Richard Hajdu

Metabolism of Thienamycin and Related Carbapenem Antibiotics by the Renal Dipeptidase, Dehydropeptidase-I

Thienamycin (THM), the N-formimidoyl thienamycin derivative MK0787, and related carbapenem antibiotics were metabolized extensively in mice, rats, rabbits, dogs, rhesus monkeys, and chimpanzees. Urinary recovery of THM ranged from a low of 5% in dogs to 58% in rhesus monkeys. Renal clearance rates in dogs and chimpanzees were unusually low, less than glomerular filtration rates. The reduction in clearance of THM and MK0787 from plasma of rats and rabbits after ligation of renal arteries indicate that the kidneys are responsible for 35 and 92%, respectively, of metabolic drug clearance. Degradation was detected only in kidney homogenates. The enzyme activity was membrane bound and sensitive to inhibitors of Zn-metalloenzymes such as EDTA. A renal dipeptidase, dehydropeptidase-I (DHP-I), EC 3.4.13.11, was found to be responsible for the metabolism of the THM-class antibiotics, which exhibit a structural homology to dehydropeptides. A parallel increase in specific activity against THM and the substrate of DHP-I, glycyldehydrophenylalanine, was observed during solubilization and purification of the enzyme from porcine and human renal cortex. DHP-I was found to catalyze the hydrolysis of the beta-lactam ring in THM and MK0787. The products of the enzyme reaction were identical by high-powered liquid chromatography to their respective metabolites found in the urine. Nonbasic N-acylated THM and natural N-acylated carbapenems (epithienamycins and olivanic acids) were degraded 4- to 50-fold faster than THM when exposed to the enzymatic hydrolysis of DHP-I. Good correlations were obtained between the increased susceptibility of the carbapenem antibiotics to DHP-I as measured in the in vitro enzyme assay and the generally lower recoveries of active antibiotic in the urine of test animals. Despite this unusual degree of metabolism localized in the kidney, the plasma half-life of MK0787 and its efficacy against experimental systemic infections in animals remain satisfactory.

Sphingosine 1‐phosphate receptor agonist FTY720‐phosphate causes marginal zone B cell displacement

FTY720 is an immunosuppressive agent that modulates lymphocyte trafficking. It is phosphorylated in vivo to FTY720‐phosphate (FTY‐P) and binds to a family of G protein‐coupled receptors recognizing sphingosine 1‐phosphate (S1P) as the natural ligand. It has previously been reported that FTY‐P blocks egress of lymphocytes from the thymus and lymph nodes, resulting in peripheral blood lymphopenia. We now report that FTY‐P also causes displacement of marginal zone (MZ) B cells to the splenic follicles, an effect that is similar to that observed after in vivo administration of lipopolysaccharide. This effect is specific to B cells in the MZ, as treatment with FTY‐P does not cause redistribution of the resident macrophage population. A small but statistically significant decrease in the expression of β1 integrin on MZ B cells was observed with FTY‐P treatment. The redistribution of MZ B cells from the MZ sinuses does not abolish the ability of these cells to respond to the T‐independent antigen, trinitrophenol‐Ficoll. It has been proposed that the displacement of MZ B cells to the follicles is an indication of cell activation. Consistent with this, FTY‐P caused an increase in percentage of MZ B cells expressing activation markers CD9, CD1d, and CD24. These results suggest that S1P receptors on MZ B cells are responsible for their mobilization to follicles.

1,3,8-Triazaspiro[4.5]decane-2,4-diones as efficacious pan-inhibitors of hypoxia-inducible factor prolyl hydroxylase 1-3 (HIF PHD1-3) for the treatment of anemia.

The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors). 1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original spiroindole hit. A new set of general conditions for C-N coupling, developed using a high-throughput experimentation (HTE) technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved. Spirohydantoins represent a class of highly efficacious, short-acting PHD1-3 inhibitors causing a robust erythropoietin (EPO) upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors with the potential for treatment of anemia.

Discovery of MK-4409, a Novel Oxazole FAAH Inhibitor for the Treatment of Inflammatory and Neuropathic Pain

We report herein the identification of MK-4409, a potent and selective fatty acid amide hydrolase (FAAH) inhibitor. Starting from a high throughput screening (HTS) hit, medicinal chemistry efforts focused on optimizing of FAAH inhibition in vitro potency, improving the pharmacokinetic (PK) profile, and increasing in vivo efficacy in rodent inflammatory and neuropathic pain assays.

Discovery of MK-3168: A PET Tracer for Imaging Brain Fatty Acid Amide Hydrolase.

We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.

Discovery of Benzimidazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys

We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Discovery of Triazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys

Hit-to-lead efforts resulted in the discovery of compound 19, a potent CYP11B2 inhibitor that displays high selectivity vs related CYPs, good pharmacokinetic properties in rat and rhesus, and lead-like physical properties. In a rhesus pharmacodynamic model, compound 19 displays robust, dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Lipopeptide antifungal agents: Amine conjugates of the semi-synthetic pneumocandins L-731,373 and L-733,560

Abstract Amine conjugates of the semi-synthetic 1,3-β-(D)-glucan synthesis inhibitors L-731,373 (3) and L-733,560 (4) were prepared and evaluated for in vitro and in vivo antifungal activity. Tricationic analogs were more potent than the dicationic which were more potent than the monocationic. The L-ornithine conjugate of 4 possessed excellent pharmacokinetic parameters but lacked sufficient antifungal spectrum for development.

Pharmacokinetics of L-671,329