Native Oxyntomodulin Has Significant Glucoregulatory Effects Independent of Weight Loss in Obese Humans With and Without Type 2 Diabetes.

Oxyntomodulin (OXM), an enteroendocrine hormone, causes appetite suppression, increased energy expenditure, and weight loss in obese humans via activation of GLP-1 and glucagon receptors. However, the effects of OXM on glucose homeostasis remain ill defined. To address this gap, we evaluated the effects of an i.v. infusion of native OXM on insulin secretion rates (ISRs) and glycemic excursion in a graded glucose infusion (GGI) procedure in two separate randomized, placebo (PBO)-controlled, single-dose crossover trials in 12 overweight and obese subjects without diabetes and in 12 obese subjects with type 2 diabetes mellitus (T2DM), using the GLP-1 analog liraglutide (LIRA) as a comparator in T2DM. In both groups, in the GGI, 3.0 pmol/kg/min of OXM significantly increased ISR and blunted glycemic excursion relative to PBO. In T2DM, the effects of OXM were comparable to those of LIRA, including restoration of ß-cell glucose responsiveness to that of nonobese subjects without diabetes. Our findings indicate that native OXM significantly augments glucose-dependent insulin secretion acutely in obese subjects with and without diabetes, with effects comparable to pharmacologic GLP-1 receptor activation and independent of weight loss. Native OXM has potential to improve hyperglycemia via complementary and independent induction of insulin secretion and weight loss.

Safety assessment of drug metabolites: implications of regulatory guidance and potential application of genetically engineered mouse models that express human P450s.

Species differences in drug metabolism present two challenges that may confound the nonclinical safety assessment of candidate drugs. The first challenge is encountered when metabolites are formed uniquely or disproportionately in humans. Another challenge is understanding the human relevance of toxicities associated with metabolites formed uniquely or disproportionately in a nonclinical species. One potential approach to minimize the impact of metabolite related challenges is to consider genetically engineered mouse models that express human P450 enzymes. Human P450 expressing mouse models may have the ability to generate major human metabolites and eliminate or reduce the formation of mouse specific metabolites. Prior to determining the utility of any particular model, it is important to qualify by characterizing protein expression, establishing whether the model generates an in vivo metabolite profile more closely related to that of humans than the wild-type mouse, verifying genetic stability, and evaluating animal health. When compared to the current strategy for handling metabolite challenges (i.e., direct administration of metabolite), identifying an appropriate human P450 expressing model could provide a number of benefits. Such benefits include improved scientific relevance of the evaluation, decreased resource needs, and a possible reduction in the number of animals used. These benefits may ultimately improve the quality and speed by which promising new drug candidates are developed and delivered to patients.

CYP2C75-involved autoinduction of metabolism in rhesus monkeys of methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), a bradykinin B1 receptor antagonist.

After oral treatment (once daily) for 4 weeks with the potent bradykinin B(1) receptor antagonist methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), rhesus monkeys (Macaca mulatta) exhibited significantly reduced systemic exposure of the compound in a dose-dependent manner, suggesting an occurrence of autoinduction of MK-0686 metabolism. This possibility is supported by two observations. 1) MK-0686 was primarily eliminated via biotransformation in rhesus monkeys, with oxidation on the chlorophenyl ring as one of the major metabolic pathways. This reaction led to appreciable formation of a dihydrodiol (M11) and a hydroxyl (M13) product in rhesus liver microsomes supplemented with NADPH. 2) The formation rate of these two metabolites determined in liver microsomes from MK-0686-treated groups was > or = 2-fold greater than the value for a control group. Studies with recombinant rhesus P450s and monoclonal antibodies against human P450 enzymes suggested that CYP2C75 played an important role in the formation of M11 and M13. The induction of this enzyme by MK-0686 was further confirmed by a concentration-dependent increase of its mRNA in rhesus hepatocytes, and, more convincingly, the enhanced CYP2C proteins and catalytic activities toward CYP2C75 probe substrates in liver microsomes from MK-0686-treated animals. Furthermore, a good correlation was observed between the rates of M11 and M13 formation and hydroxylase activities toward probe substrates determined in a panel of liver microsomal preparations from control and MK-0686-treated animals. Therefore, MK-0686, both a substrate and inducer for CYP2C75, caused autoinduction of its own metabolism in rhesus monkeys by increasing the expression of this enzyme.

Evaluation of ubiquinone concentration and mitochondrial function relative to cerivastatin-induced skeletal myopathy in rats.

As a class, hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors can potentially cause skeletal myopathy. One statin, cerivastatin, has recently been withdrawn from the market due to an unacceptably high incidence of rhabdomyolysis. The mechanism underlying statin-induced myopathy is unknown. This paper sought to investigate the relationship among statin-induced myopathy, mitochondrial function, and muscle ubiquinone levels. Rats were administered cerivastatin at 0.1, 0.5, and 1.0 (mg/kg)/day or dose vehicle (controls) by oral gavage for 15 days. Samples of type I-predominant skeletal muscle (soleus) and type II-predominant skeletal muscle [quadriceps and extensor digitorum longus (EDL)], and blood were collected on study days 5, 10, and 15 for morphological evaluation, clinical chemistry, mitochondrial function tests, and analysis of ubiquinone levels. No histological changes were observed in any of the animals on study days 5 or 10, but on study day 15, mid- and high-dose animals had necrosis and inflammation in type II skeletal muscle. Elevated creatine kinase (CK) levels in blood (a clinical marker of myopathy) correlated with the histopathological diagnosis of myopathy. Ultrastructural characterization of skeletal muscle revealed disruption of the sarcomere and altered mitochondria only in myofibers with degeneration, while adjacent myofibers were unaffected and had normal mitochondria. Thus, mitochondrial effects appeared not to precede myofiber degeneration. Mean coenzyme Q9 (CoQ9) levels in all dose groups were slightly decreased relative to controls in type II skeletal muscle, although the difference was not significantly different in most cases. Mitochondrial function in skeletal muscle was not affected by the changes in ubiquinone levels. The ubiquinone levels in high-dose-treated animals exhibiting myopathy were not significantly different from low-dose animals with no observable toxic effects. Furthermore, ubiquinone levels did not correlate with circulating CK levels in treated animals. The results of this study suggest that neither mitochondrial injury, nor a decrease in muscle ubiquinone levels, is the primary cause of skeletal myopathy in cerivastatin-dosed rats.

