Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in ...tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.
Early stage chemical development presents numerous challenges, and achieving a functional balance is a major hurdle, with many early compounds not meeting the clinical requirements for advancement ...benchmarks due to issues like poor oral bioavailability. There is a need to develop strategies for achieving the desired systemic concentration for these compounds. This will enable further evaluation of the biological response upon a compound-target interaction, providing deeper insight into the postulated biological pathways. Our study elucidates alternative drug delivery paradigms by comparing formulation strategies across oral (PO), intraperitoneal (IP), subcutaneous (SC), and intravenous (IV) routes. While each modality boasts its own set of merits and constraints, it is the drug's formulation that crucially influences its pharmacokinetic (PK) trajectory and the maintenance of its therapeutic levels. Our examination of model compounds G7883 and G6893 highlighted their distinct physio-chemical attributes. By harnessing varied formulation methods, we sought to fine-tune their PK profiles. PK studies showcased G7883's extended half-life using an SC oil formulation, resulting in a 4.5-fold and 2.5-fold enhancement compared with the IP and PO routes, respectively. In contrast, with G6893, we achieved a prolonged systemic coverage time above the desired target concentration through a different approach using an IV infusion pump. These outcomes underscore the need for tailored formulation strategies, which are dictated by the compound's innate properties, to reach the optimal in vivo systemic concentrations. Prioritizing formulation and delivery optimization early on is pivotal for effective systemic uptake, thereby facilitating a deeper understanding of biological pathways and expediting the overall clinical drug development timeline.
Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50years. The multifactorial nature of events that precede severe liver injury in human ...patients is difficult to model in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine if serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50mg/kg troglitazone. Minimal serum transaminase elevations (approximately two-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2hours (h) after dosing and returned to baseline values before 24h. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be poorly detected by traditional toxicity endpoints.
•Troglitazone is a potent BSEP inhibitor and perturbs bile acid homeostasis in rats.•Taurine-conjugated bile acids had the best dynamic range of profiled bile acids.•Serum bile acids identify mechanistic DILI that cannot be detected with routine toxicity testing.•The study design captures transient changes in bile acids in context with in vivo exposures.
The subcutaneous administration of therapeutic peptides would provide significant benefits to patients. However, subcutaneous injections are limited in dosing volume, potentially resulting in high ...peptide concentrations that can incur significant challenges with solubility limitations, high viscosity, and stability liabilities. Herein, we report on the discovery that low-shear resonant acoustic mixing can be used as a general method to prepare stable nanoparticles of a number of peptides of diverse molecular weights and structures in water without the need for extensive amounts of organic solvents or lipid excipients. This approach avoids the stability issues observed with typical high-shear, high-intensity milling methods. The resultant peptide nanosuspensions exhibit low viscosity even at high concentrations of >100 mg/mL while remaining chemically and physically stable. An example nanosuspension of cyclosporine nanoparticles was dosed in rats via a subcutaneous injection and exhibited sustained release behavior. This suggests that peptide nanosuspension formulations can be one approach to overcome the challenges with high-concentration peptide formulations.
The PI3K/AKT/mTOR pathway has been shown to play an important role in cancer. Starting with compounds 1 and 2 (GDC-0941) as templates, (thienopyrimidin-2-yl)aminopyrimidines were discovered as potent ...inhibitors of PI3K or both PI3K and mTOR. Structural information derived from PI3Kγ−ligand cocrystal structures of 1 and 2 were used to design inhibitors that maintained potency for PI3K yet improved metabolic stability and oral bioavailability relative to 1. The addition of a single methyl group to the optimized 5 resulted in 21, which had significantly reduced potency for mTOR. The lead compounds 5 (GNE-493) and 21 (GNE-490) have good pharmacokinetic (PK) parameters, are highly selective, demonstrate knock down of pathway markers in vivo, and are efficacious in xenograft models where the PI3K pathway is deregulated. Both compounds were compared in a PI3Kα mutated MCF7.1 xenograft model and were found to have equivalent efficacy when normalized for exposure.
Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to ...the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.
Organic anion transporting polypeptide (Oatp) 1a/1b knockout and OATP1B1 and -1B3 humanized mouse models are promising tools for studying the roles of these transporters in drug disposition. Detailed ...characterization of these models will help to better understand their utility for predicting clinical outcomes. To advance this approach, we carried out a comprehensive analysis of these mouse lines by evaluating the compensatory changes in mRNA expression, quantifying the amounts of OATP1B1 and -1B3 protein by liquid chromatography-tandem mass spectrometry, and studying the active uptake in isolated hepatocytes and the pharmacokinetics of some prototypical substrates including statins. Major outcomes from these studies were 1) mostly moderate compensatory changes in only a few genes involved in drug metabolism and disposition, 2) a robust hepatic expression of OATP1B1 and -1B3 proteins in the respective humanized mouse models, and 3) functional activities of the human transporters in hepatocytes isolated from the humanized models with several substrates tested in vitro and with pravastatin in vivo. However, the expression of OATP1B1 and -1B3 in the humanized models did not significantly alter liver or plasma concentrations of rosuvastatin and pitavastatin compared with Oatp1a/1b knockout controls under the conditions used in our studies. Hence, although the humanized OATP1B1 and -1B3 mice showed in vitro and/or in vivo functional activity with some statins, further characterization of these models is required to define their potential use and limitations in the prediction of drug disposition and drug-drug interactions in humans.
Efforts to identify potent small molecule inhibitors of PI3 kinase and mTOR led to the evaluation of tetrasubstituted thienopyrimidines. These molecules generally have reduced in vivo clearance ...relative to the trisubstituted thienopyrimidines culminating in the identification of
GNE-477 (
8).
Efforts to identify potent small molecule inhibitors of PI3 kinase and mTOR led to the discovery of the exceptionally potent 6-aryl morpholino thienopyrimidine
6. In an effort to reduce the melting point in analogs of
6, the thienopyrimidine was modified by the addition of a methyl group to disrupt planarity. This modification resulted in a general improvement in in vivo clearance. This discovery led to the identification of
GNE-477 (
8), a potent and efficacious dual PI3K/mTOR inhibitor.
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