Sara Belli

A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents

We report the discovery of a new monomeric peptide that reduces body weight and diabetic complications in rodent models of obesity by acting as an agonist at three key metabolically-related peptide hormone receptors: glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors. This triple agonist demonstrates supraphysiological potency and equally aligned constituent activities at each receptor, all without cross-reactivity at other related receptors. Such balanced unimolecular triple agonism proved superior to any existing dual coagonists and best-in-class monoagonists to reduce body weight, enhance glycemic control and reverse hepatic steatosis in relevant rodent models. Various loss-of-function models, including genetic knockout, pharmacological blockade and selective chemical knockout, confirmed contributions of each constituent activity in vivo. We demonstrate that these individual constituent activities harmonize to govern the overall metabolic efficacy, which predominantly results from synergistic glucagon action to increase energy expenditure, GLP-1 action to reduce caloric intake and improve glucose control, and GIP action to potentiate the incretin effect and buffer against the diabetogenic effect of inherent glucagon activity. These preclinical studies suggest that, so far, this unimolecular, polypharmaceutical strategy has potential to be the most effective pharmacological approach to reversing obesity and related metabolic disorders.

Fibroblast Growth Factor 21 Mediates Specific Glucagon Actions

Glucagon, an essential regulator of glucose homeostasis, also modulates lipid metabolism and promotes weight loss, as reflected by the wasting observed in glucagonoma patients. Recently, coagonist peptides that include glucagon agonism have emerged as promising therapeutic candidates for the treatment of obesity and diabetes. We developed a novel stable and soluble glucagon receptor (GcgR) agonist, which allowed for in vivo dissection of glucagon action. As expected, chronic GcgR agonism in mice resulted in hyperglycemia and lower body fat and plasma cholesterol. Notably, GcgR activation also raised hepatic expression and circulating levels of fibroblast growth factor 21 (FGF21). This effect was retained in isolated primary hepatocytes from wild-type (WT) mice, but not GcgR knockout mice. We confirmed this link in healthy human volunteers, where injection of natural glucagon increased plasma FGF21 within hours. Functional relevance was evidenced in mice with genetic deletion of FGF21, where GcgR activation failed to induce the body weight loss and lipid metabolism changes observed in WT mice. Taken together, these data reveal for the first time that glucagon controls glucose, energy, and lipid metabolism at least in part via FGF21-dependent pathways.

Cholesterol-Mediated Activation of P-Glycoprotein: Distinct Effects on Basal and Drug-Induced ATPase Activities

Cholesterol promotes basal and verapamil-induced ATPase activity of P-glycoprotein (P-gp). We investigated whether these effects are related to each other and to the impact of the sterol on bilayer fluidity and verapamil membrane affinity. P-gp was reconstituted in egg-phosphatidylcholine (PhC) liposomes with or without cholesterol, 1,2-dipalmitoyl-phosphatidylcholine (DPPC), alpha-tocopherol (alpha-Toc) or 2,2,5,7,8-pentamethyl-6-chromanol (PMC). Basal and verapamil-induced ATPase activities were studied with an enzymatic assay. Membrane fluidity was characterized with diphenyl-hexatriene anisotropy measurements and membrane affinity by equilibrium dialysis. DPPC (70% mol/mol) decreased the fluidity of PhC bilayers to the same level as 20% cholesterol. PMC (20%) and alpha-Toc (20%) decreased the fluidity to lesser extents. alpha-Toc and PMC, but not DPPC increased the verapamil membrane affinity. While 20% cholesterol strikingly enhanced the basal ATPase activity, none of the other constituents had a similar effect. In contrast, verapamil stimulation of P-gp ATPase activity was not only enabled by cholesterol but also by alpha-Toc and DPPC. PMC had no effect. In conclusion, cholesterol exerts distinct effects on basal and verapamil-induced ATPase activity. The influence on basal ATPase activity is sterol-specific while its effect on verapamil-induced ATPase activity is unspecific and not related to its influence on membrane fluidity and on verapamil membrane affinity.

Expression Profiles of Metabolic Enzymes and Drug Transporters in the Liver and along the Intestine of Beagle Dogs

Beagle dogs are widely used in preclinical pharmacokinetic, safety, and formulation studies. However, little is known about intestinal and hepatic distribution of major enzymes and transporters involved in oral absorption and presystemic drug metabolism. We characterized mRNA levels of CYP3A12, CYP3A26, CYP2D15, UGT1A6, ABCB1 (MDR1), ABCC1 (MRP1), ABCG2 (BCRP), SLC15A1 (PEPT1), and SLC22A1 (OCT1) in dog liver and along the intestine by real-time quantitative reverse transcription-polymerase chain reaction. Tissue protein levels of CYP2D15, MDR1, and PEPT1 were obtained by Western blot. Gene distribution and expression variability was statistically described by a generalized additive mixed model smoothing function and correspondence analysis. Results were compared with the expression pattern known for the human orthologs. Hepatic mRNA levels for metabolic enzymes were generally higher than those for membrane transporters, whereas in the intestine the opposite was observed. Hepatic mRNA levels followed the order CYP2D15 > UGT1A6 ≈ CYP3A26 > ABCB1 ≈ SLC15A1 ≈ SLC22A1 > ABCG2 > ABCC1 ≈ CYP3A12. Along the gut, the genes were differentially distributed with greatest expression in duodenum/upper jejunum (ABCG2), middle jejunum (ABCB1 and SLC15A1), or in cecum/colon (ABCC1 and CYP2D15). CYP3A12, CYP3A26, SLC22A1, and UGT1A6 had a rather uniform expression. Intestinal mRNA profiles of CYP2D15, ABCB1, and SLC15A1 correlated with the respective protein levels. Canine CYP3A12/26, CYP2D15, and ABCB1 colonic distributions differed from those of human orthologs, whereas UGT1A6, ABCC1, ABCG2, SLC15A1, and SLC22A1 were comparable to those of humans in both small and large intestine. We aim to apply these data to better interpret pharmacokinetic studies in dogs with respect to their human relevance.

P-glycoprotein in proteoliposomes with low residual detergent: the effects of cholesterol.

PurposeThere is evidence that cholesterol affects the ATPase and transport functions of P-glycoprotein (P-gp). To study the influence of cholesterol on P-gp in a well defined lipid environment, we reconstituted P-gp in egg phosphatidylcholine (PhC) and PhC/cholesterol proteoliposomes with negligible residual amounts of detergents.Materials and methodsP-gp proteoliposomes were prepared by continuous dialysis from micelles consisting of P-gp, lipids, sodium dodecyl sulfate and cholate. Basal and modulator-induced ATPase activities were studied in an established enzyme assay. Modulator affinities to P-gp and to the lipid bilayers were determined by equilibrium dialysis.ResultsIn the absence of cholesterol the basal ATPase activity was six fold lower than in the presence of 20 or 40% cholesterol, and no P-gp binding and ATPase induction was detected for the tested modulators verapamil and progesterone. In proteoliposomes containing 20 and 40% cholesterol, respectively, the modulators showed significant P-gp binding and ATPase activation. The concentration of the modulators for half maximal activation of the ATPase was higher with 40% than with 20% cholesterol.ConclusionsCholesterol influences P-gp in three ways: (a) it enhances its basal ATPase activity, (b) it renders P-gp sensitive towards the modulators verapamil and progesterone and (c) it affects the modulator concentration at half maximal ATPase activation.

Comparative analysis of CK2 expression and function in tumor cell lines displaying sensitivity vs. resistance to chemical induced apoptosis

CK2 is a pleiotropic protein kinase, which phosphorylates many substrates and has a global role in promoting cell survival and preventing apoptosis. In this study, we investigated its involvement in the phenomenon of the drug resistance, by which tumor cells frequently become unresponsive to chemical apoptosis. By comparing the expression of CK2 subunits in four different pairs of sensitive (S) and resistant (R) cancer cell lines, we found that in three cases the resistant phenotype is accompanied by the overexpression of the CK2 catalytic α subunit, either alone or in combination with the regulatory β subunit. The degree of CK2 expression correlates with the CK2 catalytic activity, when measured toward endogenous protein substrates. All the tested R cell lines, including the one with no CK2 overexpression, can be induced to undergo death by treatment with CK2 inhibitors. We therefore conclude that, although CK2 overexpression is not an absolute requirement for the resistant phenotype, its activity is essential for cell survival and contributes to a high degree of resistance. We also found that CK2 inhibition increases the accumulation of cytotoxic drugs inside the R cells, presumably by impairing the functionality of the extrusion pump P-gp. We therefore propose that CK2 should be considered a target to counteract the pharmaco-resistant phenotype.

Determination of Ganciclovir and its prodrug Valganciclovir by hydrophilic interaction liquid chromatography–tandem mass spectrometry

This manuscript describes the determination of Ganciclovir (GCV), active component of the antiviral drug Valcyte®, and its ester prodrug Valganciclovir (VGC) in human and rat plasma, using liquid chromatography coupled to tandem mass spectrometry. Protein precipitation with acetonitrile was followed by hydrophilic interaction liquid chromatography on a silica column with 4 min run time. After electrospray ionization, the compounds were detected in positive ion selected reaction monitoring (SRM) mode. The lower limits of quantification (LLOQ) were 16 ng/mL for GCV and 4 ng/mL for VGC in human and rat plasma. Inter-day and intra-day precisions and inaccuracies were below 15% and between 85 and 115%, respectively. Five-fold deuterated GCV and VGC were used as internal standards and compensated for any matrix effect. The method was successfully applied to samples from a rat pharmacokinetic study. The feasibility of blood analysis as dried blood spots (DBS) was investigated.

When barriers ignore the “rule-of-five”

Why are a few drugs with properties beyond the rule of 5 (bRo5) absorbed across the intestinal mucosa while most other bRo5 compounds are not? Are such exceptional bRo5 compounds exclusively taken up by carrier-mediated transport or are they able to permeate the lipid bilayer (passive lipoidal diffusion)? Our experimental data with liposomes indicate that tetracycline, which violates one rule of the Ro5, and rifampicin, violating three of the rules, significantly permeate a phospholipid bilayer with kinetics similar to labetalol and metoprolol, respectively. Published data from experimental work and molecular dynamics simulations suggest that the formation of intramolecular H-bonds and the possibility to adopt an elongated shape besides the presence of a significant fraction of net neutral species facilitate lipid bilayer permeation. As an alternative to lipid bilayer permeation, carrier proteins can be targeted to improve absorption, with the potential drawbacks of drug-drug interactions and non-linear pharmacokinetics.

PK/PD assessment in CNS drug discovery: Prediction of CSF concentration in rodents for P-glycoprotein substrates and application to in vivo potency estimation

The unbound drug concentration in brain parenchyma is considered to be the relevant driver for interaction with central nervous system (CNS) biological targets. Drug levels in cerebrospinal fluid (C_CSF) are frequently used surrogates for the unbound concentrations in brain. For drugs actively transported across the blood-brain barrier (BBB), C_CSF differs from unbound plasma concentration (Cu_p) to an extent that is commonly unknown. In this study, the relationship between CSF-to-unbound plasma drug partitioning in rats and the mouse Pgp (Mdr1a) efflux ratio (ER) obtained from in vitro transcellular studies has been investigated for a set of 61 CNS compounds exhibiting substantial diversity in chemical structure and physico-chemical properties. In order to understand the in vitro-in vivo extrapolation of Pgp efflux, a mechanistic model was derived relating in vivo CNS distribution kinetics to in vitro active transport. The model was applied to predict C_CSF from Cu_p and ER data for 19 proprietary Roche CNS drug candidates. The calculated CSF concentrations were correlated with CNS pharmacodynamic responses observed in rodent models. The correlation between in vitro and in vivo potency for different pharmacological endpoints indicated that the predicted C_CSF is a valuable surrogate of the concentration at the target site. Overall, C_CSF proved superior description of PK/PD data than unbound plasma or total brain concentration for Mdr1a substrates. Predicted C_CSF can be used as a default approach to understand the PK/PD relationships in CNS efficacy models and can support the extrapolation of efficacious brain exposure for new drug candidates from rodent to man.

Role of the Intestinal Peptide Transporter PEPT1 in Oseltamivir Absorption: In Vitro and In Vivo Studies

It was reported that oseltamivir (Tamiflu) absorption was mediated by human peptide transporter (hPEPT) 1. Understanding the exact mechanism(s) of absorption is important in the context of drug-drug and diet-drug interactions. Hence, we investigated the mechanism governing the intestinal absorption of oseltamivir and its active metabolite (oseltamivir carboxylate) in wild-type [Chinese hamster ovary (CHO)-K1] and hPEPT1-transfected cells (CHO-PEPT1), in pharmacokinetic studies in juvenile and adult rats, and in healthy volunteers. In vitro cell culture studies showed that the intracellular accumulation of oseltamivir and its carboxylate into CHO-PEPT1 and CHO-K1 was always similar under a variety of experimental conditions, demonstrating that these compounds are not substrates of hPEPT1. Furthermore, neither oseltamivir nor its active metabolite was capable of inhibiting Gly-Sar uptake in CHO-PEPT1 cells. In vivo pharmacokinetic studies in juvenile and adult rats showed that the disposition of oseltamivir and oseltamivir carboxylate, after oral administration of oseltamivir, was sensitive to the feed status but insensitive to the presence of milk and Gly-Sar. Moreover, oseltamivir and oseltamivir carboxylate exhibited significantly higher exposure in rats under fasted conditions than under fed conditions. In humans, oral dosing after a high-fat meal resulted in a statistically significant but moderate lower exposure than after an overnight fasting. This change has no clinical implications. Taken together, the results do not implicate either rat Pept1 or hPEPT1 in the oral absorption of oseltamivir.