Rana Rais

Design, Synthesis, and Pharmacological Evaluation of Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl Sulfide 3 (BPTES) Analogs as Glutaminase Inhibitors

Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) is a potent and selective allosteric inhibitor of kidney-type glutaminase (GLS) that has served as a molecular probe to determine the therapeutic potential of GLS inhibition. In an attempt to identify more potent GLS inhibitors with improved drug-like molecular properties, a series of BPTES analogs were synthesized and evaluated. Our structure-activity relationship (SAR) studies revealed that some truncated analogs retained the potency of BPTES, presenting an opportunity to improve its aqueous solubility. One of the analogs, N-(5-{2-[2-(5-amino-[1,3,4]thiadiazol-2-yl)-ethylsulfanyl]-ethyl}-[1,3,4]thiadiazol-2-yl)-2-phenyl-acetamide 6, exhibited similar potency and better solubility relative to BPTES and attenuated the growth of P493 human lymphoma B cells in vitro as well as in a mouse xenograft model.

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer.

Significance There are no effective therapies currently available for advanced pancreatic cancer. We show that there are two populations of cancer cells within a pancreatic tumor that require targeting by different metabolic inhibitors for effective tumor control. Rapidly dividing cells use glutamine, and can be effectively killed by administration of a nanoparticle containing an inhibitor of glutamine metabolism. Hypoxic cells, which are slowly dividing cells, metabolize glucose and can be targeted by metformin, a drug used for the treatment of diabetes. Clinical trials are needed to determine whether combination therapy, with drugs that effectively block the metabolism of glutamine and glucose, improves the survival of patients with pancreatic cancer. Targeting glutamine metabolism via pharmacological inhibition of glutaminase has been translated into clinical trials as a novel cancer therapy, but available drugs lack optimal safety and efficacy. In this study, we used a proprietary emulsification process to encapsulate bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a selective but relatively insoluble glutaminase inhibitor, in nanoparticles. BPTES nanoparticles demonstrated improved pharmacokinetics and efficacy compared with unencapsulated BPTES. In addition, BPTES nanoparticles had no effect on the plasma levels of liver enzymes in contrast to CB-839, a glutaminase inhibitor that is currently in clinical trials. In a mouse model using orthotopic transplantation of patient-derived pancreatic tumor tissue, BPTES nanoparticle monotherapy led to modest antitumor effects. Using the HypoxCR reporter in vivo, we found that glutaminase inhibition reduced tumor growth by specifically targeting proliferating cancer cells but did not affect hypoxic, noncycling cells. Metabolomics analyses revealed that surviving tumor cells following glutaminase inhibition were reliant on glycolysis and glycogen synthesis. Based on these findings, metformin was selected for combination therapy with BPTES nanoparticles, which resulted in significantly greater pancreatic tumor reduction than either treatment alone. Thus, targeting of multiple metabolic pathways, including effective inhibition of glutaminase by nanoparticle drug delivery, holds promise as a novel therapy for pancreatic cancer.

Regional brain distribution of translocator protein using [11C]DPA-713 PET in individuals infected with HIV

Imaging the brain distribution of translocator protein (TSPO), a putative biomarker for glial cell activation and neuroinflammation, may inform management of individuals infected with HIV by uncovering regional abnormalities related to neurocognitive deficits and enable non-invasive therapeutic monitoring. Using the second-generation TSPO-targeted radiotracer, [11C]DPA-713, we conducted a positron emission tomography (PET) study to compare the brains of 12 healthy human subjects to those of 23 individuals with HIV who were effectively treated with combination antiretroviral therapy (cART). Compared to PET data from age-matched healthy control subjects, [11C]DPA-713 PET of individuals infected with HIV demonstrated significantly higher volume-of-distribution (VT) ratios in white matter, cingulate cortex, and supramarginal gyrus, relative to overall gray matter VT, suggesting localized glial cell activation in susceptible regions. Regional TSPO abnormalities were evident within a sub-cohort of neuro-asymptomatic HIV subjects, and an increase in the VT ratio within frontal cortex was specifically linked to individuals affected with HIV-associated dementia. These findings were enabled by employing a gray matter normalization approach for PET data quantification, which improved test–retest reproducibility, intra-class correlation within the healthy control cohort, and sensitivity of uncovering abnormal regional findings.

