RESUMO
Systemic therapies targeting transforming growth factor beta (TGFß) or TGFßR1 kinase (ALK5) have been plagued by toxicities including cardiac valvulopathy and bone physeal dysplasia in animals, posing a significant challenge for clinical development in pulmonary indications. The current work aims to demonstrate that systemic ALK5-associated toxicities can be mitigated through localized lung delivery. Lung-selective (THRX-144644) and systemically bioavailable (galunisertib) ALK5 inhibitors were compared to determine whether lung selectivity is sufficient to maintain local tissue concentrations while mitigating systemic exposure and consequent pathway-related findings. Both molecules demonstrated potent ALK5 activity in rat precision cut lung slices (PCLS; p-SMAD3 half-maximal inhibitory concentration [IC50], 141 nM and 1070 nM for THRX-144644 and galunisertib, respectively). In 14-day repeat-dose studies in rats, dose-related cardiac valvulopathy was recapitulated with oral galunisertib at doses ≥150 mg/kg/day. In contrast, inhaled nebulized THRX-144644 did not cause similar systemic findings up to the maximally tolerated doses in rats or dogs (10 and 1.5 mg/kg/day, respectively). THRX-144644 lung-to-plasma ratios ranged from 100- to 1200-fold in rats and dogs across dose levels. THRX-144644 lung trough (24 h) concentrations in rats and dogs ranged from 3- to 17-fold above the PCLS IC50 across tolerated doses. At a dose level exceeding tolerability (60 mg/kg/day; 76-fold above PCLS IC50) minimal heart and bone changes were observed when systemic drug concentrations reached pharmacologic levels. In conclusion, the current preclinical work demonstrates that localized pulmonary delivery of an ALK5 inhibitor leads to favorable TGFß pathway pharmacodynamic inhibition in lung while minimizing key systemic toxicities.
Assuntos
Pulmão/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Administração Oral , Animais , Cães , Feminino , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pirazóis/toxicidade , Quinolinas/toxicidade , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismoRESUMO
Activated Wnt/ß-catenin signaling is frequently associated with colorectal cancer. Wnt inhibitors, including tankyrase inhibitors, are being explored as potential anticancer agents. Wnt signaling is also critical for intestinal tissue homeostasis, and Wnt inhibitors have been shown to cause intestinal toxicity in mice by affecting intestinal stem cells. This study sought to characterize the intestinal toxicity of tankyrase inhibitors, including reversibility, and to assess their therapeutic index. Novel tankyrase inhibitor G-631 caused dose-dependent intestinal toxicity with a therapeutic index < 1 after 14 days of dosing in mice. At a tolerated subtherapeutic dose level, the intestinal toxicity was composed of enteritis characterized by villus blunting, epithelial degeneration, and inflammation, which fully reversed after 14 days of recovery. Doubled exposure showed weak antitumor activity in a xenograft colorectal cancer model but also caused more severe intestinal toxicity characterized by multifocal-regionally extensive necrotizing and ulcerative enteritis leading to morbidity or moribundity in some animals. This toxicity was only partially reversed after 14 days of recovery, with evidence of crypt and villus regeneration, mildly blunted villi, and/or scarring in association with chronic inflammation of the submucosa. Therefore, the clinical utility of tankyrase inhibitors is likely limited by the on-target intestinal toxicity and a therapeutic index < 1 in mice.
Assuntos
Antineoplásicos/toxicidade , Neoplasias Colorretais/metabolismo , Inibidores Enzimáticos/toxicidade , Intestinos/efeitos dos fármacos , Tanquirases/antagonistas & inibidores , Animais , Peso Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Mucosa Intestinal/metabolismo , Intestinos/patologia , Camundongos , Camundongos Nus , Testes de Toxicidade , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Nonclinical safety and pharmacokinetic data for MMAE and 14 vedotin ADCs were evaluated to determine patterns of toxicity, consistency of pharmacokinetic results, and species differences between rats and monkeys. Most nonclinical toxicities were antigen-independent, common across ADCs, and included hematologic, lymphoid, and reproductive toxicity related to MMAE pharmacology. Hematologic toxicity was the dose-limiting or predominant toxicity for the majority of vedotin ADCs in both species. Tissue expression of the targeted antigen of an ADC rarely correlated with dose-limiting toxicity (DLT); only two ADCs had antigen-dependent skin DLTs. For two additional ADCs, antigen-dependent delivery of MMAE in the bone marrow may have exacerbated the antigen-independent hematologic DLT. The highest tolerated doses and pharmacokinetics were similar within a given species, with rats tolerating higher doses than monkeys. Studies longer than one month in duration detected the same or fewer toxicities than one-month studies and had no additional findings that affected the human risk assessment. These data support opportunities to streamline ADC toxicity assessments without compromising human starting dose selection or target organ identification.
