AdCMV.VEGF121 and approximately 5 mm/mm3 for the limb injected with AdCMV.Null, while, for the gastrocnemius muscle, the length density of the injected limb was approximately 6 mm/mm3 for AdCMV.VEGF121 and 5 mm/mm3 for AdCMV.Null.
- FIG. 4A which is a graph of arteriole length densities in the adductor and gastrocnemius muscles of both hindlimbs, shows that the length density in the adductor muscle was approximately 11 mm/mm3 for the limb injected with AdCMV.VEGF121 and approximately 5 mm/mm3 for the limb injected with AdCMV.Null, while, for the gastrocnemius muscle, the length density of the injected
- 4B which is a graph of capillary length densities in the adductor and gastrocnemius muscles of both hindlimbs, shows that the length density in the adductor muscle was approximately 350 mm/mm3 for the limb injected with AdCMV.VEGF121 and approximately 300 mm/mm3 for the limb injected with AdCMV.Null, while, for the gastrocnemius muscle, the length density of the injected limb was approximately 250 mm/mm3 for AdCMV.VEGF121 and 275 mm/mm3 for AdCMV.Null.
- This example demonstrates the maintenance or enhancement of perfusion of blood to a nonischemic skeletal muscle at risk of being affected by, and subsequently affected by, ischemia or a vascular occlusion, after treatment with a pharmaceutical composition comprising a DNA encoding an angiogenic peptide.
Abstract
The present invention provides a method for attenuating or treating pain in nonischemic skeletal muscle, e.g., by inducing angiogenesis in a nonischemic skeletal muscle, by administration of a pharmaceutical composition comprising a DNA encoding an angiogenic peptide, such that the blood flow to the nonischemic skeletal muscle is enhanced and pain in the nonischemic skeletal muscle is attenuated. A method of treating a symptom associated with risk of ischemic damage in nonischemic skeletal muscle also is provided.
Description
- This application is a continuation of copending U.S. patent application Ser. No. 09/573,457, filed on May 17, 2000, which claims priority to U.S. Provisional Patent Application No. 60/136,612, filed on May 27, 1999.
- The present invention pertains generally to methods for inducing angiogenesis or collateral blood formation in a nonischemic skeletal muscle at risk of being affected by ischemia or a vascular occlusion, thereby maintaining or enhancing the level of perfusion of blood to the nonischemic skeletal muscle.
- Vascular atherosclerotic disease, also known as peripheral arterial occlusive disease, is a major health problem, especially in the elderly. Its prevalence increases with age from 3% in individuals younger than 60 years old to over 20% in individuals 75 years or older. Treatment of patients suffering from peripheral arterial occlusive disease remains a considerable clinical issue despite advances in both surgical and percutaneous revascularization techniques. Many patients cannot benefit from these therapies because of the anatomic extent and distribution of arterial occlusion. In such patients, new therapeutic strategies have been sought to prevent the development of disabling symptoms related to ischemia such as claudication, resting pain and loss of tissue integrity in the distal limbs. The latter can ultimately lead to limb loss.
- Angiogenesis, the growth of new blood vessels, is a complex process involving disruption of vascular basement membranes, migration and proliferation of endothelial cells, and subsequent blood vessel formation and maturation. Several mediators are known to elicit angiogenic responses, and administration of these mediators promotes revascularization of ischemic tissues. Vascular endothelial growth factor (VEGF) is one of the most specific of the known angiogenic mediators due to localization of its receptors almost exclusively on endothelial cells. Receptors for VEGF are upregulated under ischemic conditions, and the administration of recombinant VEGF augments development of collateral vessels and improves function in peripheral and myocardial ischemic tissue.
- The presence of tissue ischemia at the time of administration of an angiogenic mediator has been considered an essential precondition to evoke the desired angiogenic effect. Whether an angiogenic mediator delivered to a normoperfused tissue prior to the occurrence of ischemia could stimulate the neovascularization process and preserve blood perfusion once ischemia develops remains an unsolved issue. Studies have shown, in principle, that it was possible to induce neovascularization in vivo using adenoviral vectors encoding VEGF in nonischemic retroperitoneal adipose tissue and nonischemic subcutaneous tissue. Another study demonstrated that in vivo angiogenesis could be induced by recombinant adenoviral vectors encoding either secreted or nonsecreted forms of acidic fibroblast growth factor (aFGF). Yet, another study failed to find that endothelial cell growth factor (ECGF) had any significant angiogenic effect on vessel growth in nonischemic tissue, yet stimulated vessel growth in ischemic tissue.
- In addition to its importance in understanding the basic mechanisms involved in therapeutic angiogenesis, induction of angiogenesis in nonischemic skeletal muscle actually has clinical significance. There are many patients with peripheral arterial disease who do not have chronic ischemia but rather recurrent episodes of ischemia during physical activity. In one study, intermittent claudication was the only complaint in approximately 70% of patients with either aortoiliac or femoropopliteal atherosclerotic involvement.
- In view of the foregoing, there exists a need for an effective method of inducing angiogenesis in a nonischemic skeletal muscle. The present invention provides such a method. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
- The present invention provides a method for enhancing the level of perfusion of blood to a nonischemic skeletal muscle involving administering to a nonischemic skeletal muscle a pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) a DNA encoding an angiogenic peptide. The present invention further provides a method of treating, either therapeutically or prophylactically, a nonischemic skeletal muscle at risk of suffering from ischemic damage. Also provided is a method of treating a nonischemic skeletal muscle at risk of being affected by a vascular occlusion through induction of collateral blood vessel formation in the nonischemic skeletal muscle. Finally, a method of inducing angiogenesis is provided by the present invention.
- FIG. 1 is a graph of blood pressure ratio (BPR; ischemic/nonischemic limb) versus time after surgery (weeks) of rabbits treated with
AdCMV.VEGF121 106 pfu/ml,AdCMV.VEGF121 108 pfu/ml, AdCMV.Null (control), and saline (control). - FIG. 2 is a graph of regional blood flow ratio measured by radioactive microspheres (RBF; ischemic/nonischemic limb) versus time after surgery (days and weeks) of the gastrocnemius muscle of rabbits treated with
AdCMV.VEGF 121 106 pfu/ml,AdCMV.VEGF 121 108 pfu/ml, AdCMV.Null (control), and saline (control). - FIGS.3A-3C are graphs of 31P nuclear magnetic resonance (NMR) spectroscopy data showing the PCr/(PCr+Pi) ratio of the gastrocnemius muscle as a function of electrical stimulation protocol time (min). FIG. 3A shows data at
day 1 after surgery, FIG. 3B shows data atday 7 after surgery, and FIG. 3C show data atday 14 after surgery. In the graph of each of FIGS. 3A-3C, data are shown at rest (2 min), during stimulation (4-8 min), and during recovery (10-20 min). - FIGS. 4A and 4B are bar graphs of blood vessel length density (mm/mm3) in the adductor and gastrocnemius muscles of both injected and non-injected hindlimbs treated with AdCMV.VEGF121 and AdCMV.Null (control): FIG. 4A is a bar graph of arteriole length density, and FIG. 4B is a bar graph of capillary length density.