Inhibition of Glutamate Carboxypeptidase II (GCPII) activity as a treatment for cognitive impairment in multiple sclerosis

Half of all patients with multiple sclerosis (MS) experience cognitive impairment, for which there is no pharmacological treatment. Using magnetic resonance spectroscopy (MRS), we examined metabolic changes in the hippocampi of MS patients, compared the findings to performance on a neurocognitive test battery, and found that N-acetylaspartylglutamate (NAAG) concentration correlated with cognitive functioning. Specifically, MS patients with cognitive impairment had low hippocampal NAAG levels, whereas those with normal cognition demonstrated higher levels. We then evaluated glutamate carboxypeptidase II (GCPII) inhibitors, known to increase brain NAAG levels, on cognition in the experimental autoimmune encephalomyelitis (EAE) model of MS. Whereas GCPII inhibitor administration did not affect physical disabilities, it increased brain NAAG levels and dramatically improved learning and memory test performance compared with vehicle-treated EAE mice. These data suggest that NAAG is a unique biomarker for cognitive function in MS and that inhibition of GCPII might be a unique therapeutic strategy for recovery of cognitive function.

Pharmacokinetics of Oral d-Serine in d-Amino Acid Oxidase Knockout Mice

d-Amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-amino acids including d-serine, a full agonist at the glycine modulatory site of the N-methyl-d-aspartate (NMDA) receptor. To evaluate the significance of DAAO-mediated metabolism in the pharmacokinetics of oral d-serine, plasma d-serine levels were measured in both wild-type mice and transgenic mice lacking DAAO. Although d-serine levels were rapidly diminished in wild-type mice (t½ = 1.2 h), sustained drug levels over the course of 4 h (t½ > 10 h) were observed in mice lacking DAAO. Coadministration of d-serine with 6-chlorobenzo[d]isoxazol-3-ol (CBIO), a small-molecule DAAO inhibitor, in wild-type mice resulted in the enhancement of plasma d-serine levels, although CBIO seems to have only temporary effects on the plasma d-serine levels due to glucuronidation of the key hydroxyl group. These findings highlight the predominant role of DAAO in the clearance of d-serine from the systemic circulation. Thus, a potent DAAO inhibitor with a longer half-life should be capable of maintaining high plasma d-serine levels over a sustained period of time and might have therapeutic implications for the treatment of schizophrenia.

High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice

The dopamine D3 receptor (D3R) is a promising target for the development of pharmacotherapeutics to treat substance use disorders. Several D3R-selective antagonists are effective in animal models of drug abuse, especially in models of relapse. Nevertheless, poor bioavailability, metabolic instability, and/or predicted toxicity have impeded success in translating these drug candidates to clinical use. Herein, we report a series of D3R-selective 4-phenylpiperazines with improved metabolic stability. A subset of these compounds was evaluated for D3R functional efficacy and off-target binding at selected 5-HT receptor subtypes, where significant overlap in SAR with D3R has been observed. Several high affinity D3R antagonists, including compounds 16 (Ki = 0.12 nM) and 32 (Ki = 0.35 nM), showed improved metabolic stability compared to the parent compound, PG648 (6). Notably, 16 and the classic D3R antagonist SB277011A (2) were effective in reducing self-administration of heroin in wild-type but not D3R knockout mice.

D-Amino-Acid Oxidase Inhibition Increases D-Serine Plasma Levels in Mouse But not in Monkey or Dog.

D-serine has been shown to improve positive, negative, and cognitive symptoms when used as add-on therapy for the treatment of schizophrenia. However, D-serine has to be administered at high doses to observe clinical effects. This is thought to be due to D-serine undergoing oxidation by D-amino-acid oxidase (DAAO) before it reaches the brain. Consequently, co-administration of D-serine with a DAAO inhibitor could be a way to lower the D-serine dose required to treat schizophrenia. Early studies in rodents to evaluate this hypothesis showed that concomitant administration of structurally distinct DAAO inhibitors with D-serine enhanced plasma and brain D-serine levels in rodents compared with administration of D-serine alone. In the present work we used three potent DAAO inhibitors and confirmed previous results in mice. In a follow-up effort, we evaluated plasma D-serine levels in monkeys after oral administration of D-serine in the presence or absence of these DAAO inhibitors. Even though the compounds reached steady state plasma concentrations exceeding their Ki values by >60-fold, plasma D-serine levels remained the same as those in the absence of DAAO inhibitors. Similar results were obtained with dogs. In summary, in contrast to rodents, DAAO inhibition in monkeys and dogs did not influence the exposure to exogenously administered D-serine. Results could be due to differences in D-serine metabolism and/or clearance mechanisms and suggest that the role of DAAO in the metabolism of D-serine is different across species. These data provide caution regarding the utility of DAAO inhibition for patients with schizophrenia.

