In vitro CYP450 enzyme down-regulation by GLP-1/glucagon co-agonist does not translate to observed drug-drug interactions in the clinic.

NN1177 is a glucagon/glucagon-like peptide 1 receptor co-agonist investigated for chronic weight management and treatment of non-alcoholic steatohepatitis. Here, we show concentration-dependent down-regulation of cytochrome P450 enzymes using freshly isolated human hepatocytes treated with this linear 29-amino acid peptide. Notably, reductions in CYP3A4 mRNA expression (57.2-71.7%) and activity (18.5-51.5%) were observed with a clinically-relevant concentration of 100 nM NN1177. CYP1A2 and CYP2B6 were also affected, but to a lesser extent. Physiological-based pharmacokinetic modelling simulated effects on CYP3A4 and CYP1A2 probe substrates (midazolam and caffeine, respectively) and revealed potential safety concerns related to drug-drug interactions (DDIs). To investigate the clinical relevance of observed in vitro CYP down-regulation, a phase 1 clinical cocktail study was initiated to assess the DDI potential. The study enrolled 45 study participants (BMI 23.0-29.9 kg/m2) to receive a Cooperstown 5+1 cocktail (midazolam, caffeine, omeprazole, dextromethorphan, and S-warfarin/vitamin K) alone and following steady state NN1177 exposure. The analysis of pharmacokinetic profiles for the cocktail drugs showed no significant effect from the co-administration of NN1177 on AUC0-inf for midazolam or S-warfarin. Omeprazole, caffeine, and dextromethorphan generally displayed decreases in AUC0-inf and Cmax following NN1177 co-administration. Thus, the in vitro observations were not reflected in the clinic. These findings highlight remaining challenges associated with standard in vitro systems used to predict DDIs for peptide-based drugs as well as the complexity of DDI trial design for these modalities. Overall, there is an urgent need for better pre-clinical models to assess potential drug-drug interaction risks associated with therapeutic peptides during drug development. Significance Statement This study highlights significant challenges associated with assessing drug-drug interaction risks for therapeutic peptides using in vitro systems, since potential concerns identified by standard assays did not translate to the clinical setting. Further research is required to guide investigators involved in peptide-based drug development towards better non-clinical models in order to more accurately evaluate potential drug-drug interactions.

Population pharmacokinetics and covariate analysis of Sym004, an antibody mixture against the epidermal growth factor receptor, in subjects with metastatic colorectal cancer and other solid tumors.

Sym004 is an equimolar mixture of two monoclonal antibodies, futuximab and modotuximab, which non-competitively block the epidermal growth factor receptor (EGFR). Sym004 has been clinically tested for treatment of solid tumors. The present work characterizes the non-linear pharmacokinetics (PK) of Sym004 and its constituent antibodies and investigates two types of covariate models for interpreting the interindividual variability of Sym004 exposure. Sym004 serum concentration data from 330 cancer patients participating in four Phase 1 and 2 trials (n = 247 metastatic colorectal cancer, n = 87 various types advanced solid tumors) were pooled for non-linear mixed effects modeling. Dose regimens of 0.4-18 mg/kg Sym004 dosed by i.v. infusion weekly or every 2nd week were explored. The PK profiles for futuximab and modotuximab were parallel, and the parameter values for their population PK models were similar. The PK of Sym004 using the sum of the serum concentrations of futuximab and modotuximab was well captured by a 2-compartment model with parallel linear and saturable, Michaelis-Menten-type elimination. The full covariate model including all plausible covariates included in a single step showed no impact on Sym004 exposure of age, Asian race, renal and hepatic function, tumor type and previous anti-EGFR treatments. The reduced covariate model contained statistically and potentially clinically significant influences of body weight, albumin, sex and baseline tumor size. Population PK modeling and covariate analysis of Sym004 were feasible using the sum of the serum concentrations of the two constituent antibodies. Full and reduced covariate models provided insights into which covariates may be clinically relevant for dose modifications and thus may need further exploration.

Sym015: A Highly Efficacious Antibody Mixture against MET-Amplified Tumors.

