RESUMO
Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and ß-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.
Assuntos
Acetilcisteína/metabolismo , Injúria Renal Aguda/metabolismo , Piperazinas/metabolismo , Piperazinas/toxicidade , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Piridinas/metabolismo , Piridinas/toxicidade , Pirimidinas/metabolismo , Pirimidinas/toxicidade , Transdução de Sinais/efeitos dos fármacos , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Animais , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
Phosphatidylinositol 3-kinase (PI3K) δ is a lipid kinase primarily found in leukocytes, which regulates important cell functions. AMG2519493 was a PI3K δ-specific inhibitor in development for treatment of various inflammatory diseases. AMG2519493-related changes in the male and/or female reproductive organs were observed in the 1- and 3-month oral repeat dose toxicology studies in the rat and cynomolgus monkey. Hemorrhagic corpora lutea cysts and increased incidence of corpora lutea cysts without hemorrhage were observed in the ovaries at supra pharmacological doses in the rat. A decrease in seminiferous germ cells in the testis, indicative of spermatogenesis maturation arrest, was observed in both the rat and cynomolgus monkey. Although the characteristics were comparable, the drug systemic exposures associated with the testicular changes were very different between the 2 species. In the rat, the testicular change was only observed at supra pharmacologic exposure. Isotype assessment of PI3K signaling in rat spermatogonia in vitro indicated a role for PI3K ß, but not δ, in the c Kit/PI3K/protein kinase B signaling pathway. Therefore, changes in both the ovary and testis of the rat were considered due to off target effect as they only occurred at suprapharmacologic exposure. In contrast, the testicular changes in the cynomolgus monkey (decrease in seminiferous germ cells) occurred at very low doses associated with PI3K δ-specific inhibition, indicating that the PI3K δ isoform may be important in spermatogenesis maturation in the cynomolgus monkey. Our results suggest species-related differences in PI3K isoform-specific control on reproductive organs.
Assuntos
Ovário/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , Testículo/efeitos dos fármacos , Animais , Feminino , Macaca fascicularis , Masculino , Camundongos , Ovário/enzimologia , Ratos , Ratos Sprague-Dawley , Espermatogônias/enzimologia , Testículo/enzimologiaRESUMO
Sotorasib is a first-in-class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. A comprehensive nonclinical safety assessment package, including secondary/safety pharmacology and toxicology studies, was conducted to support the marketing application for sotorasib. Sotorasib was negative in a battery of genotoxicity assays and negative in an in vitro phototoxicity assay. Based on in vitro assays, sotorasib had no off-target effects against various receptors, enzymes (including numerous kinases), ion channels, or transporters. Consistent with the tumor-specific target distribution (ie, KRASG12C), there were no primary pharmacology-related on-target effects identified. The kidney was identified as a target organ in the rat but not the dog. Renal toxicity in the rat was characterized by tubular degeneration and necrosis restricted to a specific region suggesting that the toxicity was attributed to the local formation of a putative toxic reactive metabolite. In the 3-month dog study, adaptive changes of hepatocellular hypertrophy due to drug metabolizing enzyme induction were observed in the liver that was associated with secondary effects in the pituitary and thyroid gland. Sotorasib was not teratogenic and had no direct effect on embryo-fetal development in the rat or rabbit. Human, dog, and rat circulating metabolites, M24, M10, and M18, raised no clinically relevant safety concerns based on the general toxicology studies, primary/secondary pharmacology screening, an in vitro human ether-à-go-go-related gene assay, or mutagenicity assessment. Overall, the results of the nonclinical safety program support a high benefit/risk ratio of sotorasib for the treatment of patients with KRAS p.G12C-mutated tumors.
Assuntos
Antineoplásicos/toxicidade , Piperazinas/toxicidade , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Piridinas/toxicidade , Pirimidinas/toxicidade , Animais , Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Piperazinas/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologiaRESUMO
Phosphatidylinositol 3-kinases (PI3Ks) regulate intracellular signaling events for multiple cell surface receptors. Phosphatidylinositol 3-kinase δ, 1 of 4 class I PI3K isoforms, is primarily found in leukocytes and regulates immune cell functions. Here, we report changes in the immune and digestive systems that were associated with AMG2519493, a highly selective small-molecule PI3Kδ inhibitor. Following 1- or 3-month oral repeat dosing in the cynomolgus monkey, changes were observed in circulating B cells, lymphoid tissues (spleen, lymph nodes, gut-associated lymphoid tissue, tonsil), and the digestive tract. Decreased circulating B cells and lymphoid cellularity in B cell-rich zones in lymphoid tissues were attributed to the intended pharmacologic activity of AMG2519493. Dose- and duration-dependent digestive system toxicity was characterized by inflammation in the large intestine and secondary opportunistic infections restricted to the digestive tract. Digestive tract changes were associated with moribundity and mortality at high-dose levels, and the effect level decreased with increased duration of exposure. These observations demonstrate the role of PI3Kδ in regulation of the immune system and of host resistance to opportunistic infections of the digestive tract.
