RESUMO
Antagonism of the mGluR2 receptor has the potential to provide therapeutic benefit to cognitive disorders by elevating synaptic glutamate, the primary excitatory neurotransmitter in the brain. Selective antagonism of the mGluR2 receptor, however, has so far been elusive, given the very high homology of this receptor with mGluR3, particularly at the orthosteric binding site. Given that inhibition of mGluR3 has been implicated in undesired effects, we sought to identify selective mGluR2 negative allosteric modulators. Herein we describe the discovery of the highly potent and selective class of mGluR2 negative allosteric modulators, 4-arylquinoline-2-carboxamides, following a successful HTS campaign and medicinal chemistry optimization, showing potent in vivo efficacy in rodent.
Assuntos
Descoberta de Drogas , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Adjuvantes Anestésicos/toxicidade , Aminoácidos/farmacologia , Anfetaminas/farmacologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ácido Glutâmico/metabolismo , Ensaios de Triagem em Larga Escala , Camundongos , Estrutura Molecular , Escopolamina/toxicidade , Relação Estrutura-AtividadeRESUMO
The ß-amyloid peptides (Aß) are thought to play a critical role in the pathophysiology of Alzheimer's disease. One therapeutic strategy aimed to reduce or eliminate the production of Aß peptides is inhibition of the γ-secretase enzyme, which cleaves amyloid precursor protein to form Aß peptides. We studied the in vivo effects of the potent, orally bioavailable and brain penetrant γ-secretase inhibitor (GSI), MRK-560, on both Aßx-40 and Aßx-42 in multiple compartments (plasma, the brain and cerebrospinal fluid) of rat. Although there were differences in the time course and magnitude of the changes, the results showed that MRK-560 caused marked inhibition of both Aßx-40 and Aßx-42 in all three compartments. We identified good correlations between plasma Aßx-40 versus brain Aßx-40 (r = 0.84), and plasma Aßx-40 versus CSF Aßx-40 (r = 0.85), indicating that these pools of Aß are related dynamically. These results suggest that central Aß changes that occur following acute dosing with MRK-560 can be predicted based on plasma Aß changes and could thus serve as a useful biomarker to help accelerate decision-making during early clinical development.
Assuntos
Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Inibidores Enzimáticos/farmacologia , Sulfonamidas/farmacologia , Administração Oral , Animais , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Masculino , Ratos , Ratos Sprague-Dawley , Estatística como Assunto , Fatores de TempoRESUMO
A series of partial agonists of the Glucocorticoid Receptor were prepared targeting reduced transactivation activity, while maintaining significant transrepression activity. Incorporation of an ortho-aryl amide produced compounds with the desired in vitro profile. Bioreactors consisting of Suspension cultures of Sf21 cells co expressing a CYP3A4 and NADPH-cytochrome P450 oxireductase were used to prepare the major metabolites of these compounds and revealed that oxidative N-dealkylation provided a pathway for formation of metabolites that were more agonistic than the parent partial agonists. Oxidative N-dealkylation was blocked in a new series of compounds, however oxidation alone was capable of producing full agonist metabolites. Incorporation of an ortho-primary amide and utilization of fluorine to modulate agonism afforded partial agonist MK-5932. Synthesis of the major metabolites of MK-5932 using bioreactor technology revealed that no significant GR-active metabolites were formed. Orally administered MK-5932 displayed anti-inflammatory efficacy in a Rat Oxazolone-induced chronic dermatitis model, while sparing plasma insulin.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Reatores Biológicos , Receptores de Glucocorticoides/agonistas , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Linhagem Celular , Citocromo P-450 CYP3A/metabolismo , Remoção de Radical Alquila , Dermatite/tratamento farmacológico , Feminino , Glucocorticoides/metabolismo , Humanos , Insetos , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/metabolismoRESUMO
A novel series of potent CGRP receptor antagonists containing a central quinoline ring constraint was identified. The combination of the quinoline constraint with a tricyclic benzimidazolinone left hand fragment produced an analog with picomolar potency (14, CGRP K(i)=23 pM). Further optimization of the tricycle produced a CGRP receptor antagonist that exhibited subnanomolar potency (19, CGRP K(i)=0.52 nM) and displayed a good pharmacokinetic profile in three preclinical species.
Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Quinolinas/farmacologia , Animais , Disponibilidade Biológica , Cães , Avaliação Pré-Clínica de Medicamentos , Macaca mulatta , Quinolinas/química , Quinolinas/farmacocinética , RatosRESUMO
The primary pathophysiology of peripheral artery occlusive disease is associated with impaired perfusion to the lower extremities. The lack of effective pharmacologic agents to treat this disease emphasizes the need for well-characterized animal models that can be used to evaluate the efficacy of emerging therapies. A major limitation with the current animal models of peripheral artery occlusive disease is that the variety of surgical methods employed to reduce peripheral blood flow produce differences in the severity and time course of the resting and reserve blood flow deficits. Furthermore, the methods used to evaluate the restoration of peripheral flow are often not suitable for serial measurements. This study used laser Doppler imaging to serially evaluate resting blood flow and the development of a functional collateral circulation after the induction of hind limb ischemia in the rat. Reserve blood flow was assessed by measuring hyperemic blood flow in the hind paw after temporary arterial occlusion. The magnitude of the hyperemic response was found to be dependent upon both the duration of arterial occlusion and the measurement time after release of the occluder. After ligation of the common iliac artery, but at a time when resting blood flow was reestablished, hyperemic tests unmasked a sustained deficit in reserve blood flow capacity that persisted for at least 14 days. Therefore, the use of a noninvasive vascular occluder and laser Doppler imaging represents a sensitive and consistent technique to measure peripheral blood flow status to assess the development of functional collateral vessels. These findings will enhance the ability to effectively study pharmacologic therapies aimed at promoting the growth and development of collateral vessels.
Assuntos
Membro Posterior/irrigação sanguínea , Hiperemia/fisiopatologia , Isquemia/fisiopatologia , Músculo Esquelético/irrigação sanguínea , Animais , Circulação Colateral , Modelos Animais de Doenças , Fluxometria por Laser-Doppler , Masculino , Microcirculação , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo RegionalRESUMO
The blood pressure (BP)-lowering effects of mineralocorticoid receptor (MR) antagonists in salt-sensitive rat models of hypertension are well understood. However, studies in salt-independent models have yielded mixed results, and therefore, we measured the hemodynamic effects of MR blockade in spontaneously hypertensive rats. We treated spontaneously hypertensive rats for 8 weeks with 30-300 mg.kg.d eplerenone or 20 mg.kg.d losartan and monitored BP using radiotelemetry and performed histopathological analyses of the hearts. Eplerenone, in contrast to losartan, caused only a small reduction in systolic BP at the highest dose tested. Both reduced left ventricular wall thickness, although eplerenone was less effective than losartan. Only losartan decreased heart weight. We observed foci of cardiomyopathy characterized by combinations of infiltrating monocytes, necrotic myocytes, and interstitial fibrosis in hearts of control animals. The number of foci seemed to be decreased in hearts of losartan- and eplerenone-treated animals. In a second study, using quantitative histomorphometry, the number of foci was significantly reduced by 20 mg.kg.d losartan (by 68%) or by 300 mg.kg.d eplerenone (by 50%). Our data support the hypothesis that a direct BP-independent effect on the progression of cardiomyopathy in the heart may be one basis for the cardiac protection afforded by MR antagonism.
Assuntos
Coração/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Eplerenona , Coração/fisiopatologia , Hipertensão/sangue , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Losartan/farmacologia , Losartan/uso terapêutico , Masculino , Ratos , Ratos Endogâmicos SHR , Cloreto de Sódio na Dieta/farmacologia , Cloreto de Sódio na Dieta/uso terapêutico , Espironolactona/análogos & derivadosRESUMO
A series of tricyclic CGRP receptor antagonists was optimized in order to improve oral bioavailability. Attenuation of polar surface area and incorporation of a weakly basic indoline nitrogen led to compound 5, a potent antagonist with good oral bioavailability in three species.
Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Compostos de Espiro/farmacocinética , Administração Oral , Animais , Disponibilidade Biológica , Linhagem Celular , Cães , Haplorrinos , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Ratos , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Compostos de Espiro/administração & dosagemRESUMO
Calcitonin gene-related peptide (CGRP) is a potent neuropeptide that plays a key role in the pathophysiology of migraine headache. CGRP levels in the cranial circulation are increased during a migraine attack, and CGRP itself has been shown to trigger migraine-like headache. The correlation between CGRP release and migraine headache points to the potential utility of CGRP receptor antagonists as novel therapeutics in the treatment of migraine. Indeed, clinical proof-of-concept in the acute treatment of migraine was demonstrated with an intravenous formulation of the CGRP receptor antagonist BIBN4096BS (olcegepant). Here we report on the pharmacological characterization of the first orally bioavailable CGRP receptor antagonist in clinical development, MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide]. In vitro, MK-0974 is a potent antagonist of the human (K(i) = 0.77 nM) and rhesus (K(i) = 1.2 nM) CGRP receptors but displays >1500-fold lower affinity for the canine and rat receptors as determined via (125)I-human CGRP competition binding assays. A rhesus pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging was utilized to determine the in vivo activity of CGRP receptor antagonism. MK-0974 produced a concentration-dependent inhibition of dermal vasodilation, generated by capsaicin-induced release of endogenous CGRP, with plasma concentrations of 127 and 994 nM required to block 50 and 90% of the blood flow increase, respectively. In conclusion, MK-0974 is a highly potent, selective, and orally bioavailable CGRP receptor antagonist, which may be valuable in the acute treatment of migraine.