Purpose: Activation of the receptor tyrosine kinase MET is associated with poor clinical outcome in certain cancers. To target MET more effectively, we developed an antagonistic antibody mixture, Sym015, consisting of two humanized mAbs directed against nonoverlapping epitopes of MET.Experimental Design/Results: We screened a large panel of well-annotated human cancer cell lines and identified a subset with highly elevated MET expression. In particular, cell lines of lung cancer and gastric cancer origin demonstrated high MET expression and activation, and Sym015 triggered degradation of MET and significantly inhibited growth of these cell lines. Next, we tested Sym015 in patient- and cell line-derived xenograft models with high MET expression and/or MET exon 14 skipping alterations, and in models harboring MET amplification as a mechanism of resistance to EGFR-targeting agents. Sym015 effectively inhibited tumor growth in all these models and was superior to an analogue of emibetuzumab, a monoclonal IgG4 antibody against MET currently in clinical development. Sym015 also induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting that secondary effector functions contribute to the efficacy of Sym015.Retrospectively, all responsive, high MET-expressing models were scored as highly MET-amplified by in situ hybridization, pointing to MET amplification as a predictive biomarker for efficacy. Preclinical toxicology studies in monkeys showed that Sym015 was well tolerated, with a pharmacokinetic profile supporting administration of Sym015 every second or third week in humans.Conclusions: The preclinical efficacy and safety data provide a clear rationale for the ongoing clinical studies of Sym015 in patients with MET-amplified tumors. Clin Cancer Res; 23(19); 5923-35. ©2017 AACR.

Chronic Hyperinsulinaemic Hypoglycaemia in Rats Is Accompanied by Increased Body Weight, Hyperleptinaemia, and Decreased Neuronal Glucose Transporter Levels in the Brain.

The brain is vulnerable to hypoglycaemia due to a continuous need of energy substrates to meet its high metabolic demands. Studies have shown that severe acute insulin-induced hypoglycaemia results in oxidative stress in the rat brain, when neuroglycopenia cannot be evaded despite increased levels of cerebral glucose transporters. Compensatory measures in the brain during chronic insulin-induced hypoglycaemia are less well understood. The present study investigated how the brain of nondiabetic rats copes with chronic insulin-induced hypoglycaemia for up to eight weeks. Brain level of different substrate transporters and redox homeostasis was evaluated. Hyperinsulinaemia for 8 weeks consistently lowered blood glucose levels by 30-50% (4-6 mM versus 7-9 mM in controls). The animals had increased food consumption, body weights, and hyperleptinaemia. During infusion, protein levels of the brain neuronal glucose transporter were decreased, whereas levels of lipid peroxidation products were unchanged. Discontinued infusion was followed by transient systemic hyperglycaemia and decreased food consumption and body weight. After 4 weeks, plasma levels of lipid peroxidation products were increased, possibly as a consequence of hyperglycaemia-induced oxidative stress. The present data suggests that chronic moderate hyperinsulinaemic hypoglycaemia causes increased body weight and hyperleptinaemia. This is accompanied by decreased neuronal glucose transporter levels, which may be leptin-induced.

Toxicological Effects during and following Persistent Insulin-Induced Hypoglycaemia in Healthy Euglycaemic Rats.

New insulin analogues with a longer duration of action and a 'peakless' pharmacokinetic profile have been developed to improve efficacy, safety and convenience for patients with diabetes. During non-clinical development, according to regulatory guidelines, these analogues are tested in healthy euglycaemic rats rendering them persistently hypoglycaemic. Little is known about the effect of persistent (24 hr/day) insulin-induced hypoglycaemia (IIH) in rats, complicating interpretation of results in pre-clinical studies with new longer-acting insulin analogues. In this study, we investigated the effects of persistent IIH and their reversibility in euglycaemic rats. Histopathological changes in insulin-infused animals included partly reversible axonal and reversible myofibre degeneration in peripheral nerve and skeletal muscle tissue, respectively, as well as reversible pancreatic islet atrophy and partly reversible increase in unilocular adipocytes in brown adipose tissue. Additionally, results suggested increased gluconeogenesis. The observed hyperphagia, the pancreatic, peripheral nerve and skeletal muscle changes were considered related to the hypoglycaemia. Cessation of insulin infusion resulted in transient hyperglycaemia, decreased food consumption and body-weight loss before returning to control levels. The implications for the interpretation of non-clinical studies with long-acting insulin analogues are discussed.

Histopathological nerve and skeletal muscle changes in rats subjected to persistent insulin-induced hypoglycemia.

New insulin analogues with a longer duration of action and a flatter pharmacodynamic profile are developed to improve convenience and safety for diabetic patients. During the nonclinical development of such analogues, safety studies must be conducted in nondiabetic rats, which consequently are rendered chronically hypoglycemic. A rat comparator model using human insulin would be valuable, as it would enable differentiation between effects related to either persistent insulin-induced hypoglycemia (IIH) or a new analogue per se. Such a model could alleviate the need for an in-study-comparator and thereby reduce the number of animals used during development. Thus, the aims of the present study were i) to develop a preclinical animal model of persistent hypoglycemia in rats using human insulin infusion for four weeks and ii) to investigate histopathological changes in sciatic nerves and quadriceps femoris muscle tissue, as little is known about the response to persistent hypoglycemia in these tissues. Histopathologic changes in insulin-infused animals included axonal degeneration and myofibre degeneration. To our knowledge, this is the first study to show that persistent IIH provokes peripheral nerve and skeletal myofiber degeneration within the same animals. This suggests that the model can serve as a nonclinical comparator model during development of long-acting insulin analogues.

