The Simultaneous Inhibition of Solute Carrier Family 6 Member 19 and Breast Cancer Resistance Protein Transporters Leads to an Increase of Indoxyl Sulfate (a Uremic Toxin) in Plasma and Kidney.

Solute carrier family 6 member 19 (SLC6A19) inhibitors are being studied as therapeutic agents for phenylketonuria. In this work, a potent SLC6A19 inhibitor (RA836) elevated rat kidney uremic toxin indoxyl sulfate (IDS) levels by intensity (arbitrary unit) of 13.7 ± 7.7 compared with vehicle 0.3 ± 0.1 (P = 0.01) as determined by tissue mass spectrometry imaging analysis. We hypothesized that increased plasma and kidney levels of IDS could be caused by the simultaneous inhibition of both Slc6a19 and a kidney IDS transporter responsible for excretion of IDS into urine. To test this, we first confirmed the formation of IDS through tryptophan metabolism by feeding rats a Trp-free diet. Inhibiting Slc6a19 with RA836 led to increased IDS in these rats. Next, RA836 and its key metabolites were evaluated in vitro for inhibiting kidney transporters such as organic anion transporter (OAT)1, OAT3, and breast cancer resistance protein (BCRP). RA836 inhibits BCRP with an IC50 of 0.045 µM but shows no significant inhibition of OAT1 or OAT3. Finally, RA836 analogs with either potent or no inhibition of SLC6A19 and/or BCRP were synthesized and administered to rats fed a normal diet. Plasma and kidney samples were collected to quantify IDS using liquid chromatography-mass spectrometry. Neither a SLC6A19 inactive but potent BCRP inhibitor nor a SLC6A19 active but weak BCRP inhibitor raised IDS levels, whereas compounds inhibiting both transporters caused IDS accumulation in rat plasma and kidney, supporting the hypothesis that rat Bcrp contributes to the excretion of IDS. In summary, we identified that inhibiting Slc6a19 increases IDS formation, while simultaneously inhibiting Bcrp results in IDS accumulation in the kidney and plasma. SIGNIFICANCE STATEMENT: This is the first publication to decipher the mechanism for accumulation of indoxyl sulfate (IDS) (a uremic toxin) in rats via inhibition of both Slc6a19 and Bcrp. Specifically, inhibition of Slc6a19 in the gastrointestinal track increases IDS formation, and inhibition of Bcrp in the kidney blocks IDS excretion. Therefore, we should avoid inhibiting both solute carrier family 6 member 19 and breast cancer resistance protein simultaneously in humans to prevent accumulation of IDS, a known risk factor for cardiovascular disease, psychic anxiety, and mortality in chronic kidney disease patients.

Mitigation of reactive metabolite formation for a series of 3-amino-2-pyridone inhibitors of Bruton's tyrosine kinase (BTK).

Reactive metabolites have been putatively linked to many adverse drug reactions including idiosyncratic toxicities for a number of drugs with black box warnings or withdrawn from the market. Therefore, it is desirable to minimize the risk of reactive metabolite formation for lead molecules in optimization, in particular for non-life threatening chronic disease, to maximize benefit to risk ratio. This article describes our effort in addressing reactive metabolite issues for a series of 3-amino-2-pyridone inhibitors of BTK, e.g. compound 1 has a value of 459pmol/mg protein in the microsomal covalent binding assay. Parallel approaches were taken to successfully resolve the issues: establishment of a predictive screening assay with correlation association of covalent binding assay, identification of the origin of reactive metabolite formation using MS/MS analysis of HLM as well as isolation and characterization of GSH adducts. This ultimately led to the discovery of compound 7 (RN941) with significantly reduced covalent binding of 26pmol/mg protein.

Machine Learning guided early drug discovery of small molecules.