- The invention may best be understood with reference to the following detailed description of the preferred embodiments. The present invention provides a method for enhancing the level of perfusion of blood to a nonischemic skeletal muscle involving administering to a nonischemic skeletal muscle a pharmaceutical composition (e.g., a dose thereof) comprising (a) a pharmaceutically acceptable carrier and (b) a DNA encoding an angiogenic peptide. The present invention further provides a method of treating, either therapeutically or prophylactically, a nonischemic skeletal muscle at risk of suffering from ischemic damage. Also provided is a method of treating a nonischemic skeletal muscle at risk of being affected by a vascular occlusion through induction of collateral blood vessel formation in the nonischemic skeletal muscle. Finally, a method of inducing angiogenesis is provided by the present invention.
- Induction of Angiogenesis
- By the term “inducing angiogenesis” or “induction of angiogenesis,” it is meant that angiogenesis is either initiated or enhanced. Therefore, for example, when the nonischemic skeletal muscle is not already undergoing angiogenesis, the present method provides for initiation of angiogenesis in the nonischemic skeletal muscle. However, when the nonischemic skeletal muscle is already undergoing angiogenesis, the present method provides a means by which the level of angiogenesis is enhanced or heightened.
- Nonischemic Skeletal Muscle
- Any suitable nonischemic skeletal muscle can be subject to administration within the context of the present invention. Preferably, the nonischemic skeletal muscle comprises receptors capable of binding the angiogenic peptide encoded by the DNA; more preferably, the nonischemic skeletal muscle comprises VEGF receptors. Generally, the nonischemic skeletal muscle will be part of a discrete organ, such as a limb.
- Typically, the nonischemic skeletal muscle will be at risk of suffering from ischemic damage, which results when tissue is deprived of an adequate supply of oxygenated blood. An interruption in the supply of oxygenated blood is often caused by a vascular occlusion. Vascular atherosclerotic disease, other diseases, trauma, surgical procedures, and/or other indications can cause such vascular occlusion in nonischemic skeletal muscle.
- There are many ways to determine if nonischemic skeletal muscle is at risk of suffering ischemic damage from an undesirable vascular occlusion. Such methods are well known to physicians who treat such conditions, and include clinical evaluation (history and physical examination), Doppler, treadmill test to evaluate time to development of symptoms (e.g., pain), CT scan, NMR angiography, 31P NMR spectroscopy, and contrast angiograms. Induction of angiogenesis in nonischemic skeletal muscle at risk of being affected by a vascular occlusion is an effective means of preventing and/or attenuating any resulting ischemia. As a result, although any suitable nonischemic skeletal muscle can be targeted for the induction of angiogenesis, the target nonischemic skeletal muscle preferably is one that is at risk of being affected by a vascular occlusion.
- DNA Encoding an Angiogenic Peptide
- Any DNA encoding an angiogenic peptide operably linked to suitable expression signals can be used within the context of the present invention. Preferably, the angiogenic peptide is a VEGF protein, and more preferably, the angiogenic peptide is VEGF121, VEGF145, VEGF165, VEGF189, or a mammalian counterpart, which are variously described in U.S. Pat. Nos. 5,332,671 (Ferrara et al.), 5,240,848 (Keck et al.); and 5,219,739 (Tischer et al.). Most preferably, because of their higher biological activity, the angiogenic peptide is VEGF121 or VEGF165, particularly VEGF121. A notable difference between VEGF121 and VEGF165 is that VEGF121 does not bind to heparin with a high degree of affinity, as does VEGF165. Generally, VEGF moieties are advantageous over other angiogenic peptides because VEGF proteins do not induce the growth of tissues not involved in the production of new vasculature. Other angiogenic peptides include VEGF II, VEGF-C, fibroblast growth factors (FGFs) (e.g., aFGF, bFGF, and FGF-4), angiopoiteins, angiogenin, angiogenin-2, and P1GF, which are variously described in U.S. Pat. Nos. 5,194,596 (Tischer et al.), 5,219,739 (Tischer et al.), 5,338,840 (Bayne et al.), 5,532,343 (Bayne et al.), 5,169,764 (Shooter et al.), 5,650,490 (Davis et al.), 5,643,755 (Davis et al.), 5,879,672 (Davis et al.), 5,851,797 (Valenzuela et al.), 5,843,775 (Valenzuela et al.), and 5,821,124 (Valenzuela et al.); International Patent Application WO 95/24473 (Hu et al.); European Patent Documents 476 983 (Bayne et al.), 506 477 (Bayne et al.), and 550 296 (Sudo et al.); Japanese Patent Documents 1038100, 2117698, 2279698, and 3178996; and J. Folkman et al., A Family of Angiogenic Peptides, Nature, 329, 671 (1987).
- Administration of DNA Encoding an Angiogenic Peptide
- Induction of angiogenesis via systemic administration of a DNA encoding an angiogenic peptide, such as VEGF, can lead to promiscuous induction of angiogenesis. Promiscuous induction of angiogenesis can cause blindness, increase the aggressiveness of tumor cells, and lead to a multitude of other negative side-effects. To attenuate or prevent such negative side effects, it is desirable to induce angiogenesis only in the tissue in which it is required (i.e., the nonischemic skeletal muscle).
- The present invention involves the administration of a DNA encoding the angiogenic peptide in a localized manner to nonischemic skeletal muscle. While any suitable means of administering the DNA encoding the angiogenic peptide to the nonischemic skeletal muscle can be used within the context of the present invention, preferably, such a localized administration to the nonischemic skeletal muscle is accomplished by directly injecting the DNA encoding the angiogenic peptide into the nonischemic skeletal muscle or by topically applying the DNA encoding the angiogenic peptide to the nonischemic skeletal muscle. By the term “injecting,” it is meant that the DNA encoding the angiogenic peptide is forcefully introduced into the nonischemic skeletal muscle. Any suitable injection device can be used within the context of the present invention. However, it is desirable that whatever means of administering the DNA encoding the angiogenic peptide is chosen, the induction of angiogenesis in non-targeted tissue is minimized.
- For treatment of the hindlimb, the DNA encoding the angiogenic peptide can be delivered, for example, by intramuscular injection or a catheter inserted into the proximal portion of the femoral artery or arteries. For treatment of other nonischemic skeletal muscle, the DNA encoding the angiogenic peptide can be delivered by a catheter or like device inserted sufficiently deeply into the proximal portion of the organ- or tissue-feeding artery or arteries so that gene transfer is effected substantially only into the cells of the target organ or tissue.
- Delivery of a DNA encoding an angiogenic peptide remains a significant challenge. The half-life of many of these angiogenic peptides is very short, the administration of high doses of angiogenic peptides is associated with hypotension, and systemic administration of angiogenic peptides can cause promiscuous induction of angiogenesis in tissues other than that which has been targeted. Furthermore, the quantity of angiogenic peptide delivered is important. If too little angiogenic peptide is delivered, angiogenesis will not be induced, and a significant therapeutic benefit will not be achieved. If too much angiogenic peptide is delivered, the formation of disorganized vasculature beds, loss of function in the affected tissue, and promiscuous angiogenesis can result.
- A number of different delivery methods are available for administering a DNA encoding an angiogenic peptide, including plasmid DNA, plasmid-liposome complexes, and viral vectors. Any suitable viral vector can be used in the context of the present inventive method to administer the DNA encoding an angiogenic peptide. Examples of such suitable viral vectors are adenoviral vectors, herpes simplex viral vectors, and adeno-associated viral vectors.