A weighted method for estimation of receptor occupancy for pharmacodynamic measurements in drug development.

BACKGROUND: Flow cytometry-based receptor occupancy (RO) assessments for pharmacodynamic (PD) response measurements along with drug pharmacokinetic (PK) measurements represent a cornerstone in mechanism based PK/PD modeling of drugs against cell surface targets. This report describes the utility of using a "Free" and a "Bound" assay in combination to derive RO estimations through a weighted calculation method. METHODS: Data from a RO assay validation study in human samples was used to explore the performance of various RO data calculation methods. The calculation methods were subsequently applied to investigate the best method to generate RO data in a first in human phase 1 clinical trial. Finally, the outcome of the analysis was used for PK/PD modeling of a prospective phase 2a trial. RESULTS: The validation data assessment demonstrated that a weighted RO calculation method had a better performance in terms of precision, accuracy and dynamic range. In the phase 1 clinical trial data analysis the weighted method again demonstrated a better performance resulting in a more robust RO estimation, and subsequently also generating a more reliable PK/PD simulation for the phase 2a trial. CONCLUSIONS: This report demonstrated the utility of using a combined "Free" and "Bound" RO assessment together with a weighted calculation method to better support mechanism-based PK/PD modeling activities.

A reversible albumin-binding growth hormone derivative is well tolerated and possesses a potential once-weekly treatment profile.

CONTEXT: Human growth hormone (hGH) replacement therapy currently requires daily sc injections for years/lifetime, which may be both inconvenient and distressing for patients. NNC0195-0092 is a novel hGH derivative intended for once-weekly treatment of GH deficiency. A noncovalent albumin binding moiety is attached to the hGH backbone. Clearance is reduced as a consequence of a reversible binding to circulating serum albumin, which prolongs the pharmacodynamic (PD) effect. OBJECTIVE: To evaluate safety, local tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of a single dose (SD) and multiple doses (MD) of NNC0195-0092. SETTING AND DESIGN: Randomized, single-center, placebo-controlled, double-blind, SD/MD, dose-escalation trial of 105 healthy male subjects. NNC0195-0092 sc administration: Five cohorts of eight subjects received one dose of NNC0195-0092 (0.01-0.32 mg/kg) (n = 6) or placebo (n = 2). Sixteen subjects (equal numbers of Japanese and non-Asian) received once-weekly doses of NNC0195-0092 (0.02-0.24 mg/kg; n=12) or placebo (n=4) for 4 weeks. Blood samples were drawn for assessment of safety, PK, IGF-1, and IGF binding protein 3 profiles and anti-drug antibodies. RESULTS: SD and MD of NNC0195-0092 were well tolerated at all dose levels. No safety concerns or local tolerability issues were identified. A dose-dependent IGF-1 response was observed. IGF-1 profiles suggest that NNC0195-0092 may be suitable for once-weekly dosing, with a clinically relevant dose ≤0.08 mg/kg/week. No differences in PK and PD were observed between Japanese and non-Asian subjects. CONCLUSIONS: SD and MD of NNC0195-0092 administered to healthy Japanese and non-Asian male subjects were well tolerated at all doses. The present trial suggests that NNC0195-0092 has the potential for an efficacious, well-tolerated, once-weekly GH treatment.

Statistical evaluation and experimental design of a psoriasis xenograft transplantation model treated with cyclosporin A.

Psoriasis xenograft transplantation models where human skin is transplanted onto immune-deficient mice are generally accepted in psoriasis research. Over the last decade, they have been widely employed to screen for new therapeutics with a potential anti-psoriatic effect. However, experimental designs differ in several parameters. Especially, the number of donors and grafts per experimental design varies greatly; numbers that are directly related to the probability of detecting statistically significant drug effects. In this study, we performed a statistical evaluation of the effect of cyclosporine A, a recognized anti-psoriatic drug, to generate a statistical model employable to simulate different scenarios of experimental designs and to calculate the associated statistical study power, defined as the probability of detecting a statistically significant anti-psoriatic drug treatment effect. Results showed that to achieve a study power of 0.8, at least 20 grafts per treatment group and a minimum of five donors should be included in the chosen experimental setting. To our knowledge, this is the first time that study power calculations have been performed to evaluate treatment effects in a psoriasis xenograft transplantation model. This study was based on a defined experimental protocol, thus other parameters such as drug potency, treatment protocol, mouse strain and graft size should, also, be taken into account when designing an experiment. We propose that the results obtained in this study may lend a more quantitative support to the validity of results obtained when exploring new potential anti-psoriatic drug effects.