RESUMO
Several toxicities are clearly driven by free drug concentrations in plasma, such as toxicities related to on-target exaggerated pharmacology or off-target pharmacological activity associated with receptors, enzymes or ion channels. However, there are examples in which organ toxicities appear to correlate better with total drug concentrations in the target tissues, rather than with free drug concentrations in plasma. Here we present a case study in which a small molecule Met inhibitor, GEN-203, with significant liver and bone marrow toxicity in preclinical species was modified with the intention of increasing the safety margin. GEN-203 is a lipophilic weak base as demonstrated by its physicochemical and structural properties: high LogD (distribution coefficient) (4.3) and high measured pKa (7.45) due to the basic amine (N-ethyl-3-fluoro-4-aminopiperidine). The physicochemical properties of GEN-203 were hypothesized to drive the high distribution of this compound to tissues as evidenced by a moderately-high volume of distribution (Vd>3l/kg) in mouse and subsequent toxicities of the compound. Specifically, the basicity of GEN-203 was decreased through addition of a second fluorine in the 3-position of the aminopiperidine to yield GEN-890 (N-ethyl-3,3-difluoro-4-aminopiperidine), which decreased the volume of distribution of the compound in mouse (Vd=1.0l/kg), decreased its tissue drug concentrations and led to decreased toxicity in mice. This strategy suggests that when toxicity is driven by tissue drug concentrations, optimization of the physicochemical parameters that drive tissue distribution can result in decreased drug concentrations in tissues, resulting in lower toxicity and improved safety margins.
Assuntos
Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/toxicidade , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Feminino , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-met/metabolismo , Distribuição Aleatória , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologiaRESUMO
Pim kinases are promising targets for the development of cancer therapeutics. Among the three Pim isoforms, Pim-2 is particularly important in multiple myeloma, yet is the most difficult to inhibit due to its high affinity for ATP. We identified compound 1 via high throughput screening. Using property-based drug design and co-crystal structures with Pim-1 kinase to guide analog design, we were able to improve potency against all three Pim isoforms including a significant 10,000-fold gain against Pim-2. Compound 17 is a novel lead with low picomolar potency on all three Pim kinase isoforms.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirazóis/química , Pirimidinas/química , Animais , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Cinética , Camundongos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Pirazóis/síntese química , Pirazóis/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Dipeptidylpeptidase 4 (DPP4) inhibitors have clinical benefit in patients with type 2 diabetes mellitus by increasing levels of glucose-lowering incretin hormones, such as glucagon-like peptide -1 (GLP-1), a peptide with a short half life that is secreted for approximately 1 hour following a meal. Since drugs with prolonged binding to their target have been shown to maximize pharmacodynamic effects while minimizing drug levels, we developed a time-dependent inhibitor that has a half-life for dissociation from DPP4 close to the duration of the first phase of GLP-1 release. RESULTS: Saxagliptin and its active metabolite (5-hydroxysaxagliptin) are potent inhibitors of human DPP4 with prolonged dissociation from its active site (Ki = 1.3 nM and 2.6 nM, t1/2 = 50 and 23 minutes respectively at 37°C). In comparison, both vildagliptin (3.5 minutes) and sitagliptin ( < 2 minutes) rapidly dissociated from DPP4 at 37°C. Saxagliptin and 5-hydroxysaxagliptin are selective for inhibition of DPP4 versus other DPP family members and a large panel of other proteases, and have similar potency and efficacy across multiple species.Inhibition of plasma DPP activity is used as a biomarker in animal models and clinical trials. However, most DPP4 inhibitors are competitive with substrate and rapidly dissociate from DPP4; therefore, the type of substrate, volume of addition and final concentration of substrate in these assays can change measured inhibition. We show that unlike a rapidly dissociating DPP4 inhibitor, inhibition of plasma DPP activity by saxagliptin and 5-hydroxysaxagliptin in an ex vivo assay was not dependent on substrate concentration when substrate was added rapidly because saxagliptin and 5-hydroxysaxagliptin dissociate slowly from DPP4, once bound. We also show that substrate concentration was important for rapidly dissociating DPP4 inhibitors. CONCLUSIONS: Saxagliptin and its active metabolite are potent, selective inhibitors of DPP4, with prolonged dissociation from its active site. They also demonstrate prolonged inhibition of plasma DPP4 ex vivo in animal models, which implies that saxagliptin and 5-hydroxysaxagliptin would continue to inhibit DPP4 during rapid increases in substrates in vivo.