- Plasmids, genetically engineered circular double-stranded DNA molecules, can be designed to contain an expression cassette for delivery of a specific DNA. Although plasmids were the first method described for gene transfer of DNA encoding an angiogenic peptide, their level of efficiency is poor, compared with other techniques. By complexing the plasmid with liposomes, the efficiency of gene transfer in general is improved. While the liposomes used for plasmid-mediated gene transfer strategies have various compositions, they are typically synthetic cationic lipids. The positively charged liposome forms a complex with a negatively charged plasmid. These plasmid-liposome complexes enter target cells by fusing with the plasma membrane. Advantages of plasmid-liposome complexes include their ability to transfer large pieces of DNA encoding an angiogenic peptide and their relatively low potential to evoke immunogenic responses in the host.
- The adenovirus is a 36 kb double-stranded DNA virus that efficiently transfers DNA in vivo to a variety of different target cell types, including skeletal muscle. The virus is made suitable by deleting some of the genes required for viral replication; the expendable E3 region is also frequently deleted to provide additional room for a larger DNA insert. The resulting replication deficient adenoviral vectors can accommodate up to 7.5 kb of exogenous DNA and are capable of being produced in high titers and efficiently transferring DNA to replicating and non-replicating cells. Of particular importance for transfer of DNA to the skeletal muscle, in which the host cell is a terminally differentiated cell, is the ability of adenoviral vectors to efficiently transfer DNA to non-replicating cells. The newly transferred genetic information remains epi-chromosomal, thus eliminating the risks of random insertional mutagenesis and permanent alteration of the genotype of the target cell.
- The herpes simplex virus (HSV) is another viral vector that can be used to administer a DNA encoding an angiogenic peptide. The mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb. Most replication-deficient HSV vectors contain a deletion to remove one or more intermediate-early genes to prevent replication. Advantages of the herpes vector are its ability to enter a latent stage that could potentially result in long-term DNA expression, and its large viral DNA genome that can accommodate exogenous DNA up to 25 kb.
- Adeno-associated virus (AAV) vectors represent other viral vectors that can be used to administer a DNA encoding an angiogenic peptide. AAV is a DNA virus, which is not known to cause human disease and which requires coinfection by a helper virus (i.e., an adenovirus or a herpes virus) for efficient replication. AAV vectors used for administration of a DNA encoding an angiogenic peptide have approximately 96% of the parental genome deleted such that only the terminal repeats remain, which contain recognition signals for DNA replication and packaging. This eliminates immunologic or toxic side effects due to expression of viral genes.
- Preferably, administration of the DNA encoding an angiogenic peptide is accomplished using an adenoviral vector. The adenoviral vector is preferably deficient in at least one gene function required for viral replication. Preferably, the adenoviral vector is deficient in at least one essential gene function of the E1 region of the adenoviral genome (e.g., the E1a and/or E1b region), particularly the E1a region. More preferably, the vector is deficient in at least one essential gene function of the E1 region and part of the E3 region (e.g., an XbaI deletion of the E3 region). Alternatively, the vector is deficient in at least one essential gene function of the E1 region and at least one essential gene function of the E4 region. However, adenoviral vectors deficient in at least one essential gene function of the E2a region and adenoviral vectors deficient in all of the E3 region also are contemplated here and are well known in the art. Adenoviral vectors deleted of the entire E4 region can elicit lower host immune responses. Suitable replication deficient adenoviral vectors are disclosed in International Patent Applications WO 95/34671 and WO 97/21826. For example, suitable replication deficient adenoviral vectors include those with a partial deletion of the E1a region, a partial deletion of the E1b region, a partial deletion of the E2a region, and a partial deletion of the E3 region. Alternatively, the replication deficient adenoviral vector can have a deletion of the E1 region, a partial deletion of the E3 region, and a partial deletion of the E4 region.
- Furthermore, the adenoviral vector's coat protein can be modified so as to incorporate a specific protein binding sequence, as described in U.S. Pat. No. 5,770,442 (Wickham et al.), or the adenoviral vector's coat protein can be modified so as to decrease the adenoviral vector's ability or inability to be recognized by a neutralizing antibody directed against the wild-type coat protein, as described in International Patent Application WO 98/40509. Other suitable modifications to the adenoviral vector are described in U.S. Pat. Nos. 5,559,099 (Wickham et al.), 5,731,190 (Wickham et al.), 5,712,136 (Wickham et al.), 5,846,782 (Wickham et al.), 5,962,311 (Wickham et al.), and 6,057,155 (Wickham et al.) and International Patent Applications WO 97/20051, WO 98/07877, WO 98/54346, and WO 00/15823.
- In addition to including the DNA encoding an angiogenic peptide, the adenoviral vector also can include a DNA encoding another peptide, for example, an angiogenic peptide receptor or another angiogenic peptide. Suitable angiogenic peptide receptors include, for example, FLT-1, FLK-1, and FLT-4.
- The DNA, operably linked to expression signals and encoding the angiogenic peptide, can be inserted into any suitable region of the adenoviral vector as an expression cassette. In that respect, the skilled artisan will readily appreciate that there are certain advantages to using an adenoviral vector deficient in some essential gene region of the adenoviral genome inasmuch as such a deficiency will provide room in the vector for a transgene and will prevent the virus from replicating. Preferably, the DNA segment is inserted into the E1 region of the adenoviral vector. Whereas the DNA segment can be inserted as an expression cassette in any suitable orientation in any suitable region of the adenoviral vector, preferably, the orientation of the DNA segment is from right to left. By the expression cassette having an orientation from right to left, it is meant that the direction of transcription of the expression cassette is opposite that of the region of the adenoviral vector into which the expression cassette is inserted.
- An adenoviral vector illustrative of the present inventive vector is deficient in the E1a region, part of the E1b region, and part of the E3 region of the adenoviral genome and contains the DNA encoding human VEGF121 or human VEGF165 under the control of the CMV immediate early promoter in the E1 region of the adenoviral genome. Such a vector supports in vivo expression of VEGF that is maximized at one day following administration and is not detectable above baseline levels as little as one week after administration. This is ideal inasmuch as it is sufficient to provide substantial growth of new vasculature while minimizing adverse neovascularization at distal sites.
- Pharmaceutical Composition
- The angiogenic peptide desirably is administered to the nonischemic skeletal muscle in a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and the DNA encoding the angiogenic peptide.
- Any suitable pharmaceutically acceptable carrier can be used within the context of the present invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition is to be administered and the particular method used to administer the composition. Formulations suitable for injection include aqueous and non-aqueous solutions, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Preferably, the pharmaceutically acceptable carrier is a buffered saline solution.
- In addition, the pharmaceutical carrier also can contain peptides, for example, an angiogenic peptide receptor, an angiogenic peptide, or a factor necessary for the development of blood vessels. These additional peptides can be encoded by a DNA, which can be a plasmid or contained within a viral vector (e.g., HSV, adenovirus, or AAV). It should be appreciated that a plasmid or viral vector comprising the DNA encoding an angiogenic peptide can be the same or different than the plasmid or viral vector that comprises the DNA encoding these additional peptides.