Machine learning (ML) approaches have been widely adopted within the early stages of the drug discovery process, particularly within the context of small-molecule drug candidates. Despite this, the use of ML is still limited in the pharmacokinetic/pharmacodynamic (PK/PD) application space. Here, we describe recent progress and the role of ML used in preclinical drug discovery. We summarize the advances and current strategies used to predict ADME (absorption, distribution, metabolism and, excretion) properties of small molecules based on their structures, and predict structures based on the desired properties for molecular screening and optimization. Finally, we discuss the use of ML to predict PK to rank the ability of drug candidates to achieve appropriate exposures and hence provide important insights into safety and efficacy.

A multi-model approach to predict efficacious clinical dose for an anti-TGF-ß antibody (GC2008) in the treatment of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous group of inherited bone dysplasias characterized by reduced skeletal mass and bone fragility. Although the primary manifestation of the disease involves the skeleton, OI is a generalized connective tissue disorder that requires a multidisciplinary treatment approach. Recent studies indicate that application of a transforming growth factor beta (TGF-ß) neutralizing antibody increased bone volume fraction (BVF) and strength in an OI mouse model and improved bone mineral density (BMD) in a small cohort of patients with OI. In this work, we have developed a multitiered quantitative pharmacology approach to predict human efficacious dose of a new anti-TGF-ß antibody drug candidate (GC2008). This method aims to translate GC2008 pharmacokinetic/pharmacodynamic (PK/PD) relationship in patients, using a number of appropriate mathematical models and available preclinical and clinical data. Compartmental PK linked with an indirect PD effect model was used to characterize both pre-clinical and clinical PK/PD data and predict a GC2008 dose that would significantly increase BMD or BVF in patients with OI. Furthermore, a physiologically-based pharmacokinetic model incorporating GC2008 and the body's physiological properties was developed and used to predict a GC2008 dose that would decrease the TGF-ß level in bone to that of healthy individuals. By using multiple models, we aim to reveal information for different aspects of OI disease that will ultimately lead to a more informed dose projection of GC2008 in humans. The different modeling efforts predicted a similar range of pharmacologically relevant doses in patients with OI providing an informed approach for an early clinical dose setting.

Characterization of xenobiotic-induced hepatocellular enzyme induction in rats: anticipated thyroid effects and unique pituitary gland findings.

During routine safety evaluation of RO2910, a non-nucleoside reverse transcriptase inhibitor for HIV infection, histopathology findings concurrent with robust hepatocellular induction occurred in multiple organs, including a unique, albeit related, finding in the pituitary gland. For fourteen days, male and female rats were administered, by oral gavage vehicle, 100, 300, or 1000 mg/kg/day of RO2910. Treated groups had elevated serum thyroid-stimulating hormone and decreased total thyroxine, and hypertrophy in the liver, thyroid gland, and pituitary pars distalis. These were considered consequences of hepatocellular induction and often were dose dependent and more pronounced in males than in females. Hepatocellular centrilobular hypertrophy corresponded with increased expression of cytochrome P450s 2B1/2, 3A1, and 3A2 and UGT 2B1. Bilateral thyroid follicular cell hypertrophy occurred concurrent to increased mitotic activity and sometimes colloid depletion, which were attributed to changes in thyroid hormone levels. Males had hypertrophy of thyroid-stimulating hormone-producing cells (thyrotrophs) in the pituitary pars distalis. All findings were consistent with the well-established adaptive physiologic response of rodents to xenobiotic-induced hepatocellular microsomal enzyme induction. Although the effects on the pituitary gland following hepatic enzyme induction-mediated hypothyroidism have not been reported previously, other models of stress and thyroid depletion leading to pituitary stimulation support such a shared pathogenesis.

Challenges of non-clinical safety testing for biologics: A Report of the 9th BioSafe European Annual General Membership Meeting.

