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1.
Artigo em Inglês | MEDLINE | ID: mdl-33361306

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause developmental disorders following congenital infection and life-threatening complications among transplant patients. Potent neutralizing monoclonal antibodies (MAbs) are promising drug candidates against HCMV infection. HCMV can infect a broad range of cell types. Therefore, single neutralizing antibodies targeting one HCMV glycoprotein often lack either potency or broad cell-type coverage. We previously characterized two human-derived HCMV neutralizing MAbs. One was the broadly neutralizing MAb 3-25, which targets the antigenic domain 2 of glycoprotein B (gB). The other was the highly potent MAb 2-18, which specifically recognizes the gH/gL/pUL128/130/131 complex (pentamer). To combine the strengths of gB- and pentamer-targeting MAbs, we developed an IgG-single-chain variable fragment (scFv) bispecific antibody by fusing the 2-18 scFv to the heavy-chain C terminus of MAb 3-25. The resulting bispecific antibody showed high-affinity binding to both gB and pentamer. Functionally, the bispecific antibody demonstrated a combined neutralization breadth and potency of the parental MAbs in multiple cell lines and inhibited postinfection viral spreading. Furthermore, the bispecific antibody was easily produced in CHO cells at a yield above 1 g/liter and showed a single-dose pharmacokinetic profile comparable to that of parental MAb 3-25 in rhesus macaques. Importantly, the bispecific antibody retained broadly and potent neutralizing activity after 21 days in circulation. Taken together, our research provides a proof-of-concept study for developing bispecific neutralizing antibody therapies against HCMV infection.


Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Cricetinae , Cricetulus , Glicoproteínas , Humanos , Macaca mulatta , Proteínas do Envelope Viral
2.
Nature ; 521(7551): 222-6, 2015 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-25754330

RESUMO

Psoriasis is a chronic inflammatory skin disorder that affects approximately 2-3% of the population worldwide and has severe effects on patients' physical and psychological well-being. The discovery that psoriasis is an immune-mediated disease has led to more targeted, effective therapies; recent advances have focused on the interleukin (IL)-12/23p40 subunit shared by IL-12 and IL-23. Evidence suggests that specific inhibition of IL-23 would result in improvement in psoriasis. Here we evaluate tildrakizumab, a monoclonal antibody that targets the IL-23p19 subunit, in a three-part, randomized, placebo-controlled, sequential, rising multiple-dose phase I study in patients with moderate-to-severe psoriasis to provide clinical proof that specific targeting of IL-23p19 results in symptomatic improvement of disease severity in human subjects. A 75% reduction in the psoriasis area and severity index (PASI) score (PASI75) was achieved by all subjects in parts 1 and 3 (pooled) in the 3 and 10 mg kg(-1) groups by day 196. In part 2, 10 out of 15 subjects in the 3 mg kg(-1) group and 13 out of 14 subjects in the 10 mg kg(-1) group achieved a PASI75 by day 112. Tildrakizumab demonstrated important clinical improvement in moderate-to-severe psoriasis patients as demonstrated by improvements in PASI scores and histological samples.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoterapia , Interleucina-23/antagonistas & inibidores , Terapia de Alvo Molecular , Psoríase/tratamento farmacológico , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Método Duplo-Cego , Epitélio/efeitos dos fármacos , Epitélio/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-23/química , Interleucina-23/imunologia , Pessoa de Meia-Idade , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Psoríase/imunologia , Psoríase/metabolismo , Psoríase/patologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Pele/patologia , Resultado do Tratamento , Adulto Jovem
3.
Dev Psychobiol ; 63(6): e22181, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34423434

RESUMO

Children reared in socioeconomically disadvantaged environments are at risk for academic, cognitive, and behavioral problems. Mounting evidence suggests that childhood adversities, encountered at disproportionate rates in contexts of socioeconomic risk, shape the developing brain in ways that explain disparities. Circuitries that subserve neurocognitive functions related to memory, attention, and cognitive control are especially affected. However, most work showing altered neural function has focused on middle childhood and adolescence. Understanding alterations in brain development during foundational points in early childhood is a key next step. To address this gap, we examined functional near-infrared-spectroscopy-based neural activation during a working memory (WM) task in young children aged 4-7 years (N = 30) who varied in socioeconomic risk exposure. Children who experienced greater disadvantage (lower income to needs ratio and lower Hollingshead index) exhibited lower activation in the lateral prefrontal cortex than children who experienced less to no disadvantage. Variability in prefrontal cortex activation, but not behavioral performance on the WM task, was associated with worse executive functioning in children as reported by parents. These findings add to existing evidence that exposure to early adversity, such as socioeconomic risk, may lead to foundational changes in the developing brain, which increases risk for disparities in functioning across multiple cognitive and social domains.