- Although any suitable volume of carrier can be utilized within the context of the present invention, preferably, the DNA encoding the angiogenic peptide is administered in small volumes of carrier. Administration of small volumes is such that the tissue to be vascularized (i.e., the nonischemic skeletal muscle) is perfused with the DNA encoding the angiogenic peptide and very little or no DNA encoding the angiogenic peptide is carried by the blood, lymphatic drainage, or physical mechanisms (e.g., gravitational flow or osmotic flow) to tissues not targeted.
- Dosage
- Those of ordinary skill in the art can easily make a determination of proper dosage of the DNA encoding the angiogenic peptide. However, generally, certain factors will impact the dosage that is administered.
- The proper dosage is such that the level of perfusion is enhanced to the nonischemic skeletal muscle. Preferably, the dosage is sufficient to have a therapeutic and/or prophylactic effect on nonischemic skeletal muscle that is at risk of being affected by ischemia or a vascular occlusion. Additionally, the dosage should be such that induction of angiogenesis in non-targeted tissue is minimized. The dosage also will vary depending upon the angiogenic peptide. Specifically, the dosage will vary depending upon the particular method of administration, including the nature of the vector and DNA encoding and controlling the expression of the angiogenic peptide.
- For example, for an adenoviral vector comprising a DNA encoding an angiogenic peptide, a dose typically will be at least about 1×106 pfu (e.g., 1×106-1×1013 pfu) to the nonischemic skeletal muscle, for example, a human hindlimb. The dose preferably is at least about 1×107 pfu (e.g., about 1×107-1×1013 pfu), more preferably at least about 1×108 pfu (e.g., about 1×108-1×1011 pfu), and most preferably at least about 1×109 pfu (e.g., about 1×109-1×1010 pfu). The dose typically is for a volume of targeted tissue of about 100 cm3, more typically about 150 cm3.
- For purposes of considering the dose in terms of particle units (pu), also referred to as viral particles, it can be assumed that there are 100 particles/pfu (e.g., 1×1012 pfu is equivalent to 1×1014 pu). In a single round of vector administration, using, for example, an adenoviral vector deleted of the E1a region, part of the E1b region, and part of the E3 region of the adenoviral genome, wherein the vector carries human VEGF121 or VEGF165 under the control of a standard CMV immediate early promoter, about 107-1013 pfu, preferably about 109-1011 pfu, are administered to a targeted tissue (e.g., to a discrete organ containing the targeted nonischemic skeletal muscle) with an estimated volume of about 150 cm3. Under these conditions, a substantial level of VEGF production is achieved in the nonischemic skeletal muscle without producing detectable levels of VEGF production in distal tissues.
- The invention can be more clearly understood with reference to the following examples. The following examples further illustrate the present invention, but should not be construed as in any way limiting its scope.
- Subjects
- A total of 112 6-month-old male New Zealand White rabbits (HRP Inc. Rabbitry, Denver, Pa.), mean weight 4.0±0.2 kg, and 36 10-month-old male Wistar rats (Wistar Rats Colony, Gerontology Research Center, NIA, NIH, Baltimore, Md.), mean weight 550±50 g, were used in the experimental protocols.
- Adenovirus Vectors
- The replication-deficient recombinant adenovirus vectors containing the cDNA for VEGF121 were engineered according to a technique previously described and were supplied by GenVec, Inc. (Gaithersburg, Md.). Briefly, the AdCMV.VEGF121 is an E1a-, partial E1b-, partial E3-adenovirus vector that carries an expression cassette in the E1 position containing the CMV immediate early promoter/enhancer driving the cDNA for the 121-residue form of human VEGF. AdCMV.Null, used as a control vector in this study, is similar to AdCMV.VEGF121 but with no gene in the expression cassette.
- Intramuscular Administration of AdCMV.VEGF121
- Four weeks before the induction of ischemia, rabbits were randomly assigned to receive AdCMV.VEGF121 (106 pfu/ml or 108 pfu/ml), AdCMV.Null (as a control) (108 pfu/ml), or saline (also as a control). Rats received injections of AdCMV.VEGF121 (2×109 pfu/ml) or AdCMV.Null (as a control) (2×109 pfu/ml) two weeks before surgery. The adenovirus vectors were stored in dialysis buffer solutions at −70° C. Each solution for injection was prepared immediately before use and given intramuscularly (IM) in four different sites in the thigh (250 μl/injection, 1 ml total volume in rabbits; 125 μl/injection, 0.5 ml total volume in rats) along the projection of the femoral artery.
- Animal Model of Hindlimb Ischemia
- Rabbits were pre-anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg), intubated using a laryngoscope and an uncuffed 3.5-neonatal orotracheal tube, and placed under mechanical ventilation. Stable anesthesia was achieved using a mixture of 1.5% isoflurane and oxygen. The surgical procedure to induce unilateral hindlimb ischemia in rats was performed under intraperitoneal anesthesia with ketamine (60 mg/kg) and xylazine (10 mg/kg). Both species underwent a similar surgical procedure as described below.
- A longitudinal incision was performed in the thigh, extending distally from the inguinal ligament to a point just above the knee. The femoral artery was dissected free along its entire length, as were all its major branches including the inferior epigastric, deep femoral, lateral circumflex, and superficial epigastric arteries. After further dissecting the popliteal and saphenous arteries distally, the external iliac artery, as well as all of the above arteries, was ligated with 5-0 silk (Ethicon, Inc., Somerville, N.J.). The femoral artery was completely excised from its proximal origin as a branch of the external iliac artery to the point distally where it bifurcates into the saphenous and popliteal arteries. Rabbits received 0.9% normal saline (50 ml IV) during surgery. Rats and rabbits were given post-operatory analgesia (buprenorphine 0.04 mg/kg) twice daily for the first two days after the procedure.
- Statistical Analysis
- All results are expressed as mean±SEM. Statistical comparisons were performed using ANOVA (BMGP Statistical Software). Analysis of the qualitative angiographic data was determined using a Pearson X2 test.
- This example demonstrates angiogenesis in a nonischemic skeletal muscle by administration of an adenoviral vector comprising a DNA encoding an angiogenic peptide. Further demonstrated by this example is that perfusion of blood is maintained in the skeletal muscle upon induction of ischemia. Angiogenesis in the hindlimb of rabbits was measured physiologically by calf blood pressure and blood flow measurements with radioactive microspheres and anatomically by post-mortem contrast angiography after administration of the adenoviral vector comprising VEGF121 to the nonischemic hindlimb and subsequent induction of ischemia.
- Calf Blood Pressure Ratio
- For twelve weeks after surgery, calf blood pressure was measured weekly in both hindlimbs of thirty-three rabbits using a Doppler flowmeter (Vascular Mini-Lab III, Parks Medical Electronics, Aloha, Oreg.). On each occasion, under sedation with ketamine (50 mg/kg) and xylazine (5 mg/kg), the hindlimbs were shaved and cleaned, the pulse of the posterior tibial artery was identified using a Doppler probe, and the systolic blood pressure in both limbs was determined according to standard techniques. Briefly, a 2.5 cm wide cuff was applied over the thigh, and the Doppler probe was placed over the posterior tibial artery. The cuff was rapidly inflated to approximately 30 mm Hg above the anticipated systolic pressure and then slowly deflated. The pressure at which the Doppler flow signal reappeared was recorded as the systolic pressure. A single observer, blinded to the treatment regimen, performed all measurements. The calf blood pressure ratio (BPR) was then defined as a ratio of systolic pressure of the ischemic limb to systolic pressure of the normal limb. Thus, the lower the ratio, the more impaired the arterial perfusion of the ischemic limb.