Assuntos
Azepinas/administração & dosagem , Azepinas/química , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Imidazóis/administração & dosagem , Imidazóis/química , Transtornos de Enxaqueca/tratamento farmacológico , Administração Oral , Animais , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Feminino , Humanos , Macaca mulatta , Masculino , Transtornos de Enxaqueca/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismoRESUMO
INTRODUCTION: Mineralocorticoid receptor (MR) antagonists are useful for the treatment of hypertension and heart failure. In the present work we sought to develop a simplified protocol for measuring the acute activity of MR antagonists on urinary excretion of sodium and potassium in rats based on the original studies of mineralocorticoids in adrenalectomized rats reported by Kagawa et al. (Kagawa, C. M., & Jacobs Jr., R. S. (1960) Mineralocorticoid effects of 9 alphafluorodeoxycorticosterone in adrenalectomized rats. Proceedings of the Society for Experimental Biology and Medicine, 104, 60-62). METHODS: Rats with intact adrenal glands were treated with test compounds and challenged with a bolus oral dose of saline. Urine was collected over 4 h in metabolism cages and urinary sodium and potassium concentrations were measured. RESULTS: Aldosterone had no significant effect on sodium or potassium excretion and MR antagonists dose-dependently increased the ratio of sodium to potassium. Diuretics with distinct mechanisms of action were differentiated via their relative effects on sodium, potassium and urine volumes and the new assay protocol was used to characterize a novel MR antagonist. DISCUSSION: A facile and robust protocol for the measurement of antagonism of renal MRs was established. This protocol used fewer animals than previously described methods and did not require preparative surgery, factors which contributed favorably to cost, experimental throughput and animal use.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Antagonistas de Receptores de Mineralocorticoides , Aldosterona/farmacologia , Amilorida/farmacologia , Análise de Variância , Animais , Creatinina/urina , Diuréticos/farmacologia , Relação Dose-Resposta a Droga , Eplerenona , Hidroclorotiazida/farmacologia , Imidazóis/farmacologia , Masculino , Potássio/urina , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Mineralocorticoides/agonistas , Receptores de Mineralocorticoides/metabolismo , Sódio/urina , Espironolactona/análogos & derivados , Espironolactona/farmacologiaRESUMO
The previously disclosed spirohydantoin-based CGRP receptor antagonists were optimized for potency through modification of the benzimidazolone substituents. Compounds were identified which had minimal shift in the cAMP functional assay containing 50% human serum. Blockade of CGRP-mediated vasodilation was observed with these compounds in a rhesus pharmacodynamic assay and the in vivo potency correlated with the in vitro activity in the serum-shifted functional assay.
Assuntos
Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Indanos/química , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Animais , Benzimidazóis/sangue , Benzimidazóis/química , Técnicas de Química Combinatória , Humanos , Macaca mulatta , Estrutura Molecular , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Herein we describe optimization of CGRP receptor antagonists based on an earlier lead structure containing a (3R)-amino-(6S)-phenylcaprolactam core. Replacement of the phenylimidazolinone with an azabenzimidazolone gave stable derivatives with lowered serum shifts. Extensive SAR studies of the C-6 aryl moiety revealed the potency-enhancing effect of the 2,3-difluorophenyl group, and trifluoroethylation of the N-1 amide position resulted in improved oral bioavailabilities, ultimately leading to clinical candidate 38 (MK-0974).