Assuntos
Memória de Curto Prazo , Córtex Pré-Frontal , Adolescente , Atenção/fisiologia , Criança , Pré-Escolar , Função Executiva/fisiologia , Humanos , Memória de Curto Prazo/fisiologia , Córtex Pré-Frontal/fisiologia , Fatores Socioeconômicos
4.
Regul Toxicol Pharmacol ; 108: 104476, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31536773

RESUMO

Tildrakizumab (also known as MK-3222), is a high-affinity, humanized, immunoglobin G1κ monoclonal antibody targeting the p19 subunit of interleukin-23 recently approved for the treatment of moderate to severe plaque psoriasis in the US, Europe, and Australia. The safety profile of tildrakizumab was characterized in nonclinical studies using a pharmacologically relevant cynomolgus monkey model. In repeat-dose toxicity studies, cynomolgus monkeys were chronically treated with subcutaneous (SC) injections of 100 mg/kg of tildrakizumab every 2 weeks up to 9 months. Tildrakizumab was well tolerated, with no toxicological findings (including assessment of reproductive organs; hormonal effects; and cardiovascular, respiratory, and central nervous system function) at systemic exposures approximately 90 times higher than the recommended human dose of 100 mg. An embryofetal developmental study conducted in pregnant monkeys revealed no treatment-related effects to the developing fetus following SC administration of tildrakizumab 100 mg/kg. In a pre- and postnatal development study, 2 neonatal deaths due to potential viral infection at 100 mg/kg were considered of uncertain relationship to the treatment based on a lack of historical data on the occurrence of viral infection in neonate cynomolgus monkeys. The results of this comprehensive nonclinical safety program support the safe use of tildrakizumab.


Assuntos
Anticorpos Monoclonais Humanizados/toxicidade , Animais , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Monoclonais Humanizados/farmacocinética , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Subunidade p19 da Interleucina-23/sangue , Subunidade p19 da Interleucina-23/imunologia , Macaca fascicularis , Masculino , Troca Materno-Fetal , Leite/química , Gravidez , Psoríase/tratamento farmacológico , Testes de Toxicidade Crônica
5.
Br J Clin Pharmacol ; 84(10): 2292-2302, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29926968

RESUMO

AIMS: Tildrakizumab, an interleukin (IL)-23 inhibitor, is indicated for the treatment of moderate to severe chronic plaque psoriasis. Although tildrakizumab is not metabolized by, and does not alter, cytochrome P450 (CYP) expression in vitro, clinically significant pharmacokinetic effects through changes in systemic inflammation, which alters CYP metabolism, have been well documented. At the time of study conduct, the effect of modulation of inflammation/cytokines, including IL-23 inhibition with tildrakizumab, on CYP metabolism, and therefore the potential for disease-drug interactions, in psoriasis patients was unknown. We therefore assessed whether tildrakizumab alters CYP metabolism in subjects with moderate to severe psoriasis. METHODS: This was an open-label, fixed-sequence, two-period trial. In Period 1 (Day 1), subjects received an oral CYP probe cocktail of up to five drugs (midazolam 2 mg [3A4], caffeine 200 mg [1A2], warfarin 10 mg [2C9], omeprazole 40 mg [2C19] and dextromethorphan 30 mg [2D6]), followed by a 7-day washout. In Period 2, subjects received tildrakizumab 200 mg subcutaneously on Days 1 and 29 and a second CYP probe cocktail on Day 57. Substrate or metabolite pharmacokinetics, safety and changes in Psoriasis Severity Area Index (PASI), interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hs-CRP), were assessed. RESULTS: Twenty subjects (13 men, 7 women) were enrolled. Tildrakizumab had no clinically relevant effect on the pharmacokinetics of any of the probe substrates tested. On Day 57 of Period 2, the median percentage decrease from baseline in PASI score following tildrakizumab was ~93%. There were no clinically relevant changes in IL-6 or hs-CRP. Treatment with tildrakizumab was generally well tolerated. CONCLUSION: In subjects with moderate to severe psoriasis, tildrakizumab 200 mg did not have a discernible effect on CYP metabolism. The potential for clinically significant drug-drug interactions (DDIs) with tildrakizumab in patients with psoriasis is low. The difference in the occurrence of DDIs seen with anti-inflammatory agents in rheumatoid arthritis patients compared with psoriasis patients may be due to the much greater extent of systemic inflammation in rheumatoid arthritis as compared to psoriasis.