New challenges and other topics in non-clinical safety testing of biotherapeutics were presented and discussed at the nineth European BioSafe Annual General Membership meeting in November 2019. The session topics were selected by European BioSafe organization committee members based on recent company achievements, agency interactions and new data obtained in the non-clinical safety testing of biotherapeutics, for which data sharing would be of interest and considered as valuable information. The presented session topics ranged from strategies of in vitro testing, immunogenicity prediction, bioimaging, and developmental and reproductive toxicology (DART) assessments to first-in-human (FIH) dose prediction and bioanalytical challenges, reflecting the entire space of different areas of expertise and different molecular modalities. During the 9th meeting of the European BioSafe members, the following topics were presented and discussed in 6 main sessions (with 3 or 4 presentations per session) and in three small group breakout sessions: 1) DART assessment with biotherapeutics: what did we learn and where to go?; 2) Non-animal testing strategies; 3) Seeing is believing: new frontiers in imaging; 4) Predicting immunogenicity during early drug development: hope or despair?; 5) Challenges in FIH dose projections; and 6) Non-canonical biologics formats: challenges in bioanalytics, PKPD and biotransformation for complex biologics formats. Small group breakout sessions were organized for team discussion about 3 specific topics: 1) Testing of cellular immune function in vitro and in vivo; 2) MABEL approach (toxicology and pharmacokinetic perspective); and 3) mRNA treatments. This workshop report presents the sessions and discussions at the meeting.

Exploration of piperidine-4-yl-aminopyrimidines as HIV-1 reverse transcriptase inhibitors. N-Phenyl derivatives with broad potency against resistant mutant viruses.

Further investigation of the recently reported piperidine-4-yl-aminopyrimidine class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been carried out. Thus, preparation of a series of N-phenyl piperidine analogs resulted in the identification of 3-carboxamides as a particularly active series. Analogs such as 28 and 40 are very potent versus wild-type HIV-1 and a broad range of NNRTI-resistant mutant viruses. Synthesis, structure-activity relationship (SAR), clearance data, and crystallographic evidence for the binding motif are discussed.

2020 FDA Drug-drug Interaction Guidance: A Comparison Analysis and Action Plan by Pharmaceutical Industrial Scientists.

BACKGROUND: In January 2020, the US FDA published two final guidelines, one entitled "In vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions Guidance for Industry" and the other entitled "Clinical Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions Guidance for Industry". These were updated from the 2017 draft in vitro and clinical DDI guidance. METHODS: This study is aimed to provide an analysis of the updates along with a comparison of the DDI guidelines published by the European Medicines Agency (EMA) and Japanese Pharmaceuticals and Medical Devices Agency (PMDA) along with the current literature. RESULTS: The updates were provided in the final FDA DDI guidelines and explained the rationale of those changes based on the understanding from research and literature. Furthermore, a comparison among the FDA, EMA, and PMDA DDI guidelines are presented in Tables 1, 2 and 3. CONCLUSION: The new 2020 clinical DDI guidance from the FDA now has even higher harmonization with the guidance (or guidelines) from the EMA and PMDA. A comparison of DDI guidance from the FDA 2017, 2020, EMA, and PMDA on CYP and transporter based DDI, mathematical models, PBPK, and clinical evaluation of DDI is presented in this review.

Preclinical Antitumor Activity and Biodistribution of a Novel Anti-GCC Antibody-Drug Conjugate in Patient-derived Xenografts.

Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody-dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).

Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation.

Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy.

Discovery of triazolinone non-nucleoside inhibitors of HIV reverse transcriptase.

Novel non-nucleoside inhibitors of HIV-RT that contain pyridazinone isosteres were prepared, and a series of triazolinones were found to be potent inhibitors of HIV replication. These compounds were active against several NNRTI-resistant virus strains. Pharmacokinetic studies indicated that inhibitor 7e has good bioavailability in rats. Several fragments of inhibitor 7c were prepared, and the binding of these compounds to HIV-RT was analyzed by surface plasmon resonance spectroscopy.

Impact of low-dose ritonavir on danoprevir pharmacokinetics: results of computer-based simulations and a clinical drug-drug interaction study.