Assuntos
Adamantano/análogos & derivados , Dipeptídeos/metabolismo , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/metabolismo , Hipoglicemiantes/metabolismo , Adamantano/metabolismo , Algoritmos , Animais , Artefatos , Clonagem Molecular , Dipeptidases/metabolismo , Dipeptidil Peptidase 4/sangue , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Indicadores e Reagentes , Cinética , Macaca fascicularis , Nitrilas/metabolismo , Ligação Proteica , Pirazinas/metabolismo , Pirrolidinas/metabolismo , Fosfato de Sitagliptina , Especificidade da Espécie , Triazóis/metabolismo , VildagliptinaRESUMO
The R- and S-enantiomer of N-(4-(3-(1-ethyl-3,3-difluoropiperidin-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide are novel MET kinase inhibitors that have been investigated as potential anticancer agents. The effect of the chirality of these compounds on preclinical in vivo pharmacokinetics and toxicity was studied. The plasma clearance for the S-enantiomer was low in mice and monkeys (23.7 and 7.8 mL min(-1) kg(-1), respectively) and high in rats (79.2 mL min(-1) kg(-1)). The R/S enantiomer clearance ratio was 1.5 except in rats (0.49). After oral single-dose administration at 5 mg kg(-1) the R/S enantiomer ratio of AUC(inf) was 0.95, 1.9 and 0.41 in mice, rats and monkeys, respectively. In an oral single-dose dose-ranging study at 200 and 500 mg kg(-1) and multi-dose toxicity study in mice plasma AUC exposure was approximately 2- to 3-fold higher for the R-enantiomer compared to the S-enantiomer. Greater toxicity of the S-enantiomer was observed which appeared to be due to high plasma C(min) values and tissue concentrations approximately 24 h after the final dose. Both enantiomers showed low to moderate permeability in MDCKI cells with no significant efflux, no preferential distribution into red blood cells and similar plasma protein binding in vitro. Overall, the differences between the enantiomers with respect to low dose pharmacokinetics and in vitro properties were relatively modest. However, toxicity results warrant further development of the R-enantiomer over the S-enantiomer.