- FIG. 1, which is a graph of BPR vs. time after surgery (weeks), shows that the animals reached their final recovery ratio of approximately 0.50-0.60 after treatment with AdCMV.VEGF121 at 108 pfu/ml after 4 weeks, AdCMV.VEGF121 at 106 pfu/ml after 10 weeks, and the control groups (AdCMV.Null and saline) after 9-12 weeks. Further analysis revealed a faster rate of recovery between
weeks week 8 but became not significant thereafter. In animals treated with AdCMV.VEGF121 at 106 pfu/ml, the rate of recovery was not different from controls. Statistical analysis of BPR data revealed that there were significant differences between treatment groups (P<0.0001) and a significant effect on recovery time after surgery (P<0.0001). - Blood Flow Measurements
- The regional blood flow to skeletal muscles in both hindlimbs of sixty-four rabbits was measured using the radioactive microspheres technique at
day 1 and then atweeks - The chest was opened at the left fourth intercostal space level, the left heart chambers were exposed, and 3.3×106 radioactive microspheres (15.5 μm diameter) labeled with 51Cr (NEN Life Science Products, Boston, Mass.) were injected directly into the left ventricle within a 20-second period. Prior to injection, the vial containing the microspheres was placed in warm water (40° C.) for thirty minutes and then, immediately before injection, vigorously shaken (Daigger Vortex,
Model Genie 2, Scientific Industries, Inc., Bohemia, N.Y.) for one minute to assure proper mixing of the beads in the solution. An arterial blood reference sample was withdrawn at a constant rate of 2 ml/min starting thirty seconds before, and continued for ninety seconds after, the injection was completed. Animals then were killed with a sodium pentobarbital overdose, and the entire gastrocnemius muscles of both limbs were removed. - Each muscle was cut in three approximately equal parts (proximal, middle and distal), weighed, and put in 50 ml conical polypropylene tubes (Coming Labware & Equipment, Coming, N.Y.). Twenty ml of 2 M KOH and 10 ml of 2% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) were added to each vial for tissue digestion. After 24 hours at 50° C. in a constant temperature shaking water bath, the tissue samples were fully dissolved. All samples then were filtered using glass microfiber filters with 1.6 μm diameter pores (Whatman Filters, Whatman International Ltd., England). The filters containing the microspheres were placed into liquid scintillation vials with 10 ml of liquid scintillation cocktail (CytoScint ES, ICN Biomedical Research Products, Costa Mesa, Calif.). To prevent the occurrence of chemiluminescence in the samples, 1 ml of acetic acid was added to each vial.
- The level of radioactivity in each sample was determined using a liquid scintillation counter (Model LS5801, Beckman Coulter, Inc., Fullerton, Calif.). The regional blood flow (ml/min/100 g) was calculated using the formula: φT=100 (φR AT)/(ARWT), where φT is the blood flow in the tissue section, φR is the reference sample withdrawal rate (ml/min), AT is the activity (CPM) in the tissue, AR is the activity (CPM) in the arterial blood reference sample, and WT is the weight (g) of the tissue section.
- There were no significant differences in regional blood flow (RBF) between treatment groups or time points in nonischemic limbs (P=0.8 and P=0.6 for effects of treatment and time, respectively). Regional blood flow to ischemic limb gastrocnemius muscle versus treatment and time after surgery is presented in Table 1.
TABLE 1 Regional Blood Flow (ml/min/100 g)(mean ± SE) Day 1Week 1Week 4Week 12Saline 2.78 ± 0.43 3.93 ± 0.27 6.59 ± 0.33 6.67 ± 0.33 AdCMV.Null 2.97 ± 0.50 4.10 ± 0.21 6.30 ± 0.17 6.44 ± 0.55 AdCMV.VEGF121 5.16 ± 0.10† 7.26 ± 0.51‡ 7.96 ± 0.53* 7.87 ± 0.70* (106 pfu/ml) AdCMV.VEGF121 5.69 ± 0.40† 7.5 ± 0.95 8.74 ± 0.84* 8.79 ± 1.03* (108 pfu/ml) - FIG. 2, which is a graph of the calculated ratio between RBF in the ischemic and nonischemic gastrocnemius muscles, shows that animals reached their final recovery ratios of approximately 1.0 and 0.9, for AdCMV.VEGF121 and the controls, respectively, after treatment with AdCMV.VEGF121 (at 106 or 108 pfu/ml) after 1 week, and the controls (AdCMV.Null and saline) after 4 weeks. Significant differences were found between treatment groups and between time points for RBF in the ischemic limb (P<0.0001 for both effects of time and treatment). As seen in Table 1, RBF in the ischemic limb exhibited nearly a two-fold increase in AdCMV.VEGF121 -treated animals relative to controls as early as
day 1 after surgery (P<0.001). A significant difference in RBF as between AdCMV.VEGF121-treated animals and the control animals persisted at all subsequent time points (P<0.01). In addition, the RBF ratio atday 1 after surgery (see FIG. 2) was significantly higher in AdCMV.VEGF12 1-treated animals than in controls, and byweek 1, the RBF ratio of the AdCMV.VEGF121-treated animals was one, thereby indicating a complete restoration of tissue perfusion. - Contrast Angiography
- To anatomically evaluate the development of collateral arteries, conventional post-mortem angiograms of the ischemic limbs of fifteen rabbits were obtained after pre-treatment with intramuscular (IM) injections of AdCMV.VEGF121 at 108 pfu/ml, AdCMV.Null, or saline, as previously described. At
day 1 after surgery, animals were pre-medicated with ketamine and xylazine, as described previously, and a median laparotomy was performed under anesthesia with sodium pentobarbital. The abdominal aorta was fully exposed and a catheter (Abbocath 20G) introduced directly into the right common iliac artery. A total of 5,000 units of heparin were given to prevent clot formation. The animal was killed with an overdose of sodium pentobarbital and immediately placed under the fluoroscope (Digimax MP4000 Series III Workstation, Acomma Medical Imaging Inc., Wheeling, Ill.). A total of 5 ml contrast media (Hypaque sodium 50%, diatrizoate sodium, Nycomed Inc., Princeton, N.J.) was injected into the right common iliac artery using an infusion syringe pump (Model 848, Edco Scientific Inc., Chapel Hill, N.C.) at a constant rate of 20 ml/min. Serial images of the ischemic hindlimb were recorded and printed out for further analysis. - Quantitative assessment of new collateral vessel development in the thigh was performed using a grid overlay that comprised 2 mm squares. The films and the grid were scanned into a personal computer with the aid of image processing software (Adobe PhotoShop 5.0, Adobe Systems Incorporated) and then were edited for best quality picture. The angiographic score was determined by direct counting of the total number of contrast-opacified vessels crossing the squares divided by the total number of squares in the pre-defined area of the ischemic thigh multiplied by 100. A qualitative assessment by observation of the arterial filling in the distal leg (saphenous and popliteal arteries) also was performed. For purposes of comparison among different treatment groups, the arterial filling was noted as present or absent.