BACKGROUND AND OBJECTIVE: Danoprevir, a potent, selective inhibitor of the hepatitis C virus (HCV) NS3/4A protease, is metabolized by cytochrome P450 (CYP) 3A. Clinical studies in HCV patients have shown a potential need for a high danoprevir daily dose and/or dosing frequency. Ritonavir, an HIV-1 protease inhibitor (PI) and potent CYP3A inhibitor, is used as a pharmacokinetic enhancer at subtherapeutic doses in combination with other HIV PIs. Coadministering danoprevir with ritonavir as a pharmacokinetic enhancer could allow reduced danoprevir doses and/or dosing frequency. Here we evaluate the impact of ritonavir on danoprevir pharmacokinetics. METHODS: The effects of low-dose ritonavir on danoprevir pharmacokinetics were simulated using Simcyp, a population-based simulator. Following results from this drug-drug interaction (DDI) model, a crossover study was performed in healthy volunteers to investigate the effects of acute and repeat dosing of low-dose ritonavir on danoprevir single-dose pharmacokinetics. Volunteers received a single oral dose of danoprevir 100 mg in a fixed sequence as follows: alone, and on the first day and the last day of 10-day dosing with ritonavir 100 mg every 12 hours. RESULTS: The initial DDI model predicted that following multiple dosing of ritonavir 100 mg every 12 hours for 10 days, the danoprevir area under the plasma concentration-time curve (AUC) from time zero to 24 hours and maximum plasma drug concentration (C(max)) would increase by about 3.9- and 3.2-fold, respectively. The clinical results at day 10 of ritonavir dosing showed that the plasma drug concentration at 12 hours postdose, AUC from time zero to infinity and C(max) of danoprevir increased by approximately 42-fold, 5.5-fold and 3.2-fold, respectively, compared with danoprevir alone. The DDI model was refined with the clinical data and sensitivity analyses were performed to better understand factors impacting the ritonavir-danoprevir interaction. CONCLUSION: DDI model simulations predicted that danoprevir exposures could be successfully enhanced with ritonavir coadministration, and that a clinical study confirming this result was warranted. The clinical results demonstrate that low-dose ritonavir enhances the pharmacokinetic profile of low-dose danoprevir such that overall danoprevir exposures can be reduced while sustaining danoprevir trough concentrations.

Diphenyl ether non-nucleoside reverse transcriptase inhibitors with excellent potency against resistant mutant viruses and promising pharmacokinetic properties.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are part of the preferred treatment regimens for individuals infected with HIV. These NNRTI-based regimens are efficacious, but the most popular NNRTIs have a low genetic barrier to resistance and have been associated with adverse events. There is therefore still a need for efficacious antiviral medicines that facilitate patient adherence and allow durable suppression of viral replication. As part of an extensive program targeted toward the discovery of NNRTIs that have favorable pharmacokinetic properties, good potency against NNRTI-resistant viruses, and a high genetic barrier to drug resistance, we focused on the optimization of a series of diaryl ether NNRTIs. In the course of this effort, we employed molecular modeling to design a new set of NNRTIs that that are active against wild-type HIV and key NNRTI-resistant mutant viruses. The structure-activity relationships observed in this series of compounds provide insight into the structural features required for NNRTIs that inhibit the replication of a wide range of mutant viruses. Selected compounds have promising pharmacokinetic profiles.

Design of annulated pyrazoles as inhibitors of HIV-1 reverse transcriptase.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of HIV. These regimens are extremely effective in suppressing virus replication. Structure-based optimization of diaryl ether inhibitors led to the discovery of a new series of pyrazolo[3,4-c]pyridazine NNRTIs that bind the reverse transcriptase enzyme of human immunodeficiency virus-1 (HIV-RT) in an expanded volume relative to most other inhibitors in this class.The binding mode maintains the beta13 and beta14 strands bearing Pro236 in a position similar to that in the unliganded reverse transcriptase structure, and the distribution of interactions creates the opportunity for substantial resilience to single point mutations. Several pyrazolopyridazine NNRTIs were found to be highly effective against wild-type and NNRTI-resistant viral strains in cell culture.