Assuntos
Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Pirazóis/farmacocinética , Piridazinas/farmacocinética , Administração Oral , Animais , Proteínas Sanguíneas/metabolismo , Peso Corporal , Linhagem Celular , Permeabilidade da Membrana Celular , Cães , Avaliação Pré-Clínica de Medicamentos , Feminino , Macaca fascicularis , Masculino , Camundongos , Ligação Proteica , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Pirazóis/administração & dosagem , Pirazóis/sangue , Pirazóis/química , Piridazinas/administração & dosagem , Piridazinas/sangue , Piridazinas/química , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Fatores de TempoRESUMO
Izencitinib (TD-1473), an oral, gut-selective pan-Janus kinase (JAK) inhibitor under investigation for treatment of inflammatory bowel diseases, was designed for optimal efficacy in the gastrointestinal tract while minimizing systemic exposures and JAK-related safety findings. The nonclinical safety of izencitinib was evaluated in rat and dog repeat-dose and rat and rabbit reproductive and developmental toxicity studies. Systemic exposures were compared with JAK inhibitory potency to determine effects at or above pharmacologic plasma concentrations (≥1× plasma average plasma concentration [Cave]:JAK 50% inhibitory concentration [IC50] ratio). In rats and dogs, 1000 and 30 mg/kg/day izencitinib, respectively, produced minimal systemic findings (ie, red/white cell changes) and low systemic concentrations (approximately 1× plasma Cave:JAK IC50 ratio) with an 8× nonclinical:clinical systemic area under the curve (AUC) margin compared with exposures at the highest clinically tested dose (300 mg, quaque die, once daily, phase 1 study in healthy volunteers). In dogs, it was possible to attain sufficient systemic exposures to result in immunosuppression characteristic of systemic JAK inhibition, but at high AUC margins (43×) compared with systemic exposures observed at the highest tested dose in humans. No adverse findings were observed in the gastrointestinal tract or systemic tissues. Izencitinib did not affect male or female fertility. Izencitinib did not affect embryonic development in rats and rabbits as commonly reported with systemic JAK inhibition, consistent with low maternal systemic concentrations (2-6× plasma Cave:JAK IC50 ratio, 10-33× nonclinical:clinical AUC margin) and negligible fetal exposures. In conclusion, the izencitinib gut-selective approach resulted in minimal systemic findings in nonclinical species at pharmacologic, clinically relevant systemic exposures, highlighting the impact of organ-selectivity in reducing systemic safety findings.
Assuntos
Janus Quinases , Naftiridinas , Nitrilas , Administração Oral , Animais , Cães , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Voluntários Saudáveis , Humanos , Doenças Inflamatórias Intestinais , Janus Quinases/antagonistas & inibidores , Masculino , Naftiridinas/farmacologia , Naftiridinas/toxicidade , Nitrilas/farmacologia , Nitrilas/toxicidade , Gravidez , Coelhos , Ratos , Reprodução/efeitos dos fármacos , Testes de ToxicidadeRESUMO
Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.
Assuntos
Antineoplásicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirazóis/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Cães , Feminino , Humanos , Macaca fascicularis , Células Madin Darby de Rim Canino , Masculino , Camundongos SCID , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Pirazóis/síntese química , Pirazóis/metabolismo , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pim kinases have been identified as promising therapeutic targets for hematologic-oncology indications, including multiple myeloma and certain leukemia. Here, we describe our continued efforts in optimizing a lead series by improving bioavailability while maintaining high inhibitory potency against all three Pim kinase isoforms. The discovery of extensive intestinal metabolism and major metabolites helped refine our design strategy, and we observed that optimizing the pharmacokinetic properties first and potency second was a more successful approach than the reverse. In the resulting work, novel analogs such as 20 (GNE-955) were discovered bearing 5-azaindazole core with noncanonical hydrogen bonding to the hinge.