Highly Selective Dopamine D3 Receptor (D3R) Antagonists and Partial Agonists Based on Eticlopride and the D3R Crystal Structure: New Leads for Opioid Dependence Treatment

The recent and precipitous increase in opioid analgesic abuse and overdose has inspired investigation of the dopamine D3 receptor (D3R) as a target for therapeutic intervention. Metabolic instability or predicted toxicity has precluded successful translation of previously reported D3R-selective antagonists to clinical use for cocaine abuse. Herein, we report a series of novel and D3R crystal structure-guided 4-phenylpiperazines with exceptionally high D3R affinities and/or selectivities with varying efficacies. Lead compound 19 was selected based on its in vitro profile: D3R Ki = 6.84 nM, 1700-fold D3R versus D2R binding selectivity, and its metabolic stability in mouse microsomes. Compound 19 inhibited oxycodone-induced hyperlocomotion in mice and reduced oxycodone-induced locomotor sensitization. In addition, pretreatment with 19 also dose-dependently inhibited the acquisition of oxycodone-induced conditioned place preference (CPP) in rats. These findings support the D3R as a target for opioid dependence treatment and compound 19 as a new lead molecule for development.

Pharmacokinetics and Pharmacodynamics of the Glutamate Carboxypeptidase II Inhibitor 2-MPPA Show Prolonged Alleviation of Neuropathic Pain through an Indirect Mechanism

Glutamate carboxypeptidase II (GCP II) is a therapeutic target in neurologic disorders associated with excessive activation of glutamatergic systems. The potent, orally bioavailable GCP II inhibitor 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA) is effective in preclinical models of diseases where excess glutamate release is implicated, including neuropathic pain, and was the first GCP II inhibitor to be administered to man. The relationships between dosing regimen, pharmacokinetics, and analgesia in a neuropathic pain model were examined in rats to aid development of clinical dosing. The efficacy of oral 2-MPPA in the chronic constrictive injury model was not simply related to plasma concentrations. Even though maximal concentrations were observed within 1 hour of dosing, the analgesic effect took at least 8 days of daily dosing to become significant. The delay was not due to tissue drug accumulation since inhibitory concentrations of the drug were achieved in the nerve within 1 hour of dosing. There was also no accumulation of drug in plasma or tissue after multiple daily dosing. Effects were dependent on reaching a threshold concentration since dividing the daily dose led to a loss of effect. The analgesic effect outlasted plasma exposure and was maintained for days even after daily dosing was halted. The delayed onset, dependence on threshold plasma concentration, and sustained effects after exposure support the hypothesis that an indirect, long-lived mechanism of action exists. Although these longer lasting secondary mechanisms are not yet identified, daily clinical dosing of a GCP II inhibitor seems justified.

Quantitation of unbound sunitinib and its metabolite N-desethyl sunitinib (SU12662) in human plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study.

A rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of unbound sunitinib and its active metabolite N-desethyl sunitinib in plasma. Plasma and post-dialysis buffer samples were extracted using a liquid-liquid extraction procedure with acetonitrile-n-butylchloride (1:4, v/v). Chromatographic separation was achieved on a Waters X-Terra® MS RP(18) column with a mobile phase consisting of acetonitrile and water (60:40, v/v) containing formic acid (0.1%, v/v) using an isocratic run, at a flow-rate of 0.2 mL/min. Analytes were detected by electrospray tandem mass spectrometry in the selective reaction monitoring mode. Linear calibration curves were generated over the ranges 0.1-100 and 0.02-5 ng/mL for sunitinib and 0.2-200 and 0.04-10 ng/mL for N-desethyl sunitinib in plasma and in phosphate-buffered solution, respectively. The values for both within-day and between-day precision and accuracy were well within the generally accepted criteria for analytical methods. The analytical range was sufficient to determine the unbound and total concentrations of both analytes. The method was applied for measurement unbound concentrations in addition to total concentrations of sunitinib and its metabolite in plasma of a cancer patient receiving 50 mg daily dose.