Assuntos
Anticorpos Monoclonais/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Subunidade p19 da Interleucina-23/antagonistas & inibidores , Psoríase/tratamento farmacológico , Administração Oral , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Cafeína/administração & dosagem , Cafeína/farmacocinética , Dextrometorfano/administração & dosagem , Dextrometorfano/farmacocinética , Interações Medicamentosas , Feminino , Humanos , Injeções Subcutâneas , Subunidade p19 da Interleucina-23/imunologia , Masculino , Midazolam/administração & dosagem , Midazolam/farmacocinética , Pessoa de Meia-Idade , Omeprazol/administração & dosagem , Omeprazol/farmacocinética , Psoríase/diagnóstico , Psoríase/imunologia , Psoríase/metabolismo , Índice de Gravidade de Doença , Resultado do Tratamento , Varfarina/administração & dosagem , Varfarina/farmacocinética , Adulto Jovem
6.
Int J Clin Pharmacol Ther ; 53(2): 139-46, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25546162

RESUMO

OBJECTIVE: To evaluate the effect of ethnicity on the pharmacokinetics (PK) of tildrakizumab, a novel anti-IL-23 monoclonal antibody for the treatment of psoriasis. MATERIALS AND METHODS: This was an open-label, 2-part study in healthy adult subjects. In part 1, Japanese subjects and matched Caucasian and Chinese subjects (to Japanese) were assigned to 1 of 3 cohorts and administered tildrakizumab 50, 200, or 400 mg subcutaneously (SC). In part 2, Japanese subjects received tildrakizumab 10 mg/kg IV. Pre- and post-treatment antidrug antibodies were assessed. Safety and tolerability were assessed throughout the study. RESULTS: 59 subjects were enrolled; 53 in part 1 and 6 in part 2. Overall geometric mean AUC∞ was 6.15, 6.05, and 6.32 day×µg/mL/mg in Japanese, Caucasian, and Chinese subjects, respectively, after administration of a single SC dose. Bioavailability was ~92%. Six out of 58 evaluable subjects were positive for post-treatment ADA; 2 of these positive subjects had reduced tildrakizumab exposure. Most AEs were mild in intensity and the most frequent treatment-related AEs were injection site hematoma (15%), injection site pain (10%), and injection site erythema (8%). CONCLUSIONS: The pharmacokinetics of tildrakizumab were similar in Japanese, Caucasian, and Chinese subjects. Tildrakizumab exposure increased proportionally with dose in the range of 50-400 mg. A single SC dose of 50, 200, and 400 mg or a single IV dose of 10 mg/kg was generally well tolerated.


Assuntos
Anticorpos Monoclonais/farmacocinética , Interleucina-23/imunologia , Adulto , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Povo Asiático , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , População Branca , Adulto Jovem
7.
J Pharm Sci ; 111(4): 960-969, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35122828