Measurement of lumefantrine and its metabolite in plasma by high performance liquid chromatography with ultraviolet detection.

Artemether-lumefantrine (ARM-LUM) has in recent years become the first-line treatment for uncomplicated malaria in many Sub-Saharan African countries. Vigorous monitoring of the therapeutic efficacy of this treatment is needed. This requires high-quality studies following standard protocols; ideally, such studies should incorporate measurement of drug levels in the study patients to exclude the possibility that insufficient drug levels explain an observed treatment failure. Several methods for measuring lumefantrine (LUM) in plasma by HPLC are available; however, several of these methods have some limitations in terms of high costs and limited feasibility arising from large required sample volumes and demanding sample preparation. Therefore, we set out to develop a simpler reversed phase high performance liquid chromatography (RP-HPLC) method based on UV detection for simultaneous measurement of LUM and its major metabolite the desbutyl LUM (DL) in plasma. Halofantrine was used as an internal standard. Liquid-liquid extraction of samples was carried out using hexane-ethyl acetate (70:30, v/v). Chromatographic separation was carried out on a Synergi Polar-RP column (250 mm × 300 mm, particle size 4 µm). The mobile phase consisted of acetonitrile-0.1M ammonium acetate buffer adjusted to pH 4.9 (85:15%, v/v). Absorbance of the compounds was monitored at 335 nm using a reference wavelength of 360 nm. Absolute extraction recovery for LUM and DL were 88% and 90%, respectively. Inter- and intraday coefficients of variation for LUM and DL were ≤ 10%. The lower limits of quantification for LUM and DL were 12.5 and 6.5 ng/ml, respectively. After validation, the methodology was transferred to a local laboratory in Tanga Tanzania and samples from a small subset of malaria patients were analysed for LUM. The method appears to be applicable in settings with limited facilities.

The synthesis and SAR of 2-arylsulfanyl-phenyl piperazinyl acetic acids as glyT-1 inhibitors.

Elevation of glycine levels and activation of the NMDA receptor by inhibition of the glycine transporter 1 (GlyT-1) is a potential strategy for the treatment of schizophrenia. A novel series of GlyT-1 inhibitors have been identified containing the 2-arylsulfanyl-phenylpiperazine motif. The most prominent member of this series, (R)-4-[5-chloro-2-(4-methoxy-phenylsulfanyl)-phenyl]-2-methyl-piperazin-1-yl-acetic acid (31) is a potent glycine transporter-1 inhibitor (IC(50)=150 nM), which elevated glycine levels in rat ventral hippocampus as measured by microdialysis in vivo at doses of 1.2-4.6 mg/kg s.c.

New alpha(1)-adrenoceptor antagonists derived from the antipsychotic sertindole - carbon-11 labelling and pet examination of brain uptake in the cynomolgus monkey.

Central alpha(1)-adrenergic receptors are potential targets for recently developed antipsychotic drugs. Two new 11C labeled potent and selective alpha(1)-adrenoceptor antagonists, 1- [2- [4-[1-(4-fluorophenyl)-5-(2-[(11)C]methyl-tetrazol-5-yl)-1H-indol-3-yl]-1-piperidinyl]ethyl]-imidazolidin-2-one ([(11)C]2) and 1- [2- [4-[1-(4-fluorophenyl)-5-(1-[(11)C]methyl-(1,2,3-triazol-4-yl)-1H-indol-3-yl]-1-piperidinyl]ethyl]-imidazolidin-2-one ([(11)C]3) were prepared and evaluated for imaging of central alpha(1)-adrenergic receptors in the cynomolgus monkey brain. For both compounds, the total brain radioactivity was only about 0.6% of the radioactivity injected i.v. There was no evident binding in regions known to contain alpha(1)-adrenoceptors. This observation suggests that the affinity of the radioligands in primates in vivo is not sufficient to provide a signal for specific binding that can be differentiated from the background. In addition, active efflux by P-glycoprotein may be responsible for the low total brain-uptake of the two radioligands. Both compounds showed a highly polarised and verapamile sensitive transport across monolayers of Caco-2 cells. The total brain-uptake of [(3)H]2 was 6 times higher in mdr1a(-/-) knock-out mice lacking the gene encoding P-glycoprotein compared to wild type mice. Pretreatment of one monkey with Cyclosporin A (15 mg/kg) resulted in 40% higher brain uptake for [(11)C]3 when compared with baseline. These observations support the view that efflux by P-glycoprotein can be of quantitative importance for the total brain-uptake of some PET radioligands.