RESUMO
Bovine colostral antibodies, purified from cow's milk produced immediately after calving, have enhanced resistance to degradation by intestinal proteases relative to antibodies from human or bovine serum, making them of particular interest as orally administered therapeutic agents. However, the basis of this resistance is not well defined. We evaluated the stability of AVX-470, a bovine colostral anti-tumor necrosis factor (TNF) polyclonal antibody used in early clinical studies for treatment of ulcerative colitis, using conditions that mimic the human small intestine. AVX-470 was degraded â¼3 times more slowly than human IgG antibodies or infliximab (a monoclonal mouse-human chimeric IgG). Bovine IgG1 antibodies, the primary component of AVX-470, were slowly cleaved to F(ab')2 fragments. In contrast, bovine IgG2 and human IgG1 antibodies were cleaved rapidly into Fab and smaller fragments, pointing to specific regions where additional stability might be gained. Infliximab was modified to incorporate the sequences from these regions, including the bovine IgG1 hinge region and a predicted disulfide bonding motif linking the upper hinge region, the CH1 domain, and the light chain. This infliximab-bovine IgG1 chimera (bovinized infliximab) retained the antigen binding and neutralization activity of the WT sequence but was degraded 9-fold more slowly than the unmodified infliximab. This remarkable increase in stability with as few as 18 amino acid substitutions suggests that this bovinization process is a means to enable oral delivery of proven therapeutic antibodies as well as novel antibodies to targets that have been previously inaccessible to therapies delivered by injection.
Assuntos
Colostro/química , Imunoglobulina G/química , Intestinos/química , Proteólise , Animais , Bovinos , Feminino , Humanos , Estabilidade ProteicaRESUMO
The use of hyper-immune bovine colostrum as a human therapeutic platform is an emerging technology with potential to deliver the efficacy of antibody therapeutics with the convenience and safety of oral or topical application. It is necessary to understand how the bovine immune system responds to immunization with foreign proteins, both in terms of the serum antibody response and the transfer of antigen-specific antibodies into the colostrum to enable efficient large-scale production of therapeutic antibodies. We have immunized 25 cows with recombinant human tumor necrosis factor (rhTNF) and measured the levels of rhTNF-specific antibodies in the serum and colostrum of these animals. We observed a decline of 84±9% in serum IgG1 concentrations in the final weeks of pregnancy that presumably reflects rapid transport of IgG1 into colostrum. The serum IgG2 levels remained constant, such that the serum IgG1 to IgG2 ratio was 1:20 at parturition. We observed substantial animal-to-animal variability in the levels of anti-rhTNF antibodies in both serum and colostrum samples. In particular, a subset of 4 cows had extraordinarily high colostral anti-rhTNF antibody production. Only a weak correlation was found between the peak serum anti-rhTNF activity and the colostral anti-rhTNF activity in these animals. The 4 cows with high colostral anti-rhTNF activities trended toward higher serum IgG1 loss relative to average colostral anti-rhTNF producers, but this difference was not statistically significant in this small sample. The high-anti-rhTNF-producing cows also exhibited a greater proportion of rhTNF-specific antibodies that bound to bovine IgG1- and IgG2-specific detection antibodies relative to the total anti-rhTNF immunoglobulin population. This finding suggests that the isotype distribution of the anti-rhTNF response is varied between individuals and genetic or environmental factors may increase the yield of antigen-specific colostral antibodies.