RESUMO

Immunogenicity to biologics is often observed following dosing in human subjects during clinical trials. Both product and host specific factors may be implicated in contributing to a potential immune response. However, even if such risk factors are identified and eliminated as part of the rational quality by design approaches, the outcome in clinic can be uncertain and challenging to predict. Several tools have been employed to identify these risk factors and consequent mitigation approaches implemented prior to dosing in humans. However, the complexity of the immune system with an interplay of network of immune cells involved in driving a long- term immune response as well as patient characteristics, can make it challenging to predict the outcome in clinic. This perspective will provide an insight into recent advances in the risk assessment approaches that are utilized during preclinical stage of development of a biologic. The outputs from such tools can help to rank order and select the most optimal candidate with the least likelihood of an immune response and can further drive the development of a clinical bioanalytical and immunogenicity monitoring strategy. Such a strategy can be proactively shared with the regulators along with the proposal to streamline clinical immunogenicity and personalizing the outcome based on pharmacogenomics and other patient-related factors. This paper provides a roadmap on performing risk assessments through a systematic identification of risks and their mitigations wherever possible. Recommendations on incorporating the key components of such risk assessments as part of the new regulatory submissions are also provided. Shorter abstract Immunogenicity to biologics is common during clinical trials. Both product and host specific factors have been implicated. Several risk assessment tools can be used to identify and mitigate the risk factors responsible for immunogenicity. An insight into recent advances in the risk assessment approaches will be presented. The outputs can define a risk score and guide the clinical bioanalytical and immunogenicity monitoring strategy. A roadmap on performing risk assessments through a systematic identification of risks and their mitigations wherever possible is provided. Best practices for a risk assessment strategy and recommendations on the content for IND and the Integrated summary of Immunogenicity are also provided.


Assuntos
Produtos Biológicos , Humanos , Medição de Risco , Fatores de Risco
8.
Clin Pharmacol Drug Dev ; 10(5): 556-566, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33125189

RESUMO

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection and related morbidity and mortality in infants. Passive immunization with an RSV-neutralizing antibody can provide rapid protection to this vulnerable population. Proof-of-concept for this approach has been demonstrated by palivizumab; however, the use of this antibody is generally restricted to the highest-risk infants due to monthly dosing requirements and its cost. To address the large unmet medical need for most infants, we are evaluating MK-1654, a fully human RSV-neutralizing antibody with half-life extending mutations targeting site IV of the fusion protein. In this 2-part, placebo-controlled, double-blind, first-in-human study, 152 healthy adults were randomized 3:1 to receive a single dose of MK-1654 or placebo in 5 cohorts (100 or 300 mg as an intramuscular dose or 300, 1000, or 3000 mg as an intravenous dose). Safety, pharmacokinetics, antidrug antibodies, and RSV serum-neutralizing antibody titers were evaluated through 1 year. MK-1654 serum concentrations increased proportionally with dose and resulted in corresponding elevations in RSV serum-neutralizing antibody titers. The antibody displayed a half-life of 73 to 88 days and an estimated bioavailability of 69% at the 300-mg dose. The overall safety profile of MK-1654 was similar to placebo, and treatment-emergent antidrug antibodies were low (2.6%) with no associated adverse events. These data support the continued development of MK-1654 for the prevention of RSV disease in infants.


Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Antivirais , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/efeitos adversos , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Antivirais/farmacocinética , Disponibilidade Biológica , Estudos de Coortes , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Meia-Vida , Humanos , Infusões Intravenosas , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sinciciais Respiratórios/imunologia , Adulto Jovem
9.
AAPS J ; 22(3): 64, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32246297

RESUMO

This document highlights some relevant factors in the assessment of immunogenicity risk of fusion protein therapeutics. Our aim is to highlight specific risks associated with this type of molecule, while also aligning with general risk assessment factors, through a hypothetical case study, where the therapeutic molecule of interest is a Receptor-Fc Fusion protein (RFF) expressed within a typical manufacturing process in Chinese Hamster Ovary Cells (CHO). Given that the components are comprised of endogenous sequences, the risk of developing an ADA response to this molecule is generally considered to be low. However, the consequences of such an immune response may be more severe, specifically, if there is cross reactivity with the endogenous receptor, inducing cell lysis, or if any ADA act as an agonist to trigger receptor signaling. The risk factors described below are not meant to provide a comprehensive list, but rather a framework for factors that should be considered. Immunogenicity risk is difficult to quantify and relies on a comprehensive analysis of both product and patient-related factors. The goal is not necessarily to quantify risk, but rather to demonstrate an understanding of factors that may pose a risk, implement a strategy to minimize risk factors and then align overall risk with a bioanalytical immunogenicity monitoring strategy. The consequences resulting from unexpected outcome will likely depend on severity and impact on patient safety. An immunogenicity risk assessment is an ongoing and continuous process throughout clinical development with the goal of maximizing the safety of patients.


