RESUMO
Clofibrate is a known rodent hepatotoxicant classically associated with hepatocellular hypertrophy and increased serum activities of cellular alanine aminotransferase/aspartate aminotransferase (ALT/AST) in the absence of microscopic hepatocellular degeneration. At toxic dose, clofibrate induces liver and skeletal muscle injury. The objective of this study was to assess novel liver and skeletal muscle biomarkers following clofibrate administration in Wistar rats at different dose levels for 7 days. In addition to classical biomarkers, liver injury was assessed by cytokeratin 18 (CK18) cleaved form, high-mobility group box 1, arginase 1 (ARG1), microRNA 122 (miR-122), and glutamate dehydrogenase. Skeletal muscle injury was evaluated with fatty acid binding protein 3 (Fabp3) and myosin light chain 3 (Myl3). Clofibrate-induced hepatocellular hypertrophy and skeletal muscle degeneration (type I rich muscles) were noted microscopically. CK, Fabp3, and Myl3 elevations correlated to myofiber degeneration. Fabp3 and Myl3 outperformed CK for detection of myofiber degeneration of minimal severity. miR-122 and ARG1 results were significantly correlated and indicated the absence of liver toxicity at low doses of clofibrate, despite increased ALT/AST activities. Moreover, combining classical and novel biomarkers (Fabp3, Myl3, ARG1, and miR-122) can be considered a valuable strategy for differentiating increased transaminases due to liver toxicity from skeletal muscle toxicity.
Assuntos
Anticolesterolemiantes/efeitos adversos , Biomarcadores/sangue , Doença Hepática Induzida por Substâncias e Drogas/patologia , Clofibrato/efeitos adversos , Fígado/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Animais , Anticolesterolemiantes/administração & dosagem , Arginase/sangue , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Colesterol/sangue , Colinesterases/sangue , Clofibrato/administração & dosagem , Creatinina/sangue , Relação Dose-Resposta a Droga , Proteína 3 Ligante de Ácido Graxo/sangue , Glutamato Desidrogenase/sangue , Queratina-18/sangue , Fígado/metabolismo , Masculino , MicroRNAs/sangue , Músculo Esquelético/metabolismo , Cadeias Leves de Miosina/sangue , Ratos , Ratos Wistar , Triglicerídeos/sangueRESUMO
We present a novel approach for first-in-human (FIH) dose selection of the CD20xCD3 bispecific antibody, glofitamab, based on pharmacokinetic/pharmacodynamic (PKPD) assessment in cynomolgus monkeys to select a high, safe starting dose, with cytokine release (CR) as the PD endpoint. Glofitamab pharmacokinetics were studied in mice and cynomolgus monkeys; PKPD of IL-6, TNF-α and interferon-γ release following glofitamab, with/without obinutuzumab pretreatment (Gpt) was studied in cynomolgus monkeys. Potency differences for CR between cynomolgus monkeys and humans were determined by glofitamab incubation in whole blood of both species. The PKPD model for CR was translated to humans to project a starting dose that did not induce CR exceeding a clinically-predefined threshold. In cynomolgus monkeys, glofitamab showed a species-specific atypical high clearance, with and without B-cell debulking by Gpt. CR was related to glofitamab serum levels and B-cell counts. B-cell reduction by Gpt led to a marked decrease in CR. FIH starting dose (5 µg) was selected based on IL-6 release considering the markedly higher glofitamab in vitro potency in human vs monkey blood. This is a novel PKPD-based approach for selection of FIH starting dose for a CD20xCD3 bispecific antibody in B-cell lymphoma, evidenced in the glofitamab study, NP30179 (NCT03075696).
Assuntos
Anticorpos Biespecíficos , Linfoma de Células B , Animais , Citocinas , Humanos , Interleucina-6 , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Macaca fascicularis , CamundongosRESUMO
PURPOSE: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. EXPERIMENTAL DESIGN: FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models. RESULTS: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. CONCLUSIONS: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.
Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Antígenos CD40/agonistas , Endopeptidases/efeitos dos fármacos , Imunoterapia/métodos , Proteínas de Membrana/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Camundongos , Células Tumorais CultivadasRESUMO
The potential of 4-chloro-ortho-toluidine (4-CloT), an aromatic amine substituted on the ortho- and para-position of the amine function, to induce DNA damage in male Wistar rats was evaluated with the micronucleus test (peripheral blood), Pig-a (peripheral blood), and comet assay (peripheral blood, liver, urinary bladder, jejunum) at several time points. In addition to those markers of DNA damage, ie, gene mutation and clastogenicity, standard hematology, including methemoglobin, histopathology and immunohistochemistry of γ-H2AX and Ki-67 in liver, jejunum, and urinary bladder were performed. 4-CloT was administered orally over 28 consecutive days (days 1-28), followed by a 28-day treatment-free (days 29-56), and a second dosing phase of 3 days (days 57-59). 4-CloT showed some effects on the integrity of the DNA as measured by the comet assay in liver and urinary bladder but not in peripheral blood or jejunum. However, for liver and urinary bladder histopathological changes were observed. An increase in the frequency of micronuclei in peripheral blood was seen in parallel to a dose-dependent increase of reticulocytes and methemoglobin. Therefore, impact from a compensatory erythropoiesis on micronucleation cannot be excluded. Interestingly, no increase in the frequency of RETCD59- and RBCCD59- was observed in the Pig-a assay.
Assuntos
Dano ao DNA , Jejuno/efeitos dos fármacos , Fígado/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mutagênicos/toxicidade , Toluidinas/toxicidade , Bexiga Urinária/efeitos dos fármacos , Animais , Ensaio Cometa , Relação Dose-Resposta a Droga , Jejuno/patologia , Fígado/patologia , Masculino , Testes para Micronúcleos , Especificidade de Órgãos , Ratos Wistar , Bexiga Urinária/patologiaRESUMO
Glycine is a key rate-limiting component of heme biosynthesis in erythropoietic cells, where the high intracellular glycine demand is primarily supplied by the glycine transporter 1 (GlyT1). The impact of intracellular glycine restriction after GlyT1 inhibition on hematopoiesis and iron regulation is not well established. We investigated the effects of a potent and selective inhibitor of GlyT1, bitopertin, on erythropoiesis and iron homeostasis in rats. GlyT1 inhibition significantly affected erythroid heme biosynthesis, manifesting as microcytic hypochromic regenerative anemia with a 20% steady-state reduction in hemoglobin. Reduced erythropoietic iron utilization was characterized by down-regulation of the transferrin receptor 1 (TfR1) on reticulocytes and modest increased iron storage in the spleen. Hepatic hepcidin expression was not affected. However, under the condition of reduced heme biosynthesis with reduced iron reutilization and increased storage iron, hepcidin at the lower and higher range of normal showed a striking role in tissue distribution of iron. Rapid formation of iron-positive inclusion bodies (IBs) was observed in circulating reticulocytes, with an ultrastructure of iron-containing polymorphic mitochondrial remnants. IB or mitochondrial iron accumulation was absent in bone marrow erythroblasts. In conclusion, GlyT1 inhibition in rats induced a steady-state microcytic hypochromic regenerative anemia and a species-specific accumulation of uncommitted mitochondrial iron in reticulocytes. Importantly, this glycine-restricted anemia provides no feedback signal for increased systemic iron acquisition and the effects reported are pathogenetically distinct from systemic iron-overload anemias and erythropoietic disorders such as acquired sideroblastic anemia.