Assuntos
Colostro/imunologia , Imunoglobulina G/sangue , Fator de Necrose Tumoral alfa/imunologia , Animais , Bovinos , Colostro/química , Feminino , Humanos , Concentração de Íons de Hidrogênio , Imunização/veterinária , Imunoglobulina G/biossíntese , Modelos Lineares , Parto/imunologia , Gravidez , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
Several studies suggest that a dopamine transporter uptake inhibitor that has a slower onset and longer duration of action than cocaine in animal behavioral measures and decreases cocaine self-administration would be useful as an indirect dopamine agonist pharmacotherapy to treat cocaine addiction. In the present study, we compared five 3-phenyltropane analogs administered orally in locomotor activity in mice and drug discrimination in rats to gain information concerning relative potency, onset, and duration of action. The compounds were also evaluated for reduction of cocaine self-administration in rats after oral administration. In general, the compounds had a slower onset of action than cocaine and reduced cocaine self-administration. 3beta-(4-Chlorophenyl)-2beta-(3-(4'-methylphenyl)-isoxazol-5-yl)tropane (RTI-336) was the most potent in locomotor activity and drug discrimination; it was less stimulatory than cocaine in the first hour and had the slowest onset and longest duration of action. It also reduced self-administration of two infusion doses of cocaine in the rat.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/psicologia , Discriminação Psicológica/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Tropanos/farmacologia , Administração Oral , Animais , Condicionamento Operante/efeitos dos fármacos , Sinais (Psicologia) , Agonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Generalização Psicológica/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Masculino , Camundongos , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores de Droga/efeitos dos fármacos , Autoadministração , Relação Estrutura-AtividadeRESUMO
BACKGROUND AND AIMS: AVX-470 is an oral, polyclonal bovine-derived anti-tumour necrosis factor (TNF) antibody in development for treatment of inflammatory bowel disease (IBD). AVX-470 neutralizes TNF locally in the gastrointestinal tract, minimizing systemic exposure. This was a double-blind, placebo-controlled, first-in-human trial designed to assess the safety, pharmacokinetics, immunogenicity and preliminary efficacy of 4 weeks of AVX-470 in patients with active ulcerative colitis (UC). METHODS: Thirty-seven patients with active UC were randomized and 36 received AVX-470 (0.2, 1.6 or 3.5g/day) or placebo over 4 weeks. Endoscopic activity was assessed by colonoscopy pre- and post-treatment. The primary endpoint was safety. Secondary endpoints included pharmacokinetics and immunogenicity. Clinical and endoscopic response and remission were assessed as exploratory endpoints. RESULTS: Thirty-three (92%) patients completed treatment and follow-up. The incidence of adverse events was similar across treatment groups and no allergic reactions or opportunistic infections were reported. AVX-470 therapy did not induce human anti-bovine antibodies (HABA). Bovine immunoglobulin (Ig) with TNF binding capacity was detected in stool, while bovine Ig levels in serum were low. Across all AVX-470 doses, 25.9% of patients achieved clinical response compared with 11.1% on placebo, with greatest improvements in the 3.5g/day group associated with proximal colon endoscopic improvement and reductions in serum CRP and IL-6. CONCLUSIONS: AVX-470 was safe and well tolerated in this first-in-human trial in UC, with efficacy trends for clinical, endoscopic and biomarker endpoints in the highest dose group (3.5g/day). Results suggest benefit of an orally delivered locally active agent in moderate to severe UC. CLINICAL TRIAL REGISTRATION NUMBER: This trial was registered with Clinicaltrials.gov as study NCT01759056 and with EudraCT as study 2012-004859-27.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Anticorpos/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Administração Oral , Adolescente , Adulto , Idoso , Anti-Inflamatórios não Esteroides/imunologia , Anti-Inflamatórios não Esteroides/farmacocinética , Anticorpos/imunologia , Colite Ulcerativa/imunologia , Método Duplo-Cego , Esquema de Medicação , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND AND AIMS: AVX-470 is an orally administered, bovine-derived, anti-tumour necrosis factor (TNF) antibody with local activity in the gastrointestinal tract. In the first-in-human clinical trial of AVX-470 in active ulcerative colitis, we evaluated inflammatory biomarkers in colon tissue as measures of disease activity and early response to treatment. METHODS: Thirty-six patients received active drug (AVX-470 at 0.2, 1.6 or 3.5g/day) or placebo over 4 weeks. Colon biopsy samples were collected from 5 regions of colon at baseline and week 4. Tissue inflammatory biomarkers were evaluated by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), epithelial cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and bovine immunoglobulin by immunohistochemistry and mass spectrometry. Endoscopic activity (Ulcerative Colitis Endoscopic Index of Severity [UCEIS]) at colonoscopy was assessed in each colonic region by a central reader. RESULTS: Bovine immunoglobulin was observed in mucosal tissue before and after dosing in lamina propria and submucosal layers of biopsy tissue. Baseline levels of TNF, myeloperoxidase (MPO), CD68 and interleukin (IL)-1ß and, to a