Assuntos
Fenômenos Imunogenéticos , Proteínas Recombinantes de Fusão/imunologia , Animais , Células CHO , Estudos Clínicos como Assunto , Cricetulus , Humanos , Receptores Fc , Medição de Risco
10.
AAPS J ; 22(3): 60, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32185565

RESUMO

This article provides a theoretical case-study risk assessment report for a low-risk monoclonal antibody (mAb) therapeutic. In terms of risk, there are considerations around risks to safety, but also risks regarding effects on pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. Much of the discussion in this document is around the risk of immunogenicity incidence. A higher incidence of immunogenicity would necessitate a detailed review of the PK, efficacy and safety in anti-drug antibody (ADA) positive and ADA negative subjects, in order to evaluate potential effects. The publication is intended to provide a framework of some the current thought processes around assessing immunogenicity risk and for building strategies to mitigate those risks. For this example, we have created a hypothetical antibody, ABC-123, targeting a membrane protein on antigen presenting cells, for the treatment of rheumatoid arthritis (RA). This hypothetical antibody therapeutic is provided as an example for the purposes of risk assessment for a low risk molecule, although any application of similar approach would be case by case.


Assuntos
Anticorpos Monoclonais/imunologia , Fenômenos Imunogenéticos , Animais , Anticorpos Monoclonais/uso terapêutico , Humanos , Medição de Risco
11.
Clin Pharmacol Drug Dev ; 9(3): 330-340, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31411386

RESUMO

Bezlotoxumab is a fully human monoclonal antibody that binds and neutralizes Clostridium difficile toxin B. This analysis investigated the potential of bezlotoxumab to induce immunogenicity in healthy phase 1 trial participants and in phase 2/3 trial participants receiving oral antibacterial therapy for primary or recurrent C difficile infection. Immunogenicity to bezlotoxumab was evaluated following a single intravenous dose (≤20 mg/kg) or 2 consecutive doses (10 mg/kg) given 84 days apart in healthy participants across 3 phase 1 trials (Protocol MK-3415A-004, N = 30; Protocol CA-GCDX-05-01, N = 54; Protocol MK-3415A-006, N = 12) and following a single 10 mg/kg dose in 1 phase 2 trial (Protocol CA-GCDX-06-02, ClinicalTrials.gov identifier: NCT00350298; N = 97) and 2 phase 3 trials (Protocols MK-3415A-001 and MK-3415A-002, ClinicalTrials.gov identifiers: NCT01241552 and NCT01513239; N = 1414). No treatment-emergent antidrug antibodies were observed following single or repeated dosing of bezlotoxumab. No phase 1 participants and only 1 phase 2 participant tested positive before bezlotoxumab exposure (non-treatment-emergent positive). Nine participants tested non-treatment-emergent positive in phase 3 trials, 1 of whom was neutralizing antibody-positive. Overall, the immunogenicity potential of bezlotoxumab is considered to be low.


Assuntos
Antibacterianos/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Infecções por Clostridium/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Anticorpos Amplamente Neutralizantes/administração & dosagem , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Infecções por Clostridium/tratamento farmacológico , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto
12.
Basic Clin Pharmacol Toxicol ; 123(3): 294-300, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29510001