- Representative post-mortem angiograms obtained at
day 1 after surgery demonstrated in AdCMV.VEGF121-treated animals an increase in the number of vessels in the thigh compared to controls. For the saline group, 24 hours after femoral artery removal, there was very little collateral development, if any, visible in the thigh. In contrast, in the AdCMV.VEGF121 group there was clearly a network of newly formed vessels sprouting mainly from the internal iliac artery towards the medial thigh. The resulting angiographic score was significantly higher for AdCMV.VEGF121-treated animals showing a four-fold increase in the number of vessels compared to animals which received saline (AdCMV.VEGF121=51±1, saline=12±2, P<0.0001). Animals treated with AdCMV.Null also had a significantly higher angioscore than the saline group (Null=29±4, P<0.05 vs. saline), yet lower than the AdCMV.VEGF 121-treated group (P<0.001 vs. AdCMV.VEGF121). - The qualitative angiographic assessment showed that not only were there more vessels in AdCMV.VEGF121-treated animals, as indicated by the angiographic score, but also that these vessels invariably reestablished the flow to the more distal arteries in the leg (five out of five animals). Among animals that received AdCMV.Null, distal arterial filling in the ischemic leg was documented in two out of five animals, while none of the animals in the saline group exhibited a similar finding. Statistical analysis of these data showed that the AdCMV.VEGF121 group was significantly different from the saline (P<0.002) and AdCMV.Null (P<0.05) groups, whereas the control groups were not different from each other (P=0.2).
- This example therefore demonstrates the induction of angiogenesis or collateral blood formation in a nonischemic skeletal muscle at risk of being affected by, and subsequently affected by, ischemia or a vascular occlusion after treatment with a pharmaceutical composition comprising a DNA encoding an angiogenic peptide.
- This example demonstrates perfusion of blood in nonischemic skeletal muscle. In rats, the bioenergetic profile of the gastrocnemius muscle, as measured by 31 P-NMR spectroscopy, was used as an indirect indicator of gastrocnemius muscle perfusion. Histology was also performed to determine the capillary and arteriole length densities in the skeletal muscles of the hindlimbs, another indirect indicator of the level of blood perfusion in the gastrocnemius muscle.
-
- 31P-NMR spectroscopy was used to determine the bioenergetic profile of the gastrocnemius muscles of both hindlimbs at rest and during exercise induced by electrical stimulation of these muscles. NMR tests were conducted in twenty-two rats on
days - After animals were sedated with ketamine (60 mg/kg) and xylazine (10 mg/kg), two platinum subdermal electrodes (Grass Instruments Manufacturing, Braintree, Mass.) were inserted in the proximal head of the gastrocnemius and in the Achilles' tendon, respectively, for electrical stimulation. An elliptical radio-frequency (RF) surface coil tuned to the31P-resonance frequency, especially built for this study, was applied against the gastrocnemius. The electrodes were then connected to a high-voltage programmable stimulator (Model S-10, Grass Instruments Manufacturing, Braintree, Mass.) with an isolation transformer (Grass Instruments Manufacturing, Braintree, Mass.) via a low-pass filter. The foot of the stimulated leg was tied to a strain gauge force transducer (Grass Instruments Manufacturing, Braintree, Mass.) using a 3-0 silk suture. The force transducer was connected to a strain gauge conditioner, preamplifier, and chart recorder (Gould Instrument Systems, Inc., Cleveland, Ohio), allowing continuous monitoring of the muscle contraction force during the electrical stimulation.
- The electrical stimulation was applied as a train of pairs of pulses of 100 μs length separated by a 200 ms interval and repeated once every two seconds. The voltage of these pulses was incremented over about thirty seconds until the observed contraction force no longer increased, thereby determining the stimulation voltage for that leg. The animal was positioned in the NMR magnet, and the surface coil tuning was adjusted for exact resonance. Radio-frequency pulses were applied every two seconds with adiabatic frequency and amplitude shaping to compensate for the surface coil's RF inhomogeneity. The proton NMR signal from the coil was detected and used as a guide to magnetic field shipping for31P-spectroscopy. The exact proton resonance frequency of the water peak was used to calculate the expected frequency for 31P, based on the gyromagnetic ratios of the two nuclei. The RF transmitter was set to the calculated 31P frequency, and a preliminary 31P spectrum was recorded with a one-minute acquisition time.
- In each NMR experiment, one spectrum (requiring two minutes of data acquisition time for 64 scans) was collected immediately prior to stimulation, three 2-minute spectra were collected during stimulation, and six 2-minute NMR acquisitions were collected right after stimulation. Thus, for each leg, ten NMR spectra were recorded. After the experiment was completed, the procedure was repeated for the other leg, beginning with administration of additional anesthetic and placement of the subdermal electrodes.
- The NMR spectra resulting from these experiments were processed to yield PCr/(PCr+Pi) ratios as a function of time before, during, and after stimulation. After linebroadening and Fourier transformation, each spectrum was manually phased and its baseline was corrected using a spline fit with manual knot selection. Integration limits were selected by hand for the creatine phosphate and inorganic phosphate resonances, and an automated routine was used to generate a list of integrals, peak heights, and peak frequencies. The resulting data was used to calculate PCr/(PCr+Pi) ratios for peak heights. PCr/(PCr+Pi) data were plotted against a time axis ranging from zero to twenty minutes, beginning with the 2-minute data acquisition prior to electrical stimulation in which the control spectrum was recorded.
- FIGS.3A-C, which collectively are graphs of NMR data (PCr/(PCr+Pi)) obtained in rats as a function of time after surgery, show: At
day 1, AdCMV.VEGF121 pre-treated ischemic limbs recovered approximately 0.8 of the original PCr/(PCr+Pi), and AdCMV.Null pre-treated ischemic limbs only recovered approximately 0.3 of the original PCr/(PCr+Pi) (see FIG. 3A). Atday 7, AdCMV.VEGF121 pre-treated ischemic limbs recovered approximately 0.8 of the original PCr/(PCr+Pi), and AdCMV.Null pre-treated ischemic limbs recovered approximately 0.6 of the original PCr/(PCr+Pi) (see FIG. 3B). Atday 14, AdCMV.VEGF121 and AdCMV.Null pre-treated ischemic limbs recovered approximately 0.8 of the original PCr/(PCr+Pi) (see FIG. 3C). Animals pre-treated with AdCMV.VEGF121 showed an improved bioenergetic profile of the gastrocnemius muscle after femoral artery removal when compared to controls. Atday 1 after surgery, pre-exercise PCr/(PCr+Pi) ratio of the ischemic limb in AdCMV.VEGF121-treated animals was not different from the nonischemic limb. There also was less reduction of the PCr/(PCr+Pi) ratio during the stimulation (exercise) phase and faster and more complete restoration of that ratio in the recovery phase in AdCMV.VEGF121-treated animals than controls (P<0.0001). The faster recovery of AdCMV.VEGF121-treated animals persisted at day 7 (P<0.004) but not at day 14 (P>0.1) after surgery since the control animals eventually recovered enough to make this difference non-significant. - Histology and Morphometric Analysis
- To evaluate the angiogenic effect of AdCMV.VEGF121 in the absence of ischemia at the capillary level, fourteen rats were injected either with AdCMV.VEGF121 (2×109 pfu/ml) or AdCMV.Null (2×109 pfu/ml), as previously described. Fifteen days after injection of the viral vector, animals were anesthetized as usual, and a median laparotomy was performed. Both legs were then perfused via the abdominal aorta with 10% buffered formalin at 100 mm Hg for fifteen minutes. Subsequently, the adductor and gastrocnemius muscles were immersion-fixed in formalin for 48 hours.