lesser extent, IL-6 mRNA were 2- to 3-fold higher in distal vs proximal colon tissue, corresponding to the 2- to 3-fold differences in baseline severities of endoscopic scores. Reductions of >10-fold in TNF and, to lesser extents, in MPO and epithelial cell apoptosis were observed in proximal and distal colon biopsies after 4 weeks of AVX-470 3.5g/day treatment. Reductions in TNF scores were correlated with changes in MPO and CD3 immunohistochemistry scores. CONCLUSIONS: These results are consistent with anti-TNF activity of orally administered AVX-470 in colon mucosal tissue in ulcerative colitis patients and demonstrate the utility of tissue biomarkers in assessing disease and treatment response in early clinical studies. CLINICAL TRIAL REGISTRATION NUMBER: This trial was registered with Clinicaltrials.gov as study NCT01759056 and with EudraCT as study 2012-004859-27.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Anticorpos/uso terapêutico , Biomarcadores/metabolismo , Colite Ulcerativa/tratamento farmacológico , Colo/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Adolescente , Adulto , Idoso , Colite Ulcerativa/diagnóstico por imagem , Colite Ulcerativa/metabolismo , Colonoscopia , Método Duplo-Cego , Esquema de Medicação , Monitoramento de Medicamentos , Feminino , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de Doença , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, which is currently treated with injected monoclonal antibodies specific for tumor necrosis factor (TNF). We developed and characterized AVX-470, a novel polyclonal antibody specific for human TNF. We evaluated the oral activity of AVX-470m, a surrogate antibody specific for murine TNF, in several well-accepted mouse models of IBD. METHODS: AVX-470 and AVX-470m were isolated from the colostrum of dairy cows that had been immunized with TNF. The potency, specificity, and affinity of both AVX-470 and AVX-470m were evaluated in vitro and compared with infliximab. AVX-470m was orally administered to mice either before or after induction of colitis, and activity was measured by endoscopy, histopathology, immunohistochemistry, and quantitative measurement of messenger RNA levels. Colitis was induced using either 2,4,6-trinitrobenzene sulfonate or dextran sodium sulfate. RESULTS: AVX-470 and AVX-470m were shown to be functionally comparable in vitro. Moreover, the specificity, neutralizing potency, and affinity of AVX-470 were comparable with infliximab. Orally administered AVX-470m effectively reduced disease severity in several mouse models of IBD. Activity was comparable with that of oral prednisolone or parenteral etanercept. The antibody penetrated the colonic mucosa and inhibited TNF-driven mucosal inflammation with minimal systemic exposure. CONCLUSIONS: AVX-470 is a novel polyclonal anti-TNF antibody with an in vitro activity profile comparable to that of infliximab. Oral administration of a surrogate antibody specific for mouse TNF is effective in treating mouse models of IBD, delivering the anti-TNF to the site of inflammation with minimal systemic exposure.
Assuntos
Anticorpos/administração & dosagem , Colite/tratamento farmacológico , Modelos Animais de Doenças , Imunoglobulina G/administração & dosagem , Inflamação/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anticorpos/farmacologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Bovinos , Colite/induzido quimicamente , Colite/imunologia , Sulfato de Dextrana/toxicidade , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/farmacologia , Inflamação/etiologia , Inflamação/patologia , Infliximab , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Trinitrobenzenossulfônico/toxicidade , Fator de Necrose Tumoral alfa/imunologiaRESUMO
RATIONALE: Despite increased education regarding its dangers, cigarette smoking remains a significant public health concern due to serious associated health consequences such as cancer and respiratory and cardiovascular diseases. Most smokers fail in their attempts to quit smoking, and current pharmacological interventions have relatively low levels of efficacy and are associated with significant adverse events. We have previously reported that combinations of metyrapone and oxazepam, administered at doses that were ineffective when delivered singly, resulted in dose-related decreases in cocaine self-administration in rats while not affecting food-maintained responding during the same sessions. OBJECTIVES: The current study was designed to test the effects of the administration of a metyrapone:oxazepam combination on nicotine self-administration in rats. METHODS: Several dose combinations of metyrapone (12.5, 25 or 50 mg/kg) and oxazepam (5 or 10 mg/kg) were tested in rats trained to intravenously (IV) self-administer nicotine (0.03 mg/kg/infusion) during 1-h self-administration sessions using both fixed-ratio and progressive-ratio (PR) schedules of reinforcement. RESULTS: The administration of low doses of metyrapone and oxazepam in combination significantly decreased IV nicotine self-administration in rats. At the lowest doses of 12.5 mg/kg of metyrapone and 5 mg/kg of oxazepam, the drugs alone did not decrease IV nicotine self-administration, but the combination was effective. Varenicline was also tested using the fixed-ratio schedule, and reductions in nicotine intake were similar to those seen with the moderate dose of the combination. CONCLUSIONS: The results of this study suggest a potential utility of the combination of metyrapone and oxazepam for smoking cessation in humans.
