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Animals frequently eat less after a test-article treatment in nonclinical toxicological studies, and it can be difficult to distinguish test article-derived toxicities from secondary changes related to this reduced food intake. Therefore, in this study, we restricted the food intake of cynomolgus monkeys (Cambodian, male, n=2 or 3, 48 ± 3 months old) to 25% of the control for two weeks and evaluated the effects on toxicological parameters (general conditions, body weight, electrocardiography, urinalysis, hematology, blood chemistry, bone marrow analysis, pathological examination). After 2 weeks, the monkeys exhibited decreases in bone marrow erythropoiesis (e.g., decreases in reticulocytes and bone marrow erythrocytes), as well as glycogenesis induction (e.g., increase in aspartate aminotransferase (AST)) and malnutrition (e.g., decrease in triglyceride and systemic adipocytes atrophy). Additionally, histopathological analysis revealed granuloma and inflammatory cell infiltration in coronary fat, which had never been found in previous food restriction studies using other animal species. These findings will enable researchers to more accurately evaluate the toxicological risks of test articles that simultaneously induce food intake reduction.
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Ingestión de Alimentos , Alimentos , Masculino , Animales , Macaca fascicularis , Peso Corporal , ElectrocardiografíaRESUMEN
BACKGROUND: Emicizumab, a factor (F) VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment option for people with hemophilia A (PwHA). However, a small proportion of PwHA still experience bleeds even under emicizumab prophylaxis, as observed in the long-term outcomes of clinical studies. A more potent BsAb may be desirable for such patients. OBJECTIVES: To identify a potent BsAb to FIXa and FX, NXT007, surpassing emicizumab by in vitro and in vivo evaluation. METHODS: New pairs of light chains for emicizumab's heavy chains were screened from phage libraries, and subsequent antibody optimization was performed. For in vitro evaluation, thrombin generation assays were performed with hemophilia A plasma. In vivo hemostatic activity was evaluated in a nonhuman primate model of acquired hemophilia A. RESULTS: NXT007 exhibited an in vitro thrombin generation activity comparable to the international standard activity of FVIII (100 IU/dL), much higher than emicizumab, when triggered by tissue factor. NXT007 also demonstrated a potent in vivo hemostatic activity at approximately 30-fold lower plasma concentrations than emicizumab's historical data. In terms of dose shift between NXT007 and emicizumab, the in vitro and in vivo results were concordant. Regarding pharmacokinetics, NXT007 showed lower in vivo clearance than those shown by typical monoclonal antibodies, suggesting that the Fc engineering to enhance FcRn binding worked well. CONCLUSION: NXT007, a potent BsAb, was successfully created. Nonclinical results suggest that NXT007 would have a potential to keep a nonhemophilic range of coagulation potential in PwHA or to realize more convenient dosing regimens than emicizumab.
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Anticuerpos Biespecíficos , Hemofilia A , Hemostáticos , Humanos , Hemostáticos/farmacología , Hemostáticos/uso terapéutico , Trombina/metabolismo , Hemostasis , Coagulación Sanguínea , Factor VIIIRESUMEN
Increased incidence of adrenal pheochromocytoma is frequently encountered in rat carcinogenicity studies. In some of the studies, the finding is judged to be due to a rat-specific mechanism of carcinogenesis caused by a disturbance of calcium homeostasis. However, direct evidence that the proliferation of chromaffin cells in the adrenal medulla is induced solely by hypercalcemia is not available. In this study, calcium gluconate was intravenously infused for 7 days to rat chromaffin cells by a tail cuff method, and cumulative labeling with bromodeoxyuridine (BrdU) was carried out to evaluate the proliferative activity. The serum calcium concentration was dose-dependently increased, and a high calcium concentration was stably sustained from day 2 to 7. In the adrenal medulla, BrdU-positive chromaffin cells increased in the calcium gluconate-treated animals, and the BrdU-labeling index increased in a dose-dependent manner. In addition, an increased BrdU-labeling index of chromaffin cells was shown to correlate with the serum calcium concentration. Our results demonstrate that hypercalcemia directly enhances the proliferative activity of chromaffin cells and that the proliferative activity is correlated with the serum calcium concentration.
