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Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent monogenic human disease, but to date, only one therapy (tolvaptan) is approved to treat kidney cysts in ADPKD patients. Cyclin-dependent kinase 5 (CDK5), an atypical member of the cyclin-dependent kinase family, has been implicated as a target for treating ADPKD. However, no compounds have been disclosed to date that selectively inhibit CDK5 while sparing the broader CDK family members. Herein, we report the discovery of CDK5 inhibitors, including

Introduction A critical unmet need exists for precision therapies for chronic kidney disease. GFB-887 is a podocyte-targeting, small molecule inhibitor of transient receptor potential canonical-5 (TRPC5) designed specifically to treat patients with glomerular kidney diseases characterized by an overactivation of the TRPC5-Rac1 pathway. In a first-in-human study, GFB-887 was found to be safe and well tolerated, had a pharmacokinetic (PK) profile allowing once-daily dosing, and dose dependently decreased urinary Rac1 in healthy adults. Methods TRACTION-2 is a phase 2a, double-blind, placebo-controlled, multiple−ascending dose study of GFB-887 in patients with focal segmental glomerulosclerosis (FSGS), treatment-resistant minimal change disease (TR-MCD), or diabetic nephropathy (DN) (NCT04387448). Adult patients on stable renin−angiotensin system blockade and/or immunosuppression with persistent proteinuria will be randomized and dosed in 3 ascending dose levels to GFB-887 or placebo for 12 weeks. Cohorts may be expanded or biomarker-enriched depending upon results of an adaptive interim analysis. Results The primary objective is to evaluate the effect of increasing doses of GFB-887 on proteinuria. Safety and tolerability, quality of life, pharmacokinetic/pharmacodynamic profiles, and the potential association of urinary Rac1 with efficacy will also be evaluated. The projected sample size has 80% power to detect a treatment difference in proteinuria of 54% (FSGS/TR-MCD) or 44% (DN) compared to placebo. Conclusion TRACTION-2 will explore whether targeted blockade of the TRPC5-Rac1 pathway with GFB-887 is an efficacious and safe treatment strategy for patients with FSGS, TR-MCD, and DN and the potential value of urinary Rac1 as a predictive biomarker of treatment response., Graphical abstract

Pharmacodynamic (PD) studies are an essential component of preclinical drug discovery. Current approaches for PD studies, including the analysis of novel kidney disease targeting therapeutic agents, are limited to animal models with unclear translatability to the human condition. To address this challenge, we developed a novel approach for PD studies using transplanted, perfused human kidney organoids. We performed pharmacokinetic (PK) studies with GFB-887, an investigational new drug now in phase 2 trials. Orally dosed GFB-887 to athymic rats that had undergone organoid transplantation resulted in measurable drug exposure in transplanted organoids. We established the efficacy of orally dosed GFB-887 in PD studies, where quantitative analysis showed significant protection of kidney filter cells in human organoids and endogenous rat host kidneys. This widely applicable approach demonstrates feasibility of using transplanted human organoids in preclinical PD studies with an investigational new drug, empowering organoids to revolutionize drug discovery.

Background and Aims A major challenge in drug discovery is gaining confidence in the human relevance of pre-clinical animal studies. While human iPSC-derived organoids offer exciting opportunities to address this, concerns about applicability and scalability remain. Here, we report a high-throughput human organoid platform for assessment of kidney disease targeting compounds. Method In vitro & in vivo transplanted under athymic rat kidney capsule, differentiated organoids were characterized using single cell RNA sequencing (scRNA-Seq), NanoString, & immunofluorescence techniques. Immunofluorescence quantitative analysis of aggregated actin per in vitro organoid in a protamine sulfate (PS) podocyte injury model was used to evaluate efficacy of GFB-887, a sub-type selective, small molecule transient receptor potential canonical 5 (TRPC5) inhibitor. In pharmacokinetic studies, GFB-887 was orally administered into rats, then the presentation of GFB-887 was measured in rat plasma, rat kidney, & in vivo transplanted human organoids. In pharmacodynamic studies of transplanted human organoids, rats were co-perfused with GFB-887 & PS or orally dosed with GFB-887 prior to PS, then immunofluorescence quantitative analysis of mean synaptopodin intensity in podocytes of the in vivo transplanted organoids was used to evaluate efficacy of GFB-887. Results We confirmed platform reproducibility by scRNA-Seq and derived a NanoString panel for efficient quality control. Organoid transplantation in rats for 2 to 4 weeks promoted organoid maturation and vascularization. In functional studies, cyclosporine A (CsA), a calcineurin inhibitor clinically utilized for the treatment of nephrotic syndrome, and GFB-887, a novel sub-type selective TRPC5 inhibitor currently in clinical development, protected in vitro kidney organoids from injury. Pharmacodynamic studies with GFB-887 delivered orally to rats were also successfully performed in human transplanted organoids. Conclusion These data show how human organoids can deliver confidence in taking development candidate compounds to the clinic, fulfilling their promise to revolutionize drug discovery.