Assuntos
Infecções Oportunistas , Fosfatidilinositol 3-Quinase , Animais , Imunomodulação , Macaca fascicularis , Fosfatidilinositol 3-QuinasesRESUMO
The ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is one of the most hotly pursued targets for the treatment of Alzheimer's disease. We used a structure- and property-based drug design approach to identify 2-aminooxazoline 3-azaxanthenes as potent BACE1 inhibitors which significantly reduced CSF and brain Aß levels in a rat pharmacodynamic model. Compared to the initial lead 2, compound 28 exhibited reduced potential for QTc prolongation in a non-human primate cardiovascular safety model.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Xantenos/química , Xantenos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Animais , Linhagem Celular , Células HEK293 , Humanos , Inibidores de Proteases/síntese química , Ratos , Xantenos/síntese químicaRESUMO
Bartonella henselae P26 has been identified as an immunodominant antigen expressed during feline infection. We used antisera from cats experimentally infected with B. henselae (n = 6), B. clarridgeiae (n = 4), or B. koehlerae (n = 2) and from a sample of naturally infected cats (B. henselae, n = 34; B. clarridgeiae, n = 1) to evaluate recombinant P26 (rP26) as a serodiagnostic antigen. Immunoblots using antisera from cats infected with B. henselae and B. clarridgeiae reacted strongly with rP26, whereas B. koehlerae antisera did not. A capture ELISA was designed to evaluate the kinetics of rP26 IgG in sera from experimentally infected cats. For B. henselae and B. clarridgeiae antisera, the kinetic profiles of reactivity were similar for rP26 capture ELISA and Bartonella spp. indirect fluorescence assay. However, for B. koehlerae antisera, reactivity in rP26 capture ELISA was consistently low. The serodiagnostic potential of rP26 capture ELISA was evaluated using sera from cats with known Bartonella sp. exposure histories. All 24 (100%) uninfected cats were seronegative, and 33 of 35 (94.3%) cats bacteremic for Bartonella spp. were seropositive. We propose that rP26-based serology can serve as a useful adjunct tool for the diagnosis of feline infection with B. henselae and B. clarridgeiae, but it may not be useful for feline infection with B. koehlerae.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos Virais/imunologia , Infecções por Bartonella/veterinária , Bartonella henselae/imunologia , Doenças do Gato/diagnóstico , Testes Sorológicos/veterinária , Animais , Antígenos Virais/genética , Infecções por Bartonella/diagnóstico , Infecções por Bartonella/imunologia , Western Blotting/veterinária , Doenças do Gato/imunologia , Gatos , Ensaio de Imunoadsorção Enzimática/veterinária , Humanos , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodosRESUMO
The influence of in vitro passage on Bartonella henselae pathogenesis in cats has not been thoroughly evaluated. Our objective was to examine the bacterial kinetics and humoral immune responses in cats experimentally infected with three different in vitro passages of B. henselae F1, a genotype I strain of feline origin. The F1 strain was in vitro passaged 20 and 40 times, and each was inoculated into a group of 5 cats. The kinetics of bacteremia and the feline humoral immune response to bacterial antigens were compared to a previous study involving a group of six cats inoculated with the original F1 strain. Among the three groups of cats, the kinetics of bacteremia profiles and the humoral immune responses to B. henselae lysates were similar. The influence of passage on bacterial membrane proteins was examined. In vitro passage altered the expression of 4/17 (23.5%) bacterial membrane proteins and 6/15 (40%) bacterial membrane antigens. An association between poor seroreactivity to three lysate antigens (15-, 18- and 45kDa), prolonged bacteremia and decreased serum bactericidal activity was noted. Our data show that in vitro passage of B. henselae did not alter the kinetics of bacteremia, including the occurrence of relapsing bacteremia, in experimentally infected cats. This suggests that highly passaged strains may not be suitable for future vaccination studies. Furthermore, in vitro passage results in phenotypic and antigenic changes in the bacterial membrane protein profile, which warrants caution in the interpretation of studies involving passaged B. henselae strains.