Physiologically based pharmacokinetic (PBPK) models for nasal tissue dosimetry of organic esters: assessing the state-of-knowledge and risk assessment applications with methyl methacrylate and vinyl acetate.

Mathematical models have been developed to describe nasal epithelial tissue dosimetry with two compounds, vinyl acetate (VA) and methyl methacrylate (MMA), that cause toxicity in these tissues These models couple computational fluid dynamics (CFD) calculations that map airflow patterns within the nose with physiologically based pharmacokinetic (PBPK) models that integrate diffusion, metabolism, and tissue interactions of these compounds. Dose metrics estimated in these models for MMA and VA, respectively, were rates of MMA metabolism per volume of tissue and alterations in pH in target tissues associated with VA hydrolysis and metabolism. In this article, four scientists who have contributed significantly to development of these models describe the many similarities and relatively few differences between the MMA and VA models. Some differences arise naturally because of differences in target tissues, in the calculated measures of tissue dose, and in the modes of action for highly extracted vapors (VA) compared with poorly extracted vapors (MMA). A difference in the approach used to estimate metabolic parameters from human tissues provides insights into interindividual extrapolation and identifies opportunities for studies with human nasal tissues to enhance current risk assessments. In general, the differences in model structure for these two esters were essential for describing the biology of the observed responses and in accounting for the different measures of target tissue dose. This article is intended to serve as a guide for understanding issues of optimum model structure and optimal data sources for these nasal tissue dosimetry models. We also hope that it leads to greater international acceptance of these hybrid CFD/PBPK modeling approaches for improving risk assessment for many nasal toxicants. In general, these models predict either equivalent (VA) or lower (MMA) nasal tissue doses in humans compared with tissue doses at equivalent exposure concentrations in rats.

Use of a hybrid computational fluid dynamics and physiologically based inhalation model for interspecies dosimetry comparisons of ester vapors.

Numerous inhalation studies have demonstrated that exposure to high concentrations of a wide range of volatile acids and esters results in cytotoxicity to the nasal olfactory epithelium. Previously, a hybrid computational fluid dynamics (CFD) and physiologically based pharmacokinetic (PBPK) dosimetry model was constructed to estimate the regional tissue dose of organic acids in the rodent and human nasal cavity. This study extends this methodology to a representative volatile organic ester, ethyl acrylate (EA). An in vitro exposure of explants of rat olfactory epithelium to EA with and without an esterase inhibitor demonstrated that the organic acid, acrylic acid, released by nasal esterases is primarily responsible for the olfactory cytotoxicity. Estimates of the steady-state concentration of acrylic acid in olfactory tissue were made for the rat nasal cavity by using data from a series of short-term in vivo studies and from the results of CFD-PBPK computer modeling. Appropriate parameterization of the CFD-PBPK model for the human nasal cavity and to accommodate human systemic anatomy, metabolism, and physiology allowed interspecies dose comparisons. The CFD-PBPK model simulations indicate that the olfactory epithelium of the human nasal cavity is exposed to at least 18-fold lower tissue concentrations of acid released from EA than the olfactory epithelium of the rat nasal cavity under the same exposure conditions. The magnitude of this difference varies with the specific exposure scenario that is simulated and with the specific dataset of human esterase activity used for the simulations. The increased olfactory tissue dose in rats relative to humans may be attributed to both the vulnerable location of the rodent olfactory tissue (comprising greater than 50% of the nasal cavity) and the high concentration of rat olfactory esterase activity (comparable to liver esterase activity) relative to human olfactory tissue. These studies suggest that the human olfactory epithelium is protected from vapors of organic esters significantly better than rat olfactory epithelium due to substantive differences in nasal anatomy, nasal and systemic metabolism, systemic physiology, and air flow. Although the accumulation of acrylic acid in the nasal tissues may be a primary concern for nasal irritation and human risk assessment, acute animal inhalation studies to evaluate lethality (LD50-type studies) conducted at very high vapor concentrations of ethyl acrylate indicated that a different mechanism is primarily responsible for mortality. The rodent studies demonstrated that systemic tissue nonprotein sulfhydryl depletion is a primary cause of death at exposure concentrations more than two orders of magnitude above the concentrations that induce nasal irritation. The CFD-PBPK model adequately simulated the severe depletion of glutathione in systemic tissues (e.g., liver and lung) associated with acute inhalation exposures in the 500-1000 ppm range. These results indicate that the CFD-PBPK model can simulate both the low-dose nasal tissue dosimetry associated with irritation and the high-dose systemic tissue dosimetry associated with mortality. In addition, the comparison of simulation results for ethyl acetate and acetone to nasal deposition data suggests that the CFD-PBPK model has general utility as a tool for dosimetry estimates for a wide range of other esters and slowly metabolized vapors.