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Currently, ERY974, a humanized IgG4 bispecific T cell-redirecting antibody recognizing glypican-3 and CD3, is in phase I clinical trials. After a first-in-human clinical trial of an anti-CD28 agonist monoclonal antibody resulting in severe life-threatening adverse events, the minimal anticipated biological effect level approach has been considered for determining the first-in-human dose of high-risk drugs. Accordingly, we aimed to determine the first-in-human dose of ERY974 using both the minimal anticipated biological effect level and no observed adverse effect level approaches. In the former, we used the 10% effective concentration value from a cytotoxicity assay using the huH-1 cell line with the highest sensitivity to ERY974 to calculate the first-in-human dose of 4.9 ng/kg, at which maximum drug concentration after 4 h of intravenous ERY974 infusion was equal to the 10% effective concentration value. To determine the no observed adverse effect level, we conducted a single-dose study in cynomolgus monkeys that were intravenously infused with ERY974 (0.1, 1, and 10 µg/kg). The lowest dose of 0.1 µg/kg was determined as the no observed adverse effect level, and the first-in-human dose of 3.2 ng/kg was calculated, considering body surface area and species difference. For the phase I clinical trial, we selected 3.0 ng/kg as a starting dose, which was lower than the first-in-human dose calculated from both the no observed adverse effect level and minimal anticipated biological effect level. Combining these two methods to determine the first-in-human dose of strong immune modulators such as T cell-redirecting antibodies would be a suitable approach from safety and efficacy perspectives.Clinical trial registration: JapicCTI-194805/NCT05022927.
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Anticuerpos Biespecíficos , Glipicanos , Linfocitos T , Anticuerpos Biespecíficos/administración & dosificación , Relación Dosis-Respuesta Inmunológica , Glipicanos/inmunología , Humanos , Linfocitos T/inmunologíaRESUMEN
Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.
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Adenosina Trifosfato , Neoplasias , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Neoplasias , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológico , Microambiente Tumoral , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell-redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid "on-target off-tumor" toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G-structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein-4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.
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Anticuerpos Biespecíficos/uso terapéutico , Glipicanos/inmunología , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Biespecíficos/farmacocinética , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Complejo CD3/metabolismo , Citocinas/metabolismo , Humanos , Inmunocompetencia/efectos de los fármacos , Inyecciones Intravenosas , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Macaca fascicularis , Ratones Transgénicos , Esteroides/farmacología , Esteroides/uso terapéutico , Linfocitos T/efectos de los fármacosRESUMEN
To investigate the contribution of intestinal calcium (Ca) absorption to 1,25-dihydroxyvitamin D(3) (1,25(OH) (2)D(3))-induced Ca action, we assessed parameters related to Ca metabolism after a single dosing of 1,25(OH)(2)D (3) in the total parenteral nutrition (TPN) solution or 5% D-mannitol (MAN) solution treatment with rats. Animals were divided into 6 groups (vehicle, 100 microg/kg p.o. and 25 microg/kg i.v.; n=8) in Experiment 1 and 8 groups (vehicle, 1, 10 and 100 microg/kg p.o.; n=6) in Experiment 2 at TPN or MAN solution treatment. In both experiments, the parameters related to Ca metabolisms, urinary Ca and urinary deoxypyridinoline on 0-24 hr or serum Ca, osteocalcin and parathyroid hormone at 24 hr were measured after 1,25(OH)(2)D (3) dosing. 1,25(OH)(2)D(3)-related increased urinary Ca or serum Ca were observed in both experiments. Decrease rates in change of urinary Ca in TPN solution treatment rats were 36.3% (100 microg/kg p.o.) or 47.1% (25 microg/kg i.v.) of MAN solution treatment rats in Experiment 1, and 29.0% (1 microg/kg), 56.2% (10 microg/kg) or 35.3% (100 microg/kg) of MAN solution treatment rats in Experiment 2. Decrease rates in change of serum Ca at 72 hr in TPN solution treatment rats were 57.3% (100 microg/kg p.o.) or 44.5% (25 microg/kg i.v.) of MAN solution treatment rats in Experiment 1, and were 57.0% (100 microg/kg) of MAN solution treatment rats in Experiment 2. There were no differences in the change of serum Ca in the 1,25(OH)(2)D(3) 1 or 10-microg/kg group in Experiment 2. Our results suggest that differences in urinary Ca or serum Ca between MAN solution treatment rats and TPN solution treatment rats express the contribution of intestinal Ca absorption to 1,25(OH)(2)D(3)-induced Ca action in the conditions of the study.