Assuntos
Formação de Anticorpos , Bartonella henselae/patogenicidade , Doenças do Gato/imunologia , Doenças do Gato/microbiologia , Doença da Arranhadura de Gato/veterinária , Animais , Antígenos de Bactérias/sangue , Bacteriemia/epidemiologia , Bacteriemia/veterinária , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Bartonella henselae/crescimento & desenvolvimento , Western Blotting/veterinária , Doenças do Gato/prevenção & controle , Doença da Arranhadura de Gato/imunologia , Doença da Arranhadura de Gato/microbiologia , Doença da Arranhadura de Gato/prevenção & controle , Gatos , Eletroforese em Gel de Poliacrilamida/veterinária , Genótipo , Cinética , Testes de Neutralização/veterinária , Organismos Livres de Patógenos Específicos , Vacinas de Produtos Inativados/imunologiaRESUMO
The medical records of 24 dogs with histologically confirmed mast cell tumors (MCT) of the muzzle were retrospectively evaluated to determine their biologic behavior and prognostic factors. Information on signalment, tumor grade and stage, treatment methods, and pattern of and time to failure and death was obtained from the medical record. Twenty-three dogs were treated with combinations of radiotherapy, surgery, and chemotherapy; 1 dog received no treatment. There were 2 Grade 1, 15 Grade 11, and 7 Grade III tumors. Tumors were stage 0 (n = 8), stage 1 (5), stage 2 (6), stage 3 (4), and stage 4 (1). Mean and median survival times of treated dogs were 36 and 30 months, respectively. Prognostic factors affecting survival time included tumor grade and presence of metastasis at diagnosis. Dogs with Grade I and II tumors survived longer than dogs with Grade III tumors. Variables, including sex, age, gross versus microscopic disease, and treatment type were not found to affect survival. Local control rate was 75% at 1 year and 50% at 3 years. Tumor grade was the only variable found to affect local control. Dogs with Grade I tumors had longer disease-free intervals than those with Grade II tumors, and dogs with Grade II tumors had longer disease-free intervals than dogs with Grade III tumors. Eight of 9 dogs dying of MCT had local or regional disease progression. Muzzle MCT a rebiologically aggressive tumors with higher regional metastatic rates than previously reported for MCT in other sites.
Assuntos
Doenças do Cão/mortalidade , Sarcoma de Mastócitos/veterinária , Recidiva Local de Neoplasia/veterinária , Neoplasias Cutâneas/veterinária , Animais , California/epidemiologia , Intervalo Livre de Doença , Doenças do Cão/diagnóstico , Doenças do Cão/patologia , Doenças do Cão/terapia , Cães , Feminino , Masculino , Sarcoma de Mastócitos/mortalidade , Metástase Neoplásica , Recidiva Local de Neoplasia/mortalidade , Estadiamento de Neoplasias , Nariz , Prognóstico , Registros/veterinária , Estudos Retrospectivos , Neoplasias Cutâneas/mortalidade , Análise de SobrevidaRESUMO
A 22-year-old female Welsh-cross pony was evaluated because of intermittent colic, signs of depression, pyrexia, anorexia, muscle wasting with abdominal distention, and weight gain over the preceding 12 months. A large abdominal mass was detected and surgically removed; the hemodynamic alterations and complications caused by the dramatic fluid losses and shifts that can occur in association with removal of a large abdominal mass required extensive postoperative management. Monitoring of clinical and hematologic variables such as attitude, heart rate, mucous membrane color, mean arterial blood pressure, PCV, and plasma total protein concentration provided useful information for successful management of the patient after surgery. On removal, the tumor weighed 19% of the pony's body weight and was characterized as a myofibroblastic tumor. Myofibroblastic tumors should be considered as a differential for large internal abdominal masses in horses, and surgical removal may be feasible and life extending with appropriate postoperative care.