IDX184 is a phosphoramidate prodrug of 2′-methylguanosine-5′-monophosphate, developed to treat patients infected with hepatitis C virus. A mass balance study of radiolabeled IDX184 and pharmacokinetic studies of IDX184 in portal vein-cannulated monkeys revealed relatively low IDX184 absorption but higher exposure of IDX184 in the portal vein than in the systemic circulation, indicating >90 % of the absorbed dose was subject to hepatic extraction. Systemic exposures to the main metabolite, 2′-methylguanosine (2′-MeG), were used as a surrogate for liver levels of the pharmacologically active entity 2′-MeG triphosphate, and accordingly, systemic levels of 2′-MeG in the monkey were used to optimize formulations for further clinical development of IDX184. Capsule formulations of IDX184 delivered acceptable levels of 2′-MeG in humans; however, the encapsulation process introduced low levels of the genotoxic impurity ethylene sulfide (ES), which necessitated formulation optimization. Animal pharmacokinetic data guided the development of a tablet with trace levels of ES and pharmacokinetic performance equal to that of the clinical capsule in the monkey. Under fed conditions in humans, the new tablet formulation showed similar exposure to the capsule used in prior clinical trials.

AZT-P-ddI is an antiviral heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (AZT) and one molecule of 2',3'-dideoxyinosine (ddI) linked through their 5' positions by a phosphate bond. The metabolic fate of the dimer was studied with isolated rat, monkey, and human hepatocytes and was compared with that of its component monomers AZT and ddI. Upon incubation of double-labeled [14C]AZT-P-[3H]ddI in freshly isolated rat hepatocytes in suspension at a final concentration of 10 microM, the dimer was taken up intact by cells and then rapidly cleaved to AZT, AZT monophosphate, ddI, and ddI monophosphate. AZT and ddI so formed were then subject to their respective catabolisms. High-performance liquid chromatography analyses of the extracellular medium and cell extracts revealed the presence of unchanged dimer, AZT, 3'-azido-3'-deoxy-5'-beta-D-glucopyranosylthymidine (GAZT), 3'-amino-3'-deoxythymidine (AMT), ddI, and a previously unrecognized derivative of the dideoxyribose moiety of ddI, designated ddI-M. Trace extracellular but substantial intracellular levels of the glucuronide derivative of AMT (3'-amino-3'-deoxy-5'-beta-D-glucopyranosylthymidine [GAMT]) were also detected. Moreover, the extent of the formation of AMT, GAZT, and ddI-M from the dimer was markedly lower than that with AZT and ddI alone by the hepatocytes. With hepatocytes in primary culture obtained from rat, monkey, and human, large interspecies variations in the metabolism of AZT-P-ddI were observed. While GAZT and ddI-M, metabolites of AZT and ddI, respectively, as well as AZT 5'-monophosphate (MP) and ddI-MP were detected in the extracellular media of all species, AMT and GAMT were produced only by rat and monkey hepatocytes. No such metabolites were formed by human hepatocytes. The metabolic fate of the dimer by human hepatocytes was consistent with in vivo data recently obtained from human immunodeficiency virus-infected patients.