Assuntos
Azepinas/síntese química , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Imidazóis/síntese química , Transtornos de Enxaqueca/tratamento farmacológico , Administração Oral , Animais , Azepinas/farmacocinética , Azepinas/farmacologia , Disponibilidade Biológica , Células CACO-2 , Permeabilidade da Membrana Celular , Cães , Humanos , Imidazóis/farmacocinética , Imidazóis/farmacologia , Macaca mulatta , Ratos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Pele/irrigação sanguínea , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The recent discovery of several nonpeptide CGRP antagonists have led to significant advances in our understanding of CGRP receptor pharmacology. Specifically, these antagonists have demonstrated a clear species selectivity with >100-fold greater affinity for human CGRP receptor compared to receptors from other species, such as rat, rabbit and guinea pig. Therefore, nonhuman primate models are required to accurately assess the in vivo activity of these antagonists. The commonly used model in marmosets involves electrical stimulation of the trigeminal ganglia and is a technically difficult and terminal procedure. In this report, we describe a noninvasive pharmacodynamic model in which topical application of capsaicin is utilized to induce the release of endogenous CGRP and a vasodilatory response which can be measured using laser Doppler imaging. Using the potent and selective CGRP antagonist Compound 3, which is an analog of the well-characterized compound BIBN4096BS, we demonstrated 62% inhibition with 300 microg/kg, i.v., in the rat. When tested in the rhesus monkey, only 30 microg/kg of Compound 3 was needed to produce complete inhibition, suggesting that the rhesus CGRP receptor shares a pharmacological profile similar to marmoset and human receptors. Two separate measurements were obtained in this model to provide an indication of both the acute inhibitory effect as well as the prophylactic effect of the CGRP antagonist. At the doses studied, Compound 3 was equally effective on both the acute and prophylactic inhibition of CGRP-mediated vasodilation in rat and rhesus. In conclusion, this is the first report to describe and validate a noninvasive model in nonhuman primates that allows rapid evaluation of CGRP antagonist activity against endogenous CGRP.
Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Capsaicina/farmacologia , Piperazinas/farmacologia , Quinazolinas/farmacologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Vasodilatação/efeitos dos fármacos , Administração Tópica , Sequência de Aminoácidos , Animais , Bioensaio , Peptídeo Relacionado com Gene de Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/administração & dosagem , Relação Dose-Resposta a Droga , Epiderme/anatomia & histologia , Epiderme/efeitos dos fármacos , Cobaias , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fluxometria por Laser-Doppler , Macaca mulatta , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Piperazinas/química , Piperazinas/farmacocinética , Quinazolinas/química , Quinazolinas/farmacocinética , Coelhos , Ratos , Proteínas Modificadoras da Atividade de Receptores , Alinhamento de SequênciaRESUMO
This study used structural equation modeling to test a theory-based model of the pathways by which exposure to the "truth" counterindustry media campaign influenced beliefs, attitudes, and smoking behavior in national random-digit-dial telephone surveys of 16,000 12- to 17-year-olds before, 8 months after, and 15 months after campaign launch. Consistent with concepts from the theory of reasoned action, youth in markets with higher levels of campaign exposure had more negative beliefs about tobacco industry practices and more negative attitudes toward the tobacco industry. Models also provided support for a social inoculation effect, because negative industry attitudes were associated with lower receptivity to protobacco advertising and with less progression along a continuum of smoking intentions and behavior.
Assuntos
Comportamento do Adolescente/psicologia , Publicidade/métodos , Promoção da Saúde/métodos , Prevenção do Hábito de Fumar , Indústria do Tabaco , Confiança/psicologia , Adolescente , Conscientização , Criança , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Inquéritos Epidemiológicos , Humanos , Entrevistas como Assunto , Masculino , Marketing/organização & administração , Marketing/normas , Modelos Teóricos , Avaliação de Programas e Projetos de Saúde , Fumar/psicologia , Abandono do Hábito de Fumar , Indústria do Tabaco/éticaRESUMO
UNLABELLED: Recent investigations have established the feasibility of using exogenously delivered angiogenic growth factors to increase collateral artery development in animal models of myocardial and hindlimb ischemia. OBJECTIVE: Our aim was to evaluate the ability of a stabilized form of acidic fibroblast growth factor (aFGF-S117) to stimulate collateralization and arteriogenesis in the rabbit hindlimb following the surgical induction of ischemia by femoral artery extraction. A secondary objective was to examine angiogenic and arteriogenic effects of aFGF-S117 in the absence of a peripheral blood flow deficit. METHODS AND RESULTS: Five days after femoral artery removal, aFGF-S117 (1, 3, or 30 microg/kg) was intramuscularly delivered into the hindlimb, three times per week for 2 consecutive weeks. End-point measurements performed on day 20 found that hindlimb reserve blood flow was significantly improved in rabbits that received 3 or 30 microg/kg of aFGF-S117, with no difference in efficacy between these two doses. These hemodynamic results were supported by angiographic evidence showing enhanced density of collateral vessels in the medial thigh region and histological findings of increased capillary density within the gastrocnemius muscle from rabbits treated with aFGF-S117. When an efficacious dose of 3 microg/kg of aFGF-S117 was administered to sham-operated rabbits with intact femoral arteries, there was no change in any of the blood flow, angiographic or histological parameters measured. CONCLUSIONS: These findings demonstrate that a stabilized form of aFGF stimulated the development of functional collateral arteries in the rabbit hindlimb, an effect which was dependent upon removal of the femoral artery. These results suggest that aFGF-S117 may have therapeutic potential for the treatment of arterial occlusive disorders.