Assuntos
Indazóis/química , Indazóis/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Humanos , Indazóis/metabolismo , Indazóis/farmacocinética , Mucosa Intestinal/metabolismo , Simulação de Acoplamento Molecular , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , RatosRESUMO
Improved small molecule bioanalytical sensitivity and concomitant decreased sample volume requirements provide an opportunity to reconsider how toxicokinetic (TK) data are collected in rat toxicity studies. Often, satellite groups of rats are designated to separate procedural effects of TK blood collection from the primary toxicity evaluation. Blood microsampling (i.e., ≤50 µL) decreases the blood volume collected such that TK samples can be collected from toxicity groups without impacting toxicity assessment. Small plasma sampling uses slightly higher blood volumes (i.e., 200 µL) with comparable technical feasibility and, importantly, allows multiple analyses with no negative impact on study interpretation. Our "base case" study designs utilize sparse TK sampling from sample toxicity group rats (1-2 samples/rat). Alternate designs with satellite animals may still be warranted based on study objectives (e.g., biomarkers), intolerability, or smaller rat strains; however, we propose these as exceptions rather than standard practice and with a focus to use the fewest animals possible. We review the state of knowledge in bioanalytical and blood sampling techniques and support the paradigm whereby TK sampling of main study animals significantly decreases the overall number of rats required for toxicity assessments and refines study interpretation with additional data options. These efforts maintain a commitment to the 3Rs (replacement, reduction, and refinement) while maintaining high-quality TK evaluations on toxicity studies.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Toxicocinética , Animais , Biomarcadores/sangue , Coleta de Amostras Sanguíneas/instrumentação , RatosRESUMO
PURPOSE: Antibody-drug conjugates (ADC) selectively deliver a cytotoxic drug to cells expressing an accessible antigenic target. Here, we have appended monomethyl auristatin E (MMAE) to an antibody recognizing the SLC34A2 gene product NaPi2b, the type II sodium-phosphate cotransporter, which is highly expressed on tumor surfaces of the lung, ovary, and thyroid as well as on normal lung pneumocytes. This study evaluated its efficacy and safety in preclinical studies. EXPERIMENTAL DESIGN: The efficacy of anti-NaPi2b ADC was evaluated in mouse ovarian and non-small cell lung cancer (NSCLC) tumor xenograft models, and its toxicity was assessed in rats and cynomolgus monkeys. RESULTS: We show here that an anti-NaPi2b ADC is effective in mouse ovarian and NSCLC tumor xenograft models and well-tolerated in rats and cynomolgus monkeys at levels in excess of therapeutic doses. Despite high levels of expression in normal lung of non-human primate, the cross-reactive ADC exhibited an acceptable safety profile with a dose-limiting toxicity unrelated to normal tissue target expression. The nonproliferative nature of normal pneumocytes, together with the antiproliferative mechanism of MMAE, likely mitigates the potential liability of this normal tissue expression. CONCLUSIONS: Overall, our preclinical results suggest that the ADC targeting NaPi2b provides an effective new therapy for the treatment of NSCLC and ovarian cancer and is currently undergoing clinical developments.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Oligopeptídeos/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Imunoconjugados/administração & dosagem , Macaca fascicularis , Masculino , Camundongos , Oligopeptídeos/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Ratos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Acute administration of cadmium (Cd) in rats results in hepatotoxicity that appears to involve the activation of Kupffer cells and the subsequent production of proinflammatory chemokines and cytokines. However, the importance of these endogenous mediators in Cd-induced hepatotoxicity is unknown. Therefore, this study was conducted to define and utilize a rat strain difference in sensitivity to Cd-induced hepatotoxicity to elucidate the role of cytokines and chemokines in Cd-induced hepatotoxicity. Doses were selected from a dose-response study of the effect of Cd on serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities. Hepatotoxic doses of 2.0 mg Cd/kg in Fischer 344 (F344) rats and 3.0 mg Cd/kg in Sprague-Dawley (SD) rats, as well as a relatively nontoxic dose of 2.0 mg Cd/kg in SD rats, were chosen for the time-course experiment. Blood and liver from F344 (saline or 2.0 mg Cd/kg iv) and SD rats (saline or 2.0 or 3.0 mg Cd/kg iv) were collected at 0, 1, 3, 6, 10, 18, 24, and 48 h after Cd administration. Cadmium treatment caused an increase in serum ALT and SDH by 3 h and peaked between 18 and 24 h in both strains. Hepatic Cd content, metallothionein (MT) induction, and nonprotein sulfhydryl (NPSH) content were quantified and determined to be consistent with dosing rather than strain differences. Total RNA samples isolated from liver samples were analyzed for chemokine (CINC-1 and MCP-1) and cytokine (TNF-alpha, IL-1beta, IL-6, and IL-10) mRNA levels by the Quantigene branched DNA signal amplification assay. Lipopolysaccharide treatment served as a positive control for chemokine and cytokine induction. After Cd administration, F344 rat livers did not contain higher levels or earlier induction of chemokine and cytokine mRNAs than SD rats. Therefore, this study demonstrates a strain difference in sensitivity to Cd-induced hepatotoxicity that appears to be unrelated to Cd, MT, NPSH, or cytokine expression.