RESUMO

Tildrakizumab, a high-affinity humanized IgG1k antibody that selectively binds interleukin (IL)-23 p19 subunit of cytokine IL-23 and neutralizes its function, is under investigation for treatment of moderate-to-severe chronic plaque psoriasis. The objective of this analysis was to assess the pharmacokinetics, bioavailability and safety/tolerability of single ascending doses of tildrakizumab after intravenous (IV) and subcutaneous (SC) dosing in healthy subjects. P05661 was a phase 1, single-dose, randomized, placebo-controlled study of tildrakizumab IV doses of 0.1, 0.5, 3 and 10 mg/kg, or placebo. P05776 was a phase 1, single-dose, randomized, placebo-controlled study of tildrakizumab SC doses of 50 or 200 mg, or placebo. After either single IV or SC dosing, tildrakizumab exhibited slow systemic clearance (CL), limited volume of distribution and a long t1/2 . Both the Cmax and the area under the curve (AUC) increased proportionally with doses from 0.1 to 10 mg/kg, or 50-200 mg. The bioavailability of SC dosing was ~80% (90% CI: 62-103%) for 50 mg and ~73% (90% CI: 46-115%) for 200 mg, respectively, versus 0.5 and 3 mg/kg IV. Across both studies, six of 43 evaluable subjects were positive for post-dose antidrug antibodies; two of these were positive for neutralizing antibodies. Most adverse events (AEs) were mild; the most frequent AEs included upper respiratory tract infection and headache. Single doses of tildrakizumab 0.1, 0.5, 3 and 10 mg/kg administered IV or single doses of 50 and 200 mg administered SC were safe and well tolerated in healthy adult subjects.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/imunologia , Fármacos Dermatológicos/administração & dosagem , Interleucina-23/imunologia , Adolescente , Adulto , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Área Sob a Curva , Disponibilidade Biológica , Fármacos Dermatológicos/efeitos adversos , Fármacos Dermatológicos/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Meia-Vida , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Masculino , Pessoa de Meia-Idade , Distribuição Tecidual , Adulto Jovem
13.
Mol Biol Cell ; 14(8): 3437-48, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12925775

RESUMO

ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD. Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al. J. Biol. Chem. 274, 3453-3460). The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate. Peptide-stimulated ATPase activity was also reduced, suggesting that the interdomain coupling in Kar2-1p is partially compromised. In contrast, Hsp40 cochaperone-activation of Kar2-1p's ATPase activity was unaffected. Consistent with UPR induction in kar2-1 yeast, an ERAD substrate aggregated in microsomes prepared from this strain but not from wild-type yeast. Overexpression of wild-type BiP increased substrate solubility in microsomes obtained from the mutant, but the ERAD defect was exacerbated, suggesting that simply retaining ERAD substrates in a soluble, retro-translocation-competent conformation is insufficient to support polypeptide transit to the cytoplasm.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Leveduras/metabolismo , Animais , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP70/genética , Mutagênese/genética , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes , Leveduras/genética
14.
J Pharmacol Toxicol Methods ; 53(3): 215-8, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16181794

RESUMO

INTRODUCTION: Previously, we have shown that a novel Caco-2/human hepatocyte system is a useful model for the prediction of oral bioavailability in humans. In this study, we attempted to use a similar system in a high-throughput screening mode for the selection of new compound entities (NCE) in drug discovery. METHODS: A total of 72 compounds randomly selected from three different chemotypes were dosed orally in rats. In vivo plasma area under the concentration versus time curve (AUC) from 0-6 h of the parent compound was determined. The same compounds were also tested in the Caco-2/rat hepatocyte system. In vitro AUC from 0-3 h in the Caco-2 rat hepatocyte system was determined. RESULTS: The predictive usefulness of the Caco-2/rat hepatocyte system was evaluated by comparing the in vivo plasma AUC and the in vitro AUC. Linear regression analysis showed a reasonable correlation (R2 = 0.5) between the in vivo AUC and the in vitro AUC. Using 0.4 microM h in vivo AUC as a cut-off, compounds were categorized as either low or high AUC. The in vitro AUC successfully matched the corresponding in vivo category for sixty-three out of seventy-two compounds. DISCUSSION: The results presented in this study suggest that the Caco-2/rat hepatocyte system may be used as a high-throughput screen in drug discovery for pharmacokinetic behaviors of compounds in rats.


Assuntos
Hepatócitos/efeitos dos fármacos , Células Híbridas/efeitos dos fármacos , Xenobióticos/sangue , Xenobióticos/farmacocinética , Administração Oral , Animais , Área Sob a Curva , Bioensaio , Células CACO-2 , Técnicas de Cultura de Células , Células Cultivadas , Hepatócitos/metabolismo , Humanos , Células Híbridas/metabolismo , Cinética , Modelos Lineares , Valor Preditivo dos Testes , Ratos , Ratos Sprague-Dawley , Xenobióticos/administração & dosagem , Xenobióticos/química
15.
Comb Chem High Throughput Screen ; 8(4): 341-6, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16101010