Numerous studies have reported effects of antiviral nucleoside analogs on mitochondrial function, but they have not correlated well with the observed toxic side effects. By comparing the effects of the five Food and Drug Administration-approved anti-human immunodeficiency virus nucleoside analogs, zidovudine (3′-azido-3′-deoxythymidine) (AZT), 2′,3′-dideoxycytidine (ddC), 2′,3′-dideoxyinosine (ddI), 2′,3′-didehydro-2′,3′-deoxythymidine (d4T), and β-L-2′,3′-dideoxy-3′-thiacytidine (3TC), as well as the metabolite of AZT, 3′-amino-3′-deoxythymidine (AMT), on mitochondrial function in a human hepatoma cell line, this issue has been reexamined. Evidence for a number of mitochondrial defects with AZT, ddC, and ddI was found, but only AZT induced a marked rise in lactic acid levels. Only in mitochondria isolated from AZT (50 μM)-treated cells was significant inhibition of cytochrome c oxidase and citrate synthase found. Our investigations also demonstrated that AZT, d4T, and 3TC did not affect the synthesis of the 11 polypeptides encoded by mitochondrial DNA, while ddC caused 70% reduction of total polypeptide content and ddI reduced by 43% the total content of 8 polypeptides (including NADH dehydrogenase subunits 1, 2, 4, and 5, cytochrome c oxidase subunits I to III, and cytochrome b ). We hypothesize that in hepatocytes the reserve capacity for mitochondrial respiration is such that inhibition of respiratory enzymes is unlikely to become critical. In contrast, the combined inhibition of the citric acid cycle and electron transport greatly enhances the dependence of the cell on glycolysis and may explain why apparent mitochondrial dysfunction is more prevalent with AZT treatment.

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3′-azido-3′-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3′-amino-3′-deoxythymidine (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low K m (58–74 μM) and V max (107–142 pmol AMT formed/min/mg protein) and the other with a high K m (4.33–5.88 mM) and V max (1804–2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b 5 ( b 5 ) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b 5 content ( r 2 = 0.96) as well as with aggregate P450 content ( r 2 = 0.97). Upon reconstitution, human liver b 5 plus NADH: b 5 reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT K m of 54 μM and V max of 301 pmol/min for b 5 plus NADH: b 5 reductase and an AZT K m of 103 μM and V max of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b 5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b 5 to reduce AZT may be a function of their heme prothestic groups.

3'-Azido-3'-deoxythymidilyl-(5',5')-2',3'-dideoxy-5'-inosinic acid (AZT-P-ddI,IVX-E-59. Scriptene) is a heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (zidovudine or AZT) and one molecule of 2',3'-dideoxyinosine (didanosine or ddl) linked through their 5' positions by a phosphate bond. AZT-P-ddl exhibits enhanced antiviral activity and selectivity in vitro compared with AZT and ddl alone. The pharmacokinetics of AZT-P-ddI were studied in 12 patients with human immunodeficiency virus (HIV) who had CD4 + cell counts higher than 200 cells/mm 3 . Isotopic preparations of ( 14 C)-AZT-P-( 3 H)-ddI were administered intravenously (50 mg and 100 mg) to eight patients; 1 month later these patients were crossed over to oral administration (100 mg and 200 mg). A second group of patients (n = 4) received only a 450-mg oral dose of AZT-P-ddl. Plasma levels of unchanged AZT-P-ddI after intravenous infusion declined rapidly and were undetectable 0.75 hours after the end of infusion, whereas the parent compound was not detected after oral administration, indicative of a very rapid metabolism. The parent entity was enzymatically cleaved in viva yielding the two constituent drugs AZT and ddI, which were subsequently subjected to their respective pharmacokinetic and metabolic processes. The β-glucuronide derivatide of AZT (GAZT) represented the major metabolite of AZT, but there were no detectable levels of the toxic metabolite 3'-amino-3'-deoxythymidine (AMT). A major and previously unrecognized in vivo metabolite of ddI, referred as ddl-M, was detected in plasma and urine. Analysis by high-field proton nuclear magnetic resonance and mass spectrometry led to the identification of ddI-M as being R(-)-dihydro-5-(hydroxymethyl)-2(3H)-furanone. The formation of AZT and ddl metabolites was increased after oral administration of AZT-P-ddI compared with the intravenous infusion, with an area under the concentration- time curve (AUC) ratio of metabolite to AZT and metabolite to ddl being 7.7 and 5.7 (oral) and 3.8 and 1.1 (intravenous), respectively. The newly identified ddI-M exhibited sustained plasma levels for extended time periods with an apparent elimination half-life (t 1/2 ) of approximately 10 hours after oral administration of AZT-P-ddl. Recovery of radioactivity associated with 3 H and 14 C in urine was essentially complete within 48 hours after oral and intravenous administration of AZT-P-ddI. The oral bioavailability of AZT (64.7-67.3 %) and ddI (33.6-42.9 %) and the other pharmacokinetic parameters were consistent with previous data reported with each nucleoside analog alone or in combination therapy.