Assuntos
Circulação Colateral , Fator 1 de Crescimento de Fibroblastos/farmacologia , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Animais , Arteriopatias Oclusivas/tratamento farmacológico , Arteriopatias Oclusivas/fisiopatologia , Artérias , Capilares , Artéria Femoral/cirurgia , Masculino , Modelos Animais , Neovascularização Fisiológica , Coelhos , Fluxo Sanguíneo Regional , Estimulação QuímicaRESUMO
Glucocorticoids are used widely in the treatment of inflammatory diseases, but use is accompanied by a significant burden of adverse effects. It has been hypothesized that gene- and cell-specific regulation of the glucocorticoid receptor by small molecule ligands could be translated into a therapeutic with an improved risk-benefit profile. MK-5932 is a highly selective glucocorticoid receptor modulator that is anti-inflammatory in vivo with an improved profile on glucose metabolism: Bungard et al. (2011). Bioorg. Med. Chem. 19, 7374-7386. Here we describe the full biological profile of MK-5932. Cytokine production following lipopolysaccharide (LPS) challenge was blocked by MK-5932 in both rat and human whole blood. Oral administration reduced inflammatory cytokine levels in the serum of rats challenged with LPS. MK-5932 was anti-inflammatory in a rat contact dermatitis model, but was differentiated from 6-methylprednisolone by a lack of elevation of fasting insulin or glucose levels after 7 days of dosing, even at high exposure levels. In fact, animals in the vehicle group were consistently hyperglycemic at the end of the study, and MK-5932 normalized glucose levels in a dose-dependent manner. MK-5932 was also anti-inflammatory in the rat collagen-induced arthritis and adjuvant-induced arthritis models. In healthy dogs, oral administration of MK-5932 exerted acute pharmacodynamic effects with potency comparable to prednisone, but with important differences on neutrophil counts, again suggestive of a dissociated profile. Important gaps in our understanding of mechanism of action remain, but MK-5932 will be a useful tool in dissecting the mechanisms of glucose dysregulation by therapeutic glucocortiocids.
Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Benzamidas/uso terapêutico , Dermatite de Contato/tratamento farmacológico , Edema/tratamento farmacológico , Indazóis/uso terapêutico , Receptores de Glucocorticoides/metabolismo , Animais , Anti-Inflamatórios/sangue , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Benzamidas/sangue , Benzamidas/farmacocinética , Benzamidas/farmacologia , Linhagem Celular Tumoral , Colágeno , Citocinas/sangue , Cães , Feminino , Células HeLa , Humanos , Indazóis/sangue , Indazóis/farmacocinética , Indazóis/farmacologia , Insulina , Lipopolissacarídeos , Masculino , Metilprednisolona/farmacologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-DawleyRESUMO
This study was designed to test the ability of adenovirus-delivered vascular endothelial growth factor (Ad-VEGF) to stimulate angiogenesis and arteriogenesis in the rabbit hindlimb following the induction of ischemia and to evaluate the functional changes in the collateral circulation. Ten days after the surgical induction of hindlimb ischemia, either a control virus (1 x 10(9) pfu) or an adenovirus containing the gene for VEGF(165) (1 x 10(6), 1 x 10(7), 1 x 10(8), or 1 x 10(9) pfu) was administered intramuscularly into the ischemic limb. Thirty days after administration of the adenoviral vectors, skeletal muscle capillary density was assessed and angiography was performed as markers of angiogenesis and arteriogenesis, respectively. Hindlimb blood flow was directly measured and hyperemic tests were performed to evaluate the functional improvements in collateral blood flow. Animals treated with Ad-VEGF at 1 x 10(8) and 1 x 10(9) pfu showed elevated levels of circulating VEGF and dose-dependent hindlimb edema. These doses also led to a robust angiogenic response (i.e., increase in capillary density), but failed to improve collateral blood flow. Consistent with the lack of a functional response, there was no angiographic evidence of enhanced arteriogenesis with any dose of Ad-VEGF. Following the induction of hindlimb ischemia, administration of Ad-VEGF stimulated capillary sprouting (i.e., angiogenesis), but did not increase the growth and development of larger conduit vessels (i.e., arteriogenesis) or improve collateral blood flow. These results support the concept that VEGF may not be expected to have therapeutic utility for the treatment of peripheral or myocardial ischemia.