RESUMO

Recent studies have suggested that both constitutive androstane receptor (CAR) and pregnane X-receptor (PXR) are involved in the induction of rat liver microsomal cytochrome P-450 (CYP) 2B and 3A through a mechanism called cross-talk. In this study we intend to determine if a PXR-reporter gene assay could be used for the prediction of CYP3A and/or CYP2B induction in rats. The induction of rat CYP2B and CYP3A by nineteen structurally diverse compounds was evaluated by using rat precision-cut liver slices and a rat PXR reporter-gene system. Induction of CYP2B and CYP3A mRNAs in rat liver slices was quantified by real-time polymerase chain reaction. Rat PXR activation was measured by induction of luciferase activity in rat PXR reporter-gene system. Linear regression analysis of the fold of induction of mRNA in liver slices and the fold of luciferase activity in rat PXR reporter-gene system shows that a reasonable correlation (r2 = 0.6) exists between the CYP3A induction and the rat PXR activation. A much lower correlation was observed between CYP2B induction and the rat PXR activation (r2 = 0.1). The results from this study suggest that the PXR may play a major role in the induction of rat CYP3A, but not CYP2B. Therefore, the PXR-reporter gene assay may be useful in a high-throughput screening to predict CYP3A induction in rats.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Bioensaio , Fígado/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Animais , Hidrocarboneto de Aril Hidroxilases/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases/genética , Citocromo P-450 CYP2B1/efeitos dos fármacos , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP3A , Avaliação Pré-Clínica de Medicamentos/métodos , Ativação Enzimática/efeitos dos fármacos , Genes Reporter , Técnicas In Vitro , Fígado/efeitos dos fármacos , Luciferases/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Masculino , Oxirredutases N-Desmetilantes/efeitos dos fármacos , Oxirredutases N-Desmetilantes/genética , Receptor de Pregnano X , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/efeitos dos fármacos , Receptores de Esteroides/metabolismo
16.
J Pharmacol Toxicol Methods ; 52(2): 234-43, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16125621

RESUMO

INTRODUCTION: In drug development, new chemical entities that cause cytochrome P450 induction are considered to be undesirable since P450 induction is linked to tumor formation and may compromise the evaluation of drug safety when autoinduction results in poor drug exposure. METHODS: We evaluated the use of the precision-cut liver slice as a model for measuring induction of cytochrome P450 in rats. Quantitative real-time reverse-transcription polymerase chain reaction was used to analyze the induction of selected forms of cytochrome P450 at the mRNA level. Firstly, the system was validated against known inducers of CYP2B and 3A. Subsequently, 26 proprietary compounds were tested in rat liver slices and rats in vivo for CYP2B and 3A induction. RESULTS: Exposure of liver slices to the known CYP2B inducers phenobarbital, benzoyl-pyridine, cabarmazepine, metyrapone, RU486 and dexamethasone caused elevation of CYP2B1/2 expression 10- to 40-fold compared to the control values. The CYP3A inducers PCN, dexamethasone, nicardipine, nifedipine, clotrimazole and RU486 induced a 4- to 50-fold expression of CYP3A14. For 26 proprietary compounds, a correlation with an R(2) value of 0.74 was established between the induction of CYP2B expression in liver slices and that in rats in vivo. When liver slice results were used to predict the induction of CYP2B in rats in vivo, the success rate was 91%. The induction of CYP3A in rats in vivo was analyzed by Western blot, then quantified by densitometry. There was a good correlation between CYP3A induction in liver slices and induction in vivo as assessed by Western blot, with an 86% positive prediction rate. DISCUSSION: The use of liver slices in combination with TaqMan technology provides a good model for predicting CYP induction in the rat. This method is useful for identifying compounds with CYP2B and 3A induction liability in the early phase of drug discovery.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Algoritmos , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Carbamazepina/farmacologia , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP3A , Primers do DNA/genética , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Metirapona/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Mifepristona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo
17.
Biochem Pharmacol ; 78(4): 406-13, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19406108