- After paraffin embedding, sections from each sample were cut in 3 μm thick slices so that the muscle fibers were oriented in a transverse direction, and stained with α-smooth muscle actin antibody, thereby allowing for the identification of smooth muscle cells in the vascular wall. By this approach, it was possible to identify arterioles and differentiate them from capillaries and veins, because the thin walls of these vessels do not contain smooth muscle cells.
- Sections were deparafinized, rinsed in phosphate buffered saline (PBS), incubated at 37° C. for sixty minutes with mouse monoclonal anti-α-smooth muscle actin (clone 1A4, Sigma Chemical Co., St. Louis, Mo.) diluted 1:30 in PBS, and subsequently incubated at 37° C. for sixty minutes with anti-mouse IgG tetramethyrhodamine B isothiocyyanate (TRITC) labeled antibody, diluted 1:60 in PBS. Finally, sections were rinsed in PBS and embedded in Vectashield (Vector Laboratories, Burlingame, Calif.) mounting medium.
- For the morphometric analysis, the total area of the muscle present in each section was examined at ×200 magnification. In each field examined, measurements of the profiles of any artery and arteriole included the length of its major and minor luminal diameter and wall thickness along the minor axis. The morphometric analysis allows the estimate of the length density of vessels arranged in any variety of orientations. This methodology is based on the evaluation of each vascular profile individually as it is encountered. Specifically, for n profiles counted in an area A, the length density Ld is equal to the sum of the ratio of the major or long axis to the minor or wide axis of each profile. Thus, Ld is equal to the length per unit volume in the same dimensional area: Ld=1/A Σ=(R1+R2+R3+ . . . Rn)/A, where arteriole length density was expressed per unit volume (mm/mm3) of muscle.
- The analysis of the capillary network was performed utilizing an ocular reticle (10,000 μm2 area) at ×1,000 magnification. Sections from each sample were cut in 3 μm thick slices and were stained with hematoxylin and eosin. The number of capillary profiles (ncap) was measured in an area of tissue section (A) in which muscle fibers were cut transversely. The number of transversely oriented capillaries per unit area is equal to their length per unit volume. In each section, seventy-five fields were randomly examined. The number of capillary profiles was counted to compute the capillary numerical density per mm2 of muscle. ncap/mm2=ncap in total fields/total area.
- FIG. 4A, which is a graph of arteriole length densities in the adductor and gastrocnemius muscles of both hindlimbs, shows that the length density in the adductor muscle was approximately 11 mm/mm3 for the limb injected with AdCMV.VEGF121 and approximately 5 mm/mm3 for the limb injected with AdCMV.Null, while, for the gastrocnemius muscle, the length density of the injected limb was approximately 6 mm/mm3 for AdCMV.VEGF121 and 5 mm/mm3 for AdCMV.Null. FIG. 4B, which is a graph of capillary length densities in the adductor and gastrocnemius muscles of both hindlimbs, shows that the length density in the adductor muscle was approximately 350 mm/mm3 for the limb injected with AdCMV.VEGF121 and approximately 300 mm/mm3 for the limb injected with AdCMV.Null, while, for the gastrocnemius muscle, the length density of the injected limb was approximately 250 mm/mm3 for AdCMV.VEGF121 and 275 mm/mm3 for AdCMV.Null.
- Histological analysis of the muscle sections of the ischemic limb revealed that in AdCMV.VEGF121-treated rats there was a 96% increase in the length density of arterioles 4-41 μm diameter (P<0.008). The wall thickness of these arterioles was 3.24±0.35 μm and 3.54±0.15 μm for AdCMV.VEGF121 and AdCMV.Null-injected tissues, respectively (P=ns). Additionally, there was a 29% increase in the capillary length density of the adductor muscles injected with AdCMV.VEGF121 vs. AdCMV.Null (P<0.03). It is noteworthy that in the limbs treated with AdCMV.VEGF121 the angiogenic effect was limited to the muscle tissue directly injected with the adenoviral vector and there was no evidence of an increase in arterioles and capillary length densities (P=ns) in the gastrocnemius muscle of the same limbs.
- This example demonstrates the maintenance or enhancement of perfusion of blood to a nonischemic skeletal muscle at risk of being affected by, and subsequently affected by, ischemia or a vascular occlusion, after treatment with a pharmaceutical composition comprising a DNA encoding an angiogenic peptide.
- All of the references cited herein, including patents, patent applications, and publications, are hereby incorporated in their entireties by reference.
- While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.
Claims (24)
1. A method for attenuating pain associated with risk of ischemic damage in non-ischemic skeletal muscle, wherein the method comprises administering to a nonischemic skeletal muscle a pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) a DNA encoding an angiogenic peptide, such that blood flow to the nonischemic skeletal muscle is enhanced and pain in the nonischemic skeletal muscle is attenuated.
2. The method of claim 1 , wherein pain is associated with intermittent claudication.
3. The method of claim 1 , wherein angiogenesis is induced in the nonischemic skeletal muscle.
4. The method of claim 1 , wherein the angiogenic peptide is a vascular endothelial growth factor (VEGF).
5. The method of claim 1 , wherein the DNA encoding an angiogenic peptide is in a viral vector.
6. The method of claim 5 , wherein the viral vector is an adenoviral vector.
7. The method of claim 6 , wherein the adenoviral vector is deficient in at least one essential gene function of the E1 region of the adenoviral genome.
8. The method of claim 1 , wherein the skeletal muscle comprises a portion of a human limb.
9. A method of treating a symptom associated with risk of ischemic damage in nonischemic skeletal muscle, wherein the method comprises administering to a nonischemic skeletal muscle a pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) a DNA encoding an angiogenic peptide, such that blood flow to the nonischemic skeletal muscle is enhanced and the symptom associated with risk of ischemic damage is treated.
10. The method of claim 9 , wherein the symptom associated with risk of ischemic damage is pain.
11. The method of claim 10 , wherein the pain is associated with intermittent claudication.
12. The method of claim 9 , wherein angiogenesis is induced in the nonischemic skeletal muscle.
13. The method of claim 9 , wherein the angiogenic peptide is a vascular endothelial growth factor (VEGF).
14. The method of claim 9 , wherein the DNA encoding an angiogenic peptide is in a viral vector.
15. The method of claim 14 , wherein the viral vector is an adenoviral vector.
16. The method of claim 15 , wherein the adenoviral vector is deficient in at least one essential gene function of the E1 region of the adenoviral genome.