Assuntos
Metirapona/farmacologia , Nicotina/administração & dosagem , Oxazepam/farmacologia , Abandono do Hábito de Fumar/métodos , Animais , Benzazepinas/farmacologia , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/farmacologia , Infusões Intravenosas , Masculino , Metirapona/administração & dosagem , Oxazepam/administração & dosagem , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Esquema de Reforço , Autoadministração , VareniclinaRESUMO
Although cocaine dependence affects an estimated 1.6 million people in the USA, there are currently no medications approved for the treatment of this disorder. Experiments performed in animal models have demonstrated that inhibitors of the stress response effectively reduce intravenous cocaine self-administration. This exploratory, double-blind, placebo-controlled study was designed to assess the safety and efficacy of combinations of the cortisol synthesis inhibitor metyrapone, and the benzodiazepine oxazepam, in 45 cocaine-dependent individuals. The subjects were randomized to a total daily dose of 500 mg metyrapone/20 mg oxazepam (low dose), a total daily dose of 1500 mg metyrapone/20 mg oxazepam (high dose), or placebo for 6 weeks of treatment. The outcome measures were a reduction in cocaine craving and associated cocaine use as determined by quantitative measurements of the cocaine metabolite benzoylecgonine (BE) in urine at all visits. Of the randomized subjects, 49% completed the study. The combination of metyrapone and oxazepam was well tolerated and tended to reduce cocaine craving and cocaine use, with significant reductions at several time points when controlling for baseline scores. These data suggest that further assessments of the ability of the metyrapone and oxazepam combination to support cocaine abstinence in cocaine-dependent subjects are warranted.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Agonistas GABAérgicos/uso terapêutico , Metirapona/uso terapêutico , Oxazepam/uso terapêutico , Esteroide 11-beta-Hidroxilase/antagonistas & inibidores , Síndrome de Abstinência a Substâncias/prevenção & controle , Adulto , Cocaína/análogos & derivados , Cocaína/urina , Transtornos Relacionados ao Uso de Cocaína/prevenção & controle , Transtornos Relacionados ao Uso de Cocaína/urina , Manual Diagnóstico e Estatístico de Transtornos Mentais , Relação Dose-Resposta a Droga , Método Duplo-Cego , Quimioterapia Combinada/efeitos adversos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Feminino , Agonistas GABAérgicos/administração & dosagem , Agonistas GABAérgicos/efeitos adversos , Humanos , Louisiana , Masculino , Metirapona/administração & dosagem , Metirapona/efeitos adversos , Pessoa de Meia-Idade , Oxazepam/administração & dosagem , Oxazepam/efeitos adversos , Cooperação do Paciente , Pacientes Desistentes do Tratamento , Projetos Piloto , Prevenção Secundária , Esteroide 11-beta-Hidroxilase/administração & dosagem , Esteroide 11-beta-Hidroxilase/efeitos adversosRESUMO
The discovery and preclinical development of selective dopamine reuptake inhibitors as potential pharmacotherapies for treating cocaine addiction are presented. The studies are based on the hypothesis that a dopamine reuptake inhibitor is expected to partially substitute for cocaine, thus decreasing cocaine self-administration and minimizing the craving for cocaine. This type of indirect agonist therapy has been highly effective for treating smoking addiction (nicotine replacement therapy) and heroin addiction (methadone). To be an effective pharmacotherapy for cocaine addiction, the potential drug must be safe, long-acting, and have minimal abuse potential. We have developed several 3-phenyltropane analogs that are potent dopamine uptake inhibitors, and some are selective for the dopamine transporter relative to the serotonin and norepinephrine transporters. In animal studies, these compounds substitute for cocaine, reduce the intake of cocaine in rats and rhesus monkeys trained to self-administer cocaine, and have demonstrated a slow onset and long duration of action and lack of sensitization. The 3-phenyltropane analogs were also tested in a rhesus monkey self-administration model to define their abuse potential relative to cocaine. Based on these studies, 3beta-(4-chlorophenyl)-2beta-[3-(4'-methylphenyl)isoxazol-5-yl]tropane (RTI-336) has been selected for preclinical development.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Tropanos/uso terapêutico , Animais , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Relação Dose-Resposta a Droga , Humanos , Tecnologia Farmacêutica/tendências , Tropanos/farmacologiaRESUMO
The Straw Nicotine Oral Delivery System is a novel form of nicotine replacement therapy designed to provide not only nicotine but also oral and manual stimulation and ease of dosing. The Straw is a single-use drinking straw containing loose nicotine bitartrate particles that are ingested with the first sip of a beverage. This article describes single- and repeated-dose pharmacokinetics and tolerability of The Straw in a group of 24 smokers. The subjects were randomized to receive 12, 8, or 4 mg nicotine or placebo via The Straw following overnight abstinence from smoking. On one occasion, subjects took only one dose of nicotine; a week later they took eight doses, one every 1.5 hr. The orally ingested nicotine was well tolerated with single and repeated dosing. Plasma levels of nicotine peaked between 1 and 2 hr after a single dose at levels ranging from 6 ng/ml (4-mg dose) to 20 ng/ml (8- and 12-mg doses). Nicotine levels were not different between the 8-mg and 12-mg doses, possibly related to individual variability. Dosing repeatedly with The Straw led to greater nicotine levels that continued to increase with additional dosing. Levels achieved were comparable with or greater than those of other replacement products.