Assuntos
Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Predisposição Genética para Doença , Fígado/efeitos dos fármacos , Ratos Endogâmicos F344/genética , Ratos Sprague-Dawley/genética , Animais , Cádmio/administração & dosagem , Cádmio/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Poluentes Ambientais/administração & dosagem , Poluentes Ambientais/metabolismo , Injeções Intravenosas , Fígado/metabolismo , Fígado/patologia , Masculino , Sondas de Oligonucleotídeos/química , RNA Mensageiro/metabolismo , Ratos , Especificidade da EspécieRESUMO
Acute administration of cadmium (Cd) to rats results in hepatotoxicity. Recent reports indicate that Kupffer cells, the resident macrophages of the liver, participate in the manifestation of Cd-induced hepatotoxicity. Nitric oxide (NO) is a reactive nitrogen radical produced by activated Kupffer cells via the induction of inducible nitric oxide synthase (iNOS). Nitric oxide can combine with superoxide to form peroxynitrite, a molecule that may participate in the toxic mechanisms of hepatotoxins, such as acetaminophen and bacterial endotoxin. It has been speculated that Cd also may exert its hepatotoxicity, in part, via the production of NO by iNOS. Therefore, this study was undertaken to determine whether iNOS contributes to Cd-induced hepatotoxicity. Wild-type (WT) mice were administered selective iNOS inhibitors (AMT and 1400W) concurrently and 3 h after administration of a hepatotoxic dose of Cd (4.0 mg Cd/mg). Additionally, WT and iNOS-null (iNOS-KO) mice were dosed iv with saline or 2.0, 2.5, 3.0, 3.5 or 4.0 mg Cd/kg. Serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities were quantified to assess liver injury. Administration of iNOS inhibitors failed to prevent Cd-induced hepatotoxicity. Also, Cd caused a dose-dependent increase in liver injury in both WT and iNOS-KO mice. The liver injury produced by Cd in the iNOS-KO mice was not different from that in WT at any dose. These data indicate that iNOS does not appear to mediate Cd-induced hepatotoxicity.
Assuntos
Cloreto de Cádmio/toxicidade , Doença Hepática Induzida por Substâncias e Drogas , Hepatopatias/enzimologia , Óxido Nítrico Sintase/deficiência , Alanina Transaminase/sangue , Amidinas/farmacologia , Animais , Benzilaminas/farmacologia , Cloreto de Cádmio/metabolismo , Inibidores Enzimáticos , Feminino , Histocitoquímica , L-Iditol 2-Desidrogenase/sangue , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Tiazinas/farmacologiaRESUMO
Having an understanding of drug tissue accumulation can be informative in the assessment of target organ toxicities; however, obtaining tissue drug levels from toxicology studies by bioanalytical methods is labor-intensive and infrequently performed. Additionally, there are no described methods for predicting tissue drug distribution for the experimental conditions in toxicology studies, which typically include non-steady-state conditions and very high exposures that may saturate several processes. The aim was the development of an algorithm to provide semiquantitative and quantitative estimates of tissue-to-plasma concentration ratios (Kp ) for several tissues from readily available parameters of pharmacokinetics (PK) such as volume of distribution (Vd ) and clearance of each drug, without performing tissue measurement in vivo. The computational approach is specific for the oral route of administration and non-steady-state conditions and was applied for a dataset of 29 Genentech small molecules such as neutral compounds as well as weak and strong organic bases. The maximum success rate in predicting Kp values within 2.5-fold error of observed Kp values was 82% at low doses (<100 mg/kg) in preclinical species. Prediction accuracy was relatively lower with saturation at high doses (≥100 mg/kg); however, an approach to perform low-to-high dose extrapolations of Kp values was presented and applied successfully in most cases. An approach for the interspecies scaling was also applied successfully. Finally, the proposed algorithm was used in a case study and successfully predicted differential tissue distribution of two small-molecule MET kinase inhibitors, which had different toxicity profiles in mice. This newly developed algorithm can be used to predict the partition coefficients Kp for small molecules in toxicology studies, which can be leveraged to optimize the PK drivers of tissue distribution in an attempt to decrease drug tissue level, and improve safety margins.