17. The method of claim 9 , wherein the skeletal muscle comprises a portion of a human limb.
18. A method for treating pain upon movement, wherein the method comprises administering to a nonischemic skeletal muscle a pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) a DNA encoding an angiogenic peptide, such that blood flow to the nonischemic skeletal muscle is enhanced and pain in the nonischemic skeletal muscle upon movement is attenuated.
19. The method of claim 18 , wherein angiogenesis is induced in the nonischemic skeletal muscle.
20. The method of claim 18 , wherein the angiogenic peptide is a vascular endothelial growth factor (VEGF).
21. The method of claim 18 , wherein the DNA encoding an angiogenic peptide is in a viral vector.
22. The method of claim 21 , wherein the viral vector is an adenoviral vector.
23. The method of claim 22 , wherein the adenoviral vector is deficient in at least one essential gene function of the E1 region of the adenoviral genome.
24. The method of claim 18 , wherein the skeletal muscle comprises a portion of a human limb.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/176,024 US20020187955A1 (en) | 1999-05-27 | 2002-06-20 | Method of inducing angiogenesis in nonischemic skeletal muscle |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13661299P | 1999-05-27 | 1999-05-27 | |
US09/573,457 US6440945B1 (en) | 1999-05-27 | 2000-05-17 | Method of inducing angiogenesis in nonis chemic skeletal muscle |
US10/176,024 US20020187955A1 (en) | 1999-05-27 | 2002-06-20 | Method of inducing angiogenesis in nonischemic skeletal muscle |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/573,457 Continuation US6440945B1 (en) | 1999-05-27 | 2000-05-17 | Method of inducing angiogenesis in nonis chemic skeletal muscle |
Publications (1)
Publication Number | Publication Date |
---|---|
US20020187955A1 true US20020187955A1 (en) | 2002-12-12 |
Family
ID=26834469
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/573,457 Expired - Lifetime US6440945B1 (en) | 1999-05-27 | 2000-05-17 | Method of inducing angiogenesis in nonis chemic skeletal muscle |
US10/176,024 Abandoned US20020187955A1 (en) | 1999-05-27 | 2002-06-20 | Method of inducing angiogenesis in nonischemic skeletal muscle |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/573,457 Expired - Lifetime US6440945B1 (en) | 1999-05-27 | 2000-05-17 | Method of inducing angiogenesis in nonis chemic skeletal muscle |
Country Status (1)
Country | Link |
---|---|
US (2) | US6440945B1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022109592A3 (en) * | 2020-11-20 | 2022-06-30 | Baylor College Of Medicine | Suppressing hippo signaling in the stem cell niche promotes skeletal muscle regeneration |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK1142590T3 (en) * | 1999-10-29 | 2009-01-26 | Anges Mg Inc | Gene therapy for diabetic ischemic disease |
US20040009940A1 (en) * | 2000-10-20 | 2004-01-15 | Coleman Michael E. | Gene delivery formulations and methods for treatment of ischemic conditions |
AU2002256388A1 (en) * | 2001-04-30 | 2002-11-11 | Cell Genesys, Inc. | Viral-mediated delivery and in vivo expression of polynucleotides encoding anti-angiogenic proteins |
AR027161A1 (en) * | 2001-05-15 | 2003-03-19 | Bio Sidus S A | METHOD FOR INDUCTING NEOVASCULAR PROLIFERATION AND TISSULAR REGENERATION |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5792453A (en) * | 1995-02-28 | 1998-08-11 | The Regents Of The University Of California | Gene transfer-mediated angiogenesis therapy |
US6121246A (en) * | 1995-10-20 | 2000-09-19 | St. Elizabeth's Medical Center Of Boston, Inc. | Method for treating ischemic tissue |
US6518255B2 (en) * | 1997-01-29 | 2003-02-11 | Cornell Research Foundation, Inc. | Multiple site delivery of adenoviral vector directly into muscle for the induction of angiogenesis |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996026742A1 (en) | 1995-02-28 | 1996-09-06 | The Regents Of The University Of California | Gene transfer-mediated angiogenesis therapy |
-
2000
- 2000-05-17 US US09/573,457 patent/US6440945B1/en not_active Expired - Lifetime
-
2002
- 2002-06-20 US US10/176,024 patent/US20020187955A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5792453A (en) * | 1995-02-28 | 1998-08-11 | The Regents Of The University Of California | Gene transfer-mediated angiogenesis therapy |
US6121246A (en) * | 1995-10-20 | 2000-09-19 | St. Elizabeth's Medical Center Of Boston, Inc. | Method for treating ischemic tissue |
US6518255B2 (en) * | 1997-01-29 | 2003-02-11 | Cornell Research Foundation, Inc. | Multiple site delivery of adenoviral vector directly into muscle for the induction of angiogenesis |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022109592A3 (en) * | 2020-11-20 | 2022-06-30 | Baylor College Of Medicine | Suppressing hippo signaling in the stem cell niche promotes skeletal muscle regeneration |
Also Published As
Publication number | Publication date |
---|---|
US6440945B1 (en) | 2002-08-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhou et al. | Neurotrophin-3 expressed in situ induces axonal plasticity in the adult injured spinal cord | |
US6518255B2 (en) | Multiple site delivery of adenoviral vector directly into muscle for the induction of angiogenesis | |
US20060003932A1 (en) | Method for promoting neovascularization | |
JP2008024718A (en) | Method for treating ischemic tissue | |
Hung et al. | Gene transfer of insulin-like growth factor–I providing neuroprotection after spinal cord injury in rats | |
AU2010257389A1 (en) | Plasmid encoding fibroblast growth factor for the treatment of hypercholesterolemia or diabetes associated angiogenic defects | |
Hershey et al. | Vascular endothelial growth factor stimulates angiogenesis without improving collateral blood flow following hindlimb ischemia in rabbits | |
US6329348B1 (en) | Method of inducing angiogenesis | |
US6440945B1 (en) | Method of inducing angiogenesis in nonis chemic skeletal muscle | |
Gowdak et al. | Induction of angiogenesis by cationic lipid-mediated VEGF165 gene transfer in the rabbit ischemic hindlimb model | |
Tazawa et al. | Granulocyte‐macrophage colony‐stimulating factor inhalation therapy for patients with idiopathic pulmonary alveolar proteinosis: a pilot study; and long‐term treatment with aerosolized granulocyte‐macrophage colony‐stimulating factor: a case report | |
JP2002529510A (en) | Method for preventing and regressing atherosclerosis in a mammal | |
ZA200509830B (en) | Plasmid encoding fibroblast growth factor for the treatment of hypercholesterolemia or diabetes associated angiogenic defects | |
JP7450244B2 (en) | Methods for treating ischemic tissue | |
Albertine et al. | Mesenchymal stromal cell extracellular vesicles improve lung development in mechanically ventilated preterm lambs | |
Ha et al. | Therapeutic angiogenesis induced by human hepatocyte growth factor gene in hindlimb ischemia of dogs | |
JP5973605B2 (en) | Vascular endothelial growth factor (VEGF) production inducer | |
MXPA05013055A (en) | Plasmid encoding fibroblast growth factor for the treatment of hypercholesterolemia or diabetes associated angiogenic defects | |
Ito | Ultrastructure of arterial spasms as related to atherosclerosis and hyperlipidemia | |
Takagi | ISCHEMIA-REPERFUSION INJURY IN RATS |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |