Discovery of a brain-sparing GIRK1/4 inhibitor for pharmacological cardioversion of atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia, and a significant risk factor for ischemic stroke and heart failure. Marketed anti-arrhythmic drugs can restore sinus rhythm, but with limited efficacy and significant toxicities, including potential to induce ventricular arrhythmia. Atrial-selective ion channel drugs are expected to restore and maintain sinus rhythm without risk of ventricular arrhythmia. One such atrial-selective channel target is GIRK1/4 (G-protein regulated inwardly rectifying potassium channel 1/4). Here we describe 14b, a potent GIRK1/4 inhibitor developed to cardiovert AF to sinus rhythm while minimizing central nervous system exposure - an issue with preceding GIRK1/4 clinical candidates.

Safety and Efficacy of Engineered Toxin Body MT-3724 in Relapsed or Refractory B-cell Non-Hodgkin's Lymphomas and Diffuse Large B-cell Lymphoma.

MT-3724, a novel engineered toxin body comprised of an anti-CD20 single-chain variable fragment genetically fused to Shiga-like Toxin A subunit, is capable of binding to and internalizing against CD20, inducing cell killing via permanent ribosomal inactivation. This study evaluated MT-3724 in patients with relapsed/refractory B-cell non-Hodgkin lymphoma (r/rNHL). This open-label, multiple-dose phase Ia/b trial included a dose escalation in patients with r/rNHL according to a standard 3+3 design. Primary objectives were to determine the MTD and pharmacokinetics/pharmacodynamics. In a dose expansion study at MTD in serum rituximab-negative patients with diffuse large B-cell lymphoma (DLBCL), primary objectives were safety, tolerability, and pharmacokinetics/pharmacodynamics. Twenty-seven patients enrolled. MTD was 50 µg/kg/dose with 6,000 µg/dose cap. Thirteen patients experienced at least one grade ≥3 treatment-related adverse events; the most common grade ≥3 event was myalgia (11.1%). Two patients (75 µg/kg/dose) experienced grade 2 treatment-related capillary leak syndrome. Overall objective response rate was 21.7%. In serum rituximab-negative patients with DLBCL or composite DLBCL (n = 12), overall response rate was 41.7% (complete response, n = 2; partial response, n = 3). In patients with detectable baseline peripheral B cells, treatment resulted in dose-dependent B-cell depletion. The proportion of patients with anti-drug antibodies (ADA) increased during treatment and the majority appeared to be neutralizing based on an in vitro assay; nevertheless, tumor regression and responses were observed. MT-3724 demonstrated efficacy at the MTD in this population of previously treated patients with r/rDLBCL, with mild-to-moderate immunogenic safety events. Significance: This work describes the safety and efficacy of a new pharmaceutical pathway that could provide a treatment option for a subset of patients with a critical unmet therapeutic need. The study drug, MT-3724, is capable of targeting B-cell lymphomas via a unique, potent cell-killing mechanism that appears to be promising.

Phenotypic screen identifies calcineurin-sparing FK506 analogs as BMP potentiators for treatment of acute kidney injury.

Acute kidney injury (AKI) is a life-threatening disease with no known curative or preventive therapies. Data from multiple animal models and human studies have linked dysregulation of bone morphogenetic protein (BMP) signaling to AKI. Small molecules that potentiate endogenous BMP signaling should have a beneficial effect in AKI. We performed a high-throughput phenotypic screen and identified a series of FK506 analogs that act as potent BMP potentiators by sequestering FKBP12 from BMP type I receptors. We further showed that calcineurin inhibition was not required for this activity. We identified a calcineurin-sparing FK506 analog oxtFK through late-stage functionalization and structure-guided design. OxtFK demonstrated an improved safety profile in vivo relative to FK506. OxtFK stimulated BMP signaling in vitro and in vivo and protected the kidneys in an AKI mouse model, making it a promising candidate for future development as a first-in-class therapeutic for diseases with dysregulated BMP signaling.

A Phase I Dose-Escalation Study to Evaluate the Safety and Tolerability of Evofosfamide in Combination with Ipilimumab in Advanced Solid Malignancies.

PURPOSE: As hypoxia can mediate resistance to immunotherapy, we investigated the safety, tolerability, and efficacy of combining evofosfamide, a prodrug that alleviates hypoxia, with ipilimumab, an immune checkpoint inhibitor, in immunologically "cold" cancers, which are intrinsically insensitive to immunotherapy, as well as in "hot/warm" metastatic cancers that are, atypical of such cancers, resistant to immunotherapy. PATIENTS AND METHODS: In a phase I, 3+3 dose-escalation trial (NCT03098160), evofosfamide (400-640 mg/m2) and ipilimumab (3 mg/kg) were administered in four 3-week cycles. The former was administered on days 1 and 8 of cycles 1-2, while the latter was administered on day 8 of cycles 1-4. Response was assessed using immune-related RECIST and retreatment was allowed, if deemed beneficial, after completion of cycle 4 or at progression. RESULTS: Twenty-two patients were enrolled, of whom 21 were evaluable, encompassing castration-resistant prostate cancer (n = 11), pancreatic cancer (n = 7), immunotherapy-resistant melanoma (n = 2), and human papillomavirus-negative head and neck cancer (n = 1). Drug-related hematologic toxicities, rash, fever, nausea, vomiting, and elevation of liver enzymes were observed in > 10% of patients. The most common drug-related grade 3 adverse event was alanine aminotransferase elevation (33.3%). Two patients discontinued ipilimumab and 4 required evofosfamide deescalation due to toxicity. Of 18 patients with measurable disease at baseline, 3 (16.7%) achieved partial response and 12 (66.7%) achieved stable disease. The best responses were observed at 560 mg/m2 evofosfamide. Preexisting immune gene signatures predicted response to therapy, while hypermetabolic tumors predicted progression. Responders also showed improved peripheral T-cell proliferation and increased intratumoral T-cell infiltration into hypoxia. CONCLUSIONS: No new or unexpected safety signals were observed from combining evofosfamide and ipilimumab, and evidence of therapeutic activity was noted.

Author Correction: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.

Reversible inhibition of human carboxylesterases by acyl glucuronides.

UNLABELLED: Carboxylesterases hydrolyze esters, amides, and thioesters to produce carboxylic acids and resulting alcohols, amines, and thiols, respectively. Uridine 5'-diphosphate- glucuronosyltransferases are colocalized with carboxylesterases and have the potential to further metabolize carboxylic acids to acyl glucuronides, but it is currently unknown if acyl glucuronides, being esters, also interact with carboxylesterases. OBJECTIVE: This study explores the ability of acyl glucuronides to act as substrates or inhibitors of human carboxylesterases 1 (hCES1) and 2 (hCES2). METHODS: The stability of six acyl glucuronides in the presence of hCES1, hCES2, and buffer alone (100 mM potassium phosphate, pH 7.4, 37°C) were investigated. Reversible inhibition of 4-nitrophenyl acetate hydrolysis by the acyl glucuronides was also studied. Diclofenac-ß-d-glucuronide was used to explore potential time-dependent inactivation. RESULTS: The chemical stability half-life values for CGP 47292-ß-d-glucuronide, diclofenac-ß-d-glucuronide, (R)-naproxen-ß-d-glucuronide, (S)-naproxen-ß-d-glucuronide, ibuprofen-ß-d-glucuronide (racemic), clopidogrel-ß-d-glucuronide, and valproate-ß-d-glucuronide were found to be 0.252, 0.537, 0.996, 1.77, 3.67, 5.02, and 15.2 hours, respectively. Diclofenac-ß-d-glucuronide, clopidogrel-ß-d-glucuronide, ibuprofen-ß-d-glucuronide, (R)-naproxen-ß-d-glucuronide, and (S)-naproxen-ß-d-glucuronide selectively inhibited hCES1, with Ki values of 4.32 ± 0.47, 24.8 ± 4.2, 355 ± 38, 468 ± 21, 707 ± 64 µM, respectively, but did not significantly inhibit hCES2. Valproate-ß-d-glucuronide and CGP 47292-ß-d-glucuronide did not inhibit either hCES. Time-dependent inactivation of hCES1 by diclofenac-ß-d-glucuronide was not observed. Lastly, both hCES1 and hCES2 were shown not to catalyze the hydrolysis of the acyl glucuronides studied. CONCLUSION: Drug-drug interaction studies may be warranted for drugs that metabolize to acyl glucuronides due to the potential inhibition of hCESs.

Investigation of the metabolism of rufinamide and its interaction with valproate.

Rufinamide was evaluated in vitro to determine which enzyme(s) are responsible for rufinamide hydrolysis and whether valproate, one of its metabolites (valproyl-CoA), and/or the rufinamide hydrolysis product (CGP 47292) could inhibit hydrolysis. Rufinamide hydrolysis was mediated primarily by human carboxylesterase (hCE) 1 and was nonsaturable up to 500 µM. Two-thirds of rufinamide hydrolysis was estimated to occur in human microsomes and one-third in cytosol. Valproate was a selective inhibitor for hCE1 compared to hCE2 and inhibition had a greater impact on rufinamide hydrolysis in microsomes than in cytosol. Valproyl-CoA caused similar inhibition of rufinamide hydrolysis in both microsomes and cytosol. Carboxylesterases were not significantly inhibited by CGP 47292. Inhibition of in vitro rufinamide hydrolysis by valproate could offer an explanation for the observed in vivo drug-drug interaction between the two antiepileptic drugs.

Characterization of the expression and activity of carboxylesterases 1 and 2 from the beagle dog, cynomolgus monkey, and human.

The carboxylesterases (CESs) are a family of serine hydrolases that hydrolyze compounds containing an ester, amide, or thioester. In humans, two dominant forms, CES1 and CES2, are highly expressed in organs of first-pass metabolism and play an important role in xenobiotic metabolism. The current study was conducted to better understand species-related differences in substrate selectivity and tissue expression of these enzymes. To elucidate potential similarities and differences among these enzymes, a series of 4-nitrophenyl esters and a series of gemcitabine prodrugs were evaluated using enzyme kinetics as substrates of expressed and purified CESs from beagle dog, cynomolgus monkey, and human genes. For the substrates examined, human and monkey CES2 more efficiently catalyzed hydrolysis compared with CES1, whereas CES1 was the more efficient enzyme in dog. Quantitative real-time polymerase chain reaction and Western blot analyses indicate that the pattern of CES tissue expression in monkey is similar to that of human, but the CES expression in dog is unique, with no detectable expression of CES in the intestine. Loperamide, a selective human CES2 inhibitor, was also found to be a CES2-selective inhibitor in both dog and monkey. This is the first study to examine substrate specificity among dog, human, and monkey CESs.

Characterization of recombinant human carboxylesterases: fluorescein diacetate as a probe substrate for human carboxylesterase 2.

Human carboxylesterase (CES) 1 and CES2 are members of the serine hydrolase superfamily, and both exhibit broad substrate specificity and are involved in xenobiotic and endobiotic metabolism. Although expression of CES1 and CES2 occurs in several organs, their expression in liver and small intestine is predominantly attributed to CES1 and CES2, respectively. We successfully expressed CES1 form b (CES1-b) and form c (CES1-c) as well as CES2 in baculovirus-infected High Five insect cells. With 4-nitrophenyl acetate (4-NPA) as the probe substrate, the K(m) values of recombinant CES1-b and CES2 matched those of human liver microsomes (HLM) and human intestinal microsomes (HIM) with approximately 200 and 180 µM, respectively. Bis(4-nitrophenyl) phosphate potently inhibited 4-NPA hydrolysis by HLM, CES1-b, CES1-c, HIM, and CES2 with IC(50) values less than 1 µM. With fluorescein diacetate (FD) as the substrate, the K(m) values were similar for all enzyme systems, with the exception of CES1-b, which was slightly lower; however, the V(max) values for HIM and CES2 were 39.5 and 14.6 µmol · mg(-1) · min(-1), respectively, which were at least 50-fold higher than those of CES1-b or CES1-c. Loperamide potently inhibited HLM, HIM, and CES2 with similar IC(50) values of approximately 1 µM. Substrate specificity was compared between human tissues and recombinant enzymes. The data suggest the following: 1) FD is a probe substrate for CES2; 2) CES1-b is the predominant form in human liver; and 3) recombinant CES1-b and CES2 expressed in insect cells are functionally consistent with native carboxylesterases expressed in human liver and intestine, respectively.

In vitro screening of metabolic clearance using two concentration points.

For high throughput screens, the quickest methodology possible is desirable, but a substantial amount of potentially useful information is lacking since most screens for metabolic stability are conducted at one concentration, and sometimes at one time point. Information that would benefit projects during the discovery phase are to know the metabolic rate linearity (K(m) value) and projected hepatic clearance (CL(h) value), which is possible by the addition of one more concentration. This study used the FDA-preferred probe cytochrome P450 substrates to determine K(m), V(max), and CL(int) values. The results showed that compounds with relatively high metabolic rates produced more accurate and reproducible results that match well with predicted K(m) values according to the FDA. On the other hand, compounds with relatively low metabolic rates yielded more variable results. Thus, the use of two substrate concentrations should be useful with screening assays for assessing the kinetic values for other compounds.

Genomic analysis of the carboxylesterases: identification and classification of novel forms.

Large species differences in the expression of carboxylesterases (CE) have been described, but the interrelationships of CEs across species are not well characterized. In the current analyses, sequences with genomic structures similar to human CEs were found in piscine, avian, and mammalian genomes. Analyses of these genes suggest that four CE groups existed prior to mammalian divergence, with another form occurring after eutherian-marsupial divergence, yielding five distinct mammalian CE groups. The CE1 and CE2 groupings appear to have undergone extensive gene duplication in species with herbivorous and omnivorous diets underscoring the potential importance of these two groups in xenobiotic metabolism. However, CE3, CE4, and CE5 have remained at one gene per species in almost all observed cases. In avian and piscine genomes, only two CE groupings each were observed in the currently available sequence data. Finally, this study presents considerations for a broader phylogenetic-based nomenclature that could encompass other serine hydrolases in addition to the CEs.

The biotransformation of prasugrel, a new thienopyridine prodrug, by the human carboxylesterases 1 and 2.

2-Acetoxy-5-(alpha-cyclopropylcarbonyl-2-fluorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (prasugrel) is a novel thienopyridine prodrug with demonstrated inhibition of platelet aggregation and activation. The biotransformation of prasugrel to its active metabolite, 2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid (R-138727), requires ester bond hydrolysis, forming the thiolactone 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone(R-95913), followed by cytochrome P450-mediated metabolism to the active metabolite. The presumed role of the human liver- and intestinal-dominant carboxylesterases, hCE1 and hCE2, respectively, in the conversion of prasugrel to R-95913 was determined using expressed and purified enzymes. The hydrolysis of prasugrel is at least 25 times greater with hCE2 than hCE1. Hydrolysis of prasugrel by hCE1 showed Michaelis-Menten kinetics yielding an apparent K(m) of 9.25 microM and an apparent V(max) of 0.725 nmol product/min/microg protein. Hydrolysis of prasugrel by hCE2 showed a mixture of Hill kinetics at low substrate concentrations and substrate inhibition at high concentrations. At low concentrations, prasugrel hydrolysis by hCE2 yielded an apparent K(s) of 11.1 microM, an apparent V(max) of 19.0 nmol/min/microg, and an apparent Hill coefficient of 1.42, whereas at high concentrations, an apparent IC(50) of 76.5 microM was obtained. In humans, no in vivo evidence of inhibition exists. In vitro transport studies using the intestinal Caco-2 epithelial cell model showed a high in vivo absorption potential for prasugrel and rapid conversion to R-95913. In conclusion, the human carboxylesterases efficiently mediate the conversion of prasugrel to R-95913. These data help explain the rapid appearance of R-138727 in human plasma, where maximum concentrations are observed 0.5 h after a prasugrel p.o. dose, and the rapid onset of action of prasugrel.

Effect of buffer components and carrier solvents on in vitro activity of recombinant human carboxylesterases.

INTRODUCTION: The effects of buffer and substrate solvent conditions on in vitro activity of carboxylesterases (CE) have not been previously described. Therefore, it is unknown if the many different assay conditions used by various laboratories have a substantial impact on the activity of CE enzymes. METHODS: Three human CEs were expressed and purified, and the hydrolysis of 4-nitrophenyl butyrate was measured to assess enzyme activity. Four buffers (HEPES, potassium phosphate, sodium phosphate, and Tris) were evaluated for their effects on enzyme activity at concentrations ranging from 5 to 900 mM, as well as phosphate buffered saline. Five commonly used substrate-carrier solvents (acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol) ranging from 0.25 to 6% were also assessed for their effect on enzyme activity. RESULTS: The clearances for the CEs in HEPES, potassium phosphate, sodium phosphate, and Tris up to 100 mM were similar to the CE clearances obtained with phosphate buffered saline. Higher buffer concentrations resulted in differential activity of the CEs. All three CEs tolerated the substrate solvents up to 2% as indicated by little effect of solvent on catalytic activity. At substrate solvent concentrations above 2% the CE activities were found to gradually decrease. In general, CES3 displayed substantially lower activity than CES1 and CES2. DISCUSSION: In conclusion, any of the buffers examined up to 100 mM resulted in clearance values similar to that of phosphate buffered saline for the hydrolysis of 4-nitrophenyl butyrate by the human CEs. With regard to the substrate solvents tested, acetone, acetonitrile, or dimethyl sulfoxide appear to be well tolerated by the CEs up to 2% of the total reaction volume.

The chimpanzee cytochrome P450 3A subfamily: Is our closest related species really that similar?

With the release of the chimpanzee genomic database, much work has been accomplished to understand more fully the closest related species to humans. This study investigates the cytochrome P450 3A (CYP3A) subfamily and examines differences which may be expected between chimpanzees and humans in regards to CYP3A metabolism. A previous publication had reported the presence of five putative chimpanzee CYP3A isoforms, as compared to the four in humans (Williams ET et al., Mol Phylogenet Evol 33, 300-8). Based on the previous report, the chimpanzee CYP3A5 should have had a different C-terminus than its human counterpart; therefore, CYP3A5 and CYP3A67 were cloned. The CYP3A5 clone obtained disputes the previous prediction and confirms that the nucleotide similarity between the two species is 99.7%. While CYP3A67 is most closely related to CYP3A7, with significant differences in the amino acid sequences. Also, the mRNA expression of CYP3A67 can rival the expression of CYP3A4 in the tissues analyzed. CYP3A7 was not found to be expressed in any chimpanzee tissue examined. Total CYP3A protein expression was not significantly different between chimpanzees and humans. Metabolism assays using benzphetamine and erythromycin with chimpanzee liver microsomes did not reveal major differences between chimpanzees and humans. In conclusion, adult CYP3A metabolism may not be significantly different between chimpanzees and humans.

Defining relationships between the known members of the cytochrome P450 3A subfamily, including five putative chimpanzee members.

An analysis of the cytochrome P450 3A subfamily (CYP3A) was undertaken in order to define relationships across species among subfamily members. Some members were excluded due to incomplete sequences, while others were held in abeyance because of their almost complete homology. This is the first publication of five chimpanzee CYP3A genes-CYP3A4, CYP3A5, CYP3A7, CYP3A43, and CYP3A67. This project utilized two approaches for characterizing possible relationships-phylogenetic analysis and genomic structure. For the phylogenetic analysis, both nucleotide and amino acid sequences were aligned in silico using the CLUSTAL algorithm, and then visually inspected for accuracy. Three different computer software packages were utilized: MEGA 2.1, TREECON 1.3b, and PHYLIP 3.5. Multiple methods were used: neighbor-joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML). The resulting topologies were compared against each other to define the consensus topology. In addition, the chimpanzee, human, mouse, and rat genome databases were searched for intron/exon information pertaining to the included genes. Both methods suggest the same conclusion, defining orthologs is plausible between similar species (i.e., mouse and rat), but is less useful between species of different orders (i.e., primate and rodent) or classes (i.e., mammal and avian).

Estrogen regulation of the cytochrome P450 3A subfamily in humans.

This study examines the possible role of estrogen in regulating the expression of the human CYP3A subfamily: CYP3A4, CYP3A5, CYP3A7, and CYP3A43. To accomplish this goal, mRNA was quantified from human livers and endometrial samples, and total CYP3A protein levels were evaluated by Western immunoblot analysis of the liver samples. The human endometrial samples were from premenopausal and postmenopausal women. The premenopausal endometrium was either in the proliferative or secretory phase, whereas for the postmenopausal endometrium samples, the women had been treated with either a placebo or estropipate, an estrogen substitute. After analyses, CYP3A4 mRNA was shown to have lower hepatic expression in females than in males. In the endometrium, CYP3A4 and CYP3A43 are down-regulated by estrogen, whereas CYP3A5 is expressed at higher levels during the secretory phase. CYP3A7 was not detected in the endometrium. In addition, the CYP3A subfamily showed increased mRNA expression in the liver as age increased. The expression levels of total CYP3A protein and total CYP3A mRNA showed good correlation. Despite apparent regulation of CYP3A4 mRNA expression by estrogen, the effects of estrogen may be overshadowed by additional regulators of gene expression.