Assuntos
Preparações Farmacêuticas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacocinética , Algoritmos , Animais , Cães , Camundongos , Modelos Biológicos , Ratos , Distribuição TecidualRESUMO
Dipeptidyl peptidases (DPPs) are proteolytic enzymes that regulate many physiological systems by degrading signaling peptides. DPP8 and DPP9 are distinct from DPP4 in sequence, cellular localization and expression levels, thus implying distinct functions. However, DPP8 and DPP9 expression needs further delineation. We evaluated DPP4, DPP8 and DPP9 expression using three independent methods at the mRNA, protein, and functional levels to better understand the local physiological contribution of each enzyme. Sprague Dawley rats and cynomolgus monkeys were selected for DPP4, DPP8 and DPP9 expression profiling to represent animal species commonly utilized for drug preclinical safety evaluation. A novel Xhibit assay of DPP protease activity was applied in addition to newly available antibodies for immunohistochemical localization. This combined approach can facilitate a functional evaluation of protease expression, which is important for understanding physiological relevance. Few inter-species differences were observed. Tissue mRNA and protein levels generally correlated to functional DPP4 and DPP8/9 enzymatic activity. All three proteins were seen in epithelial cells, lymphoid cells and some endothelial and vascular smooth muscle cells. Combined DPP8/DPP9 enzymatic activity was uniformly intracellular across tissues at approximately 10-fold lower levels than non-renal DPP4. Consistent levels of each DPP were detected among most non-renal tissues in rats and monkeys. DPP4 was ubiquitous, principally detected on cell membranes of epithelial and endothelial cells and was greatest in the kidney. The expression patterns suggest that DPP8 and DPP9 may act similarly across tissues, and that their actions might in part overlap with DPP4.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Rim/enzimologia , Sequência de Aminoácidos , Animais , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Feminino , Expressão Gênica , Macaca fascicularis , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Pâncreas/enzimologia , Ratos , Ratos Sprague-Dawley , Especificidade da EspécieRESUMO
Herein we report on the structure-based discovery of a C-2 hydroxyethyl moiety which provided consistently high levels of selectivity for JAK1 over JAK2 to the imidazopyrrolopyridine series of JAK1 inhibitors. X-ray structures of a C-2 hydroxyethyl analogue in complex with both JAK1 and JAK2 revealed differential ligand/protein interactions between the two isoforms and offered an explanation for the observed selectivity. Analysis of historical data from related molecules was used to develop a set of physicochemical compound design parameters to impart desirable properties such as acceptable membrane permeability, potent whole blood activity, and a high degree of metabolic stability. This work culminated in the identification of a highly JAK1 selective compound (31) exhibiting favorable oral bioavailability across a range of preclinical species and robust efficacy in a rat CIA model.
Assuntos
Antirreumáticos/síntese química , Compostos Heterocíclicos com 3 Anéis/síntese química , Imidazóis/síntese química , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Piridinas/síntese química , Pirróis/síntese química , Administração Oral , Animais , Antirreumáticos/química , Antirreumáticos/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Experimental/etiologia , Disponibilidade Biológica , Permeabilidade da Membrana Celular , Colágeno , Cristalografia por Raios X , Cães , Haplorrinos , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Imidazóis/química , Imidazóis/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Janus Quinase 1/química , Janus Quinase 2/química , Células Madin Darby de Rim Canino , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Piridinas/química , Piridinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Ratos , EstereoisomerismoRESUMO
Herein we report the discovery of the C-2 methyl substituted imidazopyrrolopyridine series and its optimization to provide potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2. The C-2 methyl substituted inhibitor 4 exhibited not only improved JAK1 potency relative to unsubstituted compound 3 but also notable JAK1 vs JAK2 selectivity (20-fold and >33-fold in biochemical and cell-based assays, respectively). Features of the X-ray structures of 4 in complex with both JAK1 and JAK2 are delineated. Efforts to improve the in vitro and in vivo ADME properties of 4 while maintaining JAK1 selectivity are described, culminating in the discovery of a highly optimized and balanced inhibitor (20). Details of the biological characterization of 20 are disclosed including JAK1 vs JAK2 selectivity levels, preclinical in vivo PK profiles, performance in an in vivo JAK1-mediated PK/PD model, and attributes of an X-ray structure in complex with JAK1.