RESUMO

Cytochromes P-450 2C11 and 2C13 are the major CYPs in rat liver microsomes. Despite a high degree of sequence identity, these two isozymes display different positional and regio-specific metabolism of steroid hormones, such as testosterone. CYP2C11 converts testosterone to 2alpha-hydroxyl and 16alpha-hydroxyl metabolites, while CYP2C13 produces primarily the 6beta-hydroxyl metabolite. Using a human CYP2C9 crystal structure as the template, homology models were generated for CYP2C11 and CYP2C13. Despite similar volume of the binding pockets for CYP2C11 and CYP2C13, the models for these two CYPs showed a substantial difference in the shape of the substrate-binding sites. Substrate docking using rigid and induced-fit methods showed that testosterone fits into the substrate-binding sites of both CYP2C11 and CYP2C13 without the need of added constraints. These docking exercises appear to support testosterone binding in both CYP2C11 and CYP2C13. A constrained docking using energy minimization is required to position testosterone for more precise positional and regio-specificity in supporting the observed metabolism. These results demonstrate the complexity of using modeling for understanding the binding of substrate to CYPs, and suggest that, as a complement to the metabolism data, modeling and docking may yield reliable structural information for the molecular interaction between the substrate and the CYPs.


Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Sítios de Ligação , Esteroide 16-alfa-Hidroxilase/química , Testosterona/química , Animais , Fenômenos Biofísicos , Domínio Catalítico/fisiologia , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A/química , Sistema Enzimático do Citocromo P-450 , Família 2 do Citocromo P450 , Humanos , Cinética , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Ratos , Especificidade por Substrato
18.
Mol Cell ; 26(1): 27-39, 2007 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-17434124

RESUMO

Hsp70 chaperones assist in protein folding, disaggregation, and membrane translocation by binding to substrate proteins with an ATP-regulated affinity that relies on allosteric coupling between ATP-binding and substrate-binding domains. We have studied single- and two-domain versions of the E. coli Hsp70, DnaK, to explore the mechanism of interdomain communication. We show that the interdomain linker controls ATPase activity by binding to a hydrophobic cleft between subdomains IA and IIA. Furthermore, the domains of DnaK dock only when ATP binds and behave independently when ADP is bound. Major conformational changes in both domains accompany ATP-induced docking: of particular importance, some regions of the substrate-binding domain are stabilized, while those near the substrate-binding site become destabilized. Thus, the energy of ATP binding is used to form a stable interface between the nucleotide- and substrate-binding domains, which results in destabilization of regions of the latter domain and consequent weaker substrate binding.


Assuntos
Regulação Alostérica , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Ligantes , Difosfato de Adenosina/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Medição da Troca de Deutério , Proteínas de Escherichia coli/química , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Modelos Químicos , Nucleotídeos/química , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
19.
Pharm Res ; 20(9): 1373-80, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14567630

RESUMO

PURPOSE: A conventional approach to assess cytochrome P450 (CYP) induction in preclinical animal models involves daily dosing for a least a week followed by Western blot and/or enzyme activity analysis. To evaluate the potential benefit of a third more specific and sensitive assay, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), with the objective of reducing the duration of the conventional 1-week study, we simultaneously assessed gene expression by qRT-PCR along with Western blots and enzyme activity assays as a time course in an in vivo model. METHODS: Rats were dosed daily for 8 days with model inducers of CYP1A, CYP2B, CYP3A, or CYP4A. Liver P450 levels were measured after 0.5, 1, 2, 4, and 8 days of dosing by qRT-PCR, Western blot, and enzyme activity. RESULTS: CYP1A, CYP3A, and CYP4A genes were maximally induced very rapidly (0.5-1 day), whereas the CYP2B gene was maximally induced after a lag time of 4 days. In all cases, fold changes in induction detected by qRT-PCR were greater than fold changes in protein levels and enzyme activities. CONCLUSIONS: Maximal persistent and larger fold changes observed by qRT-PCR either preceded or occurred simultaneously with maximal sustained fold changes in protein levels as measured by Western blots and enzyme activity assays. Our data show that qRT-PCR provides increased sensitivity and specificity over conventional assays and may be key information for reliable assessment of drug-related changes in CYP induction during the transition from discovery to toxicology studies.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Western Blotting , Clofibrato/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Masculino , Microssomos Hepáticos/enzimologia , Ratos , Ratos Sprague-Dawley , Transcrição Gênica , beta-Naftoflavona/farmacologia
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