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Calcitriol/farmacología , Calcio/metabolismo , Absorción Intestinal , Nutrición Parenteral Total , Aminoácidos/orina , Animales , Peso Corporal , Femenino , Manitol/administración & dosificación , Osteocalcina/sangre , Hormona Paratiroidea/sangre , Ratas , Ratas Sprague-DawleyRESUMEN
This study was conducted to evaluate the feasibility of total parenteral nutrition (TPN) in rats using continuous intravenous infusion (tail cuff method) via the posterior vena cava. A catheter was inserted into the posterior vena cava from the femoral vein of 10 females (Experiment 1) and 16 females (Experiment 2). The depth of the inserted catheter from the femoral vein was set at 4.5 cm for Experiment 1 and was set at 6 cm for Experiment 2. The test animals were divided into two groups in each experiment: a 5% D-Mannitol (MAN) group and a TPN group. In Experiment 1, TPN rats showed macroscopic lesions (edema in peritoneum, increased collateral vasculature, induration in perivenous tissue, and thrombus) at the tip of the catheter. The diameter of the posterior vena cava (2.86 +/- 0.16 mm, mean +/- S.D.) was significantly greater than that of the anterior vena cava (2.45 +/- 0.22 mm) in 10 rats of Experiment 1. In Experiment 2, TPN rats showed no abnormalities at necropsy. Our findings suggest that TPN administered via the posterior vena cava in Sprague-Dawley rats requires the catheter to be inserted to a depth of 6 cm from the femoral vein. We hypothesize that this is because it is inserted to the level of the renal vein branch where the diameter of the posterior vena cava may be greatest.
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Cateterismo Periférico/métodos , Nutrición Parenteral Total/métodos , Animales , Análisis Químico de la Sangre , Peso Corporal/efectos de los fármacos , Femenino , Infusiones Intravenosas , Nutrición Parenteral Total/efectos adversos , Ratas , Ratas Sprague-Dawley , Cola (estructura animal) , Vena Cava Inferior/fisiologíaRESUMEN
Previously we reported that dietary supplemental vitamin B6 (B6) reduced colon tumorigenesis and cell proliferation in mice receiving azoxymethane (AOM) for 22 weeks. This study was conducted to examine the influence of short-term consumption (5 weeks) of diets containing graded levels of B6 and fat on colonic cell proliferation in mice with or without receiving AOM. In experiment 1, mice were fed the 10% corn oil diet containing 1, 7, 14, 35 or 70 mg pyridoxine HCl/kg, and received weekly injections of AOM for the initial 3 weeks. In experiment 2, mice were fed 5 or 20% corn oil diet containing 1, 7, 14 or 35 mg pyridoxine HCl/kg, and received weekly injections of AOM or saline for the initial 3 weeks. In experiment 1, supplemental B6 caused a dose-dependent reduction of colon aberrant crypt foci and cell proliferation (BrdU-labeling index) among the 1-14 mg pyridoxine HCl/kg. There was no influence of B6 on these parameters among 14-70 mg pyridoxine HCl/kg. Immunohistochemical analysis of apoptosis labeling by TUNEL method indicated no influence of dietary B6 on colon apoptosis. In experiment 2, supplemental B6 significantly reduced colon cell proliferation regardless of AOM injection. This inhibitory effect on cell proliferation was markedly enhanced by a high-fat diet, but slightly affected by AOM treatment. The results suggest that dietary supplemental B6 inhibits colon cell proliferation from the early stage of colon carcinogenesis, and a high-fat diet markedly enhances the inhibitory effect.
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Proliferación Celular/efectos de los fármacos , Colon/efectos de los fármacos , Grasas de la Dieta/administración & dosificación , Piridoxina/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Azoximetano/administración & dosificación , Azoximetano/toxicidad , Peso Corporal/efectos de los fármacos , Bromodesoxiuridina/metabolismo , Colon/metabolismo , Colon/patología , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/patología , Neoplasias del Colon/prevención & control , Suplementos Dietéticos , Relación Dosis-Respuesta a Droga , Ingestión de Alimentos/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos ICRRESUMEN
Although vitamin B6 has been supposed to have anti-inflammatory effects, the molecular mechanism is not fully understood. To explore the mechanism of anti-inflammatory effects of vitamin B6, we have examined the effect of vitamin B6 on lipopolysaccharide (LPS)-stimulated inflammatory response in RAW 264.7 macrophages. This study demonstrated that vitamin B6 (pyridoxal) pretreatment of RAW cells inhibited LPS-induced expression of iNOS and COX-2 at the mRNA and protein levels. Vitamin B6 inhibited LPS-induced nuclear translocation of the NF-kappaB, the proinflammatory transcription factor, with reduction of the extent of LPS-induced IkappaBalpha degradation in RAW cells. Although vitamin B6 did not affect cellular proteasome activity, in vitro phosphorylation analysis with glutathione S-transferase-fused IkappaBalpha has shown that vitamin B6 suppressed LPS-induced IkappaB kinase activation. Furthermore, we demonstrated that elevating dietary vitamin B6 suppressed NO production in vivo in response to LPS administration. These observations suggest that the anti-inflammatory effect of vitamin B6 is mediated by suppression of NF-kappaB activation.
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Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , FN-kappa B/antagonistas & inhibidores , Vitamina B 6/farmacología , Animales , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Masculino , Ratones , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piridoxal/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
This review describes current research on the preventive effect of dietary vitamin B(6) against colon tumorigenesis and its possible mechanisms. Studies in cell culture have demonstrated that high levels of vitamin B(6) suppress growth of some cancer cells. From these studies it has been considered that supraphysiological doses of vitamin B(6) suppress tumor growth and metastasis. However, recent rodent study has indicated that azoxymethane-induced colon tumorigenesis in mice is suppressed by moderate doses of dietary vitamin B(6.) Epidemiological studies also support an inverse relationship between vitamin B(6) intake and colon cancer risk. Potential mechanisms underlying the preventive effect of dietary vitamin B(6) have been suggested to include the suppression of cell proliferation, oxidative stress, nitric oxide (NO) synthesis, and angiogenesis.
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División Celular/efectos de los fármacos , Neoplasias del Colon/prevención & control , Dieta , Neovascularización Fisiológica/efectos de los fármacos , Vitamina B 6/administración & dosificación , Animales , Humanos , Ratones , Óxido Nítrico , Estrés OxidativoRESUMEN
Recently we reported that the supplementation of vitamin B6 to low vitamin B6 diet caused suppression in colon tumorigenesis and cell proliferation of azoxymethane-treated mice in a dose-dependent manner among 1, 7, and 14 mg pyridoxine HCl/kg diet (J. Nutr. 131: 2204-2207, 2001). To examine the mechanism of the anticolon tumor effect of vitamin B6, male ICR mice were fed the diet containing 1, 7, 14, and 35 mg pyridoxine HCl/kg diet for 22 wk and simultaneously given a weekly injection of azoxymethane for an initial 10 wk. The supplementation of vitamin B6 to a low vitamin B6 diet (1 mg pyridoxine HCl/kg) suppressed the levels of colonic 8-hydroxyguanosine and 4-hydroxynonenal and inducible nitric oxide synthase protein. The results suggest that the preventive effect of vitamin B6 against colon tumorigenesis is at least in part mediated by reducing oxidative stress and nitric oxide production.
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Adyuvantes Inmunológicos/metabolismo , Aldehídos/metabolismo , Azoximetano/administración & dosificación , Carcinógenos/administración & dosificación , Neoplasias del Colon/dietoterapia , Guanosina/análogos & derivados , Guanosina/metabolismo , Óxido Nítrico Sintasa/metabolismo , Vitamina B 6/administración & dosificación , Análisis de Varianza , Animales , Reactivos de Enlaces Cruzados/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Óxido Nítrico Sintasa de Tipo IIRESUMEN
Totally implantable catheter animal models are considered useful for pharmacological and toxicological studies. In this report, we assessed the feasibility of using an indwelling vascular access port (VAP) in rats for long-term evaluation of repeated and intermittent dose toxicity studies. In Experiment 1, the VAP devices were implanted in male and female rats and a saline solution administered intravenously via the posterior vena cava for 2 weeks (4 ml/kg, 2 ml/min, 5 times/week, 10 times total). General conditions, body weight and blood chemistry showed no toxicological changes compared with the rats in the non-implanted, non-treated group. Hematology changes such as transient increases in peripheral blood reticulocytes and eosinophils were noted post-implantation. In pathology, proliferation of the endothelium at the site of VAP implantation and perivascular inflammatory cell infiltration including eosinophils in lung were noted at the end of the treatment period. Moreover, we found that the lumbar area is more suitable for VAP implantation than the back of neck for young, still growing rats. Experiment 2 included a 1-month intravenous intermittent dose (4 ml/kg, 2 ml/min, 1 time/week, 5 times total) toxicity study in VAP-implanted rats followed by a 1-month recovery period conducted under Good Laboratory Practice (GLP) regulations. The results suggested that an animal model with implanted VAP is useful for intermittent intravenous dosing of drugs. Moreover, VAP implantation in animals is expected to be extrapolated to use VAP in humans in clinical studies.