Assuntos
Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/química , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Animais , Bioensaio , Disponibilidade Biológica , Linhagem Celular , Cristalografia por Raios X , Cães , Hepatócitos/citologia , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Humanos , Janus Quinase 1/química , Janus Quinase 2/química , Camundongos , Modelos Moleculares , Ratos , Relação Estrutura-AtividadeRESUMO
Mice harboring mutations in the Ahr locus display a patent ductus venosus and smaller livers throughout life. We tested the hypothesis that these hepatic aberrations are secondary to a developmental defect in hepatovascular blood flow by performing a detailed analysis of hepatic development in wild-type and Ahr-/- mice. This study revealed necrotic lesions in the peripheries of Ahr-/- fetal livers as early as embryonic day 15.5, with an increasing incidence up to postnatal day 1 and resolution by 2 weeks post partum. To visualize perfusion of fetal livers, we injected fluorescein isothiocyanate-labeled dextran into the cranial artery and monitored hepatic fluorescence by microscopy. The peripheries of the median and left lobes displayed decreased perfusion in regions corresponding to those regions that displayed necrosis at later developmental times. An examination of adult Ahr-/- animals revealed that smaller livers are predominantly due to decreased sizes of the left and right lobes, corresponding to regions of decreased perfusion and hepatic necrosis observed in fetal livers. Histological aberrations in the portal vein also support a model in which perfusion is compromised in the Ahr-/- liver. Taken in sum, these results indicate that the Ahr locus is required for normal perfusion of the developing liver and that disruption of the AHR signaling pathway gives rise to fetal hepatic necrosis and consequent liver deformation which persists through adult-hood.
Assuntos
Circulação Hepática/genética , Fígado/anormalidades , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cruzamentos Genéticos , Desenvolvimento Embrionário , Feminino , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Necrose , Perfusão , Gravidez , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
The heavy metal cadmium (Cd) causes hepatotoxicity upon acute administration. Kupffer cells, the resident macrophages of the liver, have been suggested to play a role in Cd-induced hepatotoxicity. Gadolinium chloride (GdCl3) may prevent Cd-induced hepatotoxicity by suppressing Kupffer cells. However, GdCl3 also induces the Cd-binding protein, metallothionein (MT). Therefore, this study was conducted to determine whether GdCl3 prevents Cd-induced hepatotoxicity via the induction of MT. Hepatic MT and Kupffer cell counts were analyzed 24 h after wild-type (WT) mice were administered saline or 10, 30, or 60 mg GdCl3/kg. GdCl3 induced MT in a dose-dependent manner without affecting nonprotein sulfhydryl content. All examined doses of GdCl3 were effective at eliminating Kupffer cells from the liver. To examine the hepatoprotective effects of GdCl3, WT and MT-null mice were pretreated with saline or 10, 30, or 60 mg GdCl3 24 h prior to a hepatotoxic dose of Cd (2.5 mg Cd/kg). Blood and livers were removed 16 h later and analyzed for hepatotoxicity as well as MT, Cd, and Kupffer cell content. Hepatotoxicity was alleviated in both WT and MT-null mice that were pretreated with 30 or 60 mg GdCl3/kg, indicating that MT induction is not required for the hepatoprotective effects of GdCl3. Hepatic Cd content was not decreased by GdCl3, demonstrating that GdCl3 does not negatively affect Cd distribution to the liver. Kupffer cells were depleted at all three doses of GdCl3, whereas hepatoprotection was only observed at doses of 30 and 60 mg GdCl3/kg. This does not rule out Kupffer cells in the mechanism of Cd-induced hepatotoxicity, but it does suggest that GdCl3 exerts hepatoprotective effects on the liver aside from depleting Kupffer cells. In summary, these data substantially rule out MT induction and decrease the importance of Kupffer cells as mechanisms of GdCl3-induced protection from Cd-induced hepatotoxicity.