Assuntos
Neoplasias Abdominais/veterinária , Hemodinâmica/fisiologia , Doenças dos Cavalos/cirurgia , Neoplasias de Tecido Muscular/veterinária , Neoplasias Abdominais/cirurgia , Animais , Feminino , Cavalos , Neoplasias de Tecido Muscular/cirurgia , Complicações Pós-Operatórias/prevenção & controle , Complicações Pós-Operatórias/veterinária , Hemorragia Pós-Operatória/prevenção & controle , Hemorragia Pós-Operatória/veterináriaRESUMO
Cat scratch disease (CSD) has an estimated prevalence of approximately 200,000 persons in the USA, and approximately 22,000 new cases occur annually. Cats are the natural carriers of Bartonella henselae, the agent for CSD. Zoonotic transmission of B. henselae can result in CSD in immunocompetent humans and bacillary angiomatosis in immunosuppressed humans. Infection in cats often goes undetected. Development of a vaccine to prevent feline infection is warranted to reduce the prevalence of infection in the feline population and to decrease the potential for zoonotic transmission. One of the immunoreactive proteins identified from our previous study was P26. In this study, we demonstrated that B. henselae recombinant P26 (rP26) was immunogenic in cats. Four cats immunized with rP26 and four control cats were challenged with B. henselae type I and blood samples were collected for culture, PCR, and serology. Immunization with rP26 did not provide protection against B. henselae infection in cats at the doses used in this study. However, p26 PCR proved to be more sensitive for detection of infection in cats compared to gltA PCR. Furthermore, ELISA using rP26 as the substrate was more sensitive than ELISA using B. henselae type I outer membrane proteins.
Assuntos
Infecções por Bartonella/veterinária , Bartonella henselae/imunologia , Doenças do Gato/imunologia , Doenças do Gato/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/isolamento & purificação , Infecções por Bartonella/imunologia , Infecções por Bartonella/microbiologia , Infecções por Bartonella/prevenção & controle , Bartonella henselae/genética , Bartonella henselae/patogenicidade , Sequência de Bases , Doenças do Gato/prevenção & controle , Doença da Arranhadura de Gato/microbiologia , Doença da Arranhadura de Gato/transmissão , Gatos , Primers do DNA/genética , DNA Bacteriano/genética , Vetores de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização/veterinária , Masculino , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Zoonoses/microbiologia , Zoonoses/transmissãoRESUMO
In order to identify immunoreactive Bartonella henselae proteins, B. henselae antiserum from an experimentally infected cat was used to screen a B. henselae genomic DNA expression library. One immunoreactive phage clone contained a gene (p26) with significant nucleotide identity with orthologs in brucellae, bartonellae, and several plant-associated bacteria. p26 gene sequences from four B. henselae strains, one B. koehlerae strain, and one B. clarridgeiae strain were cloned. Comparative nucleotide sequence analysis showed that p26 is a potential marker for molecular diagnosis of infection, as well as for identification to species level and genotyping of Bartonella sp. isolates. Alignment of the predicted amino acid sequences illustrated conserved putative protein features including a hydrophobic transmembrane region, a peptide cleavage site, and four dominant antigenic sites. Expression of p26 in Escherichia coli produced two proteins (26 and 27.5 kDa), both of which were reactive with feline anti-B. henselae antisera. Furthermore, murine hyperimmune serum raised against either recombinant protein reacted with both proteins. No reactivity to either recombinant protein was detected in nonimmune serum, and reactivity persisted as long as 20 weeks for one cat. The p26 protein product is an immunodominant antigen that is expressed during infection in cats as a preprotein and is subsequently cleaved to form mature P26.
Assuntos
Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Bartonella henselae/genética , Expressão Gênica , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Bartonella henselae/imunologia , Western Blotting , Gatos , Clonagem Molecular , Escherichia coli/genética , Biblioteca Gênica , Genoma Bacteriano , Imunização , Camundongos , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
The objective of this work was to compare the accuracy of radiographs and magnetic resonance imaging (MRI) for estimating appendicular osteosarcoma margins. The accuracy of computed tomography (CT) and bone scintigraphy was also assessed when these studies were available. Eight dogs with appendicular osteosarcoma underwent radiographic and MRI of affected limbs. In addition, bone scintigraphy was performed in six dogs and CT examination was performed in five dogs. Two observers jointly measured tumor length on all imaging studies. Correlative gross and histologic evaluation of all affected limbs was performed to determine tumor extent as measured from the nearest articular surface. Results from imaging studies were compared to gross and microscopic morphometry findings to determine the accuracy of each modality for determining tumor boundaries. MRI images were accurate with a mean overestimation of actual tumor length of 3 +/- 13%. T1-weighted non-contrast images were superior in identifying intramedullary tumor margins in most instances whereas contrast-enhanced images provided supplemental information in two dogs. Lateromedial and craniocaudal radiographs overestimated tumor length by 17 +/- 28% and 4 +/- 26%, respectively. Scintigraphy and CT overestimated tumor margins by 14 +/- 28% and 27 +/- 36%, respectively. MRI appears to be an accurate diagnostic imaging modality in determining intramedullary osteosarcoma boundaries. MRI should be considered as part of a pre-operative assessment of appendicular osteosarcoma, particularly when a limb-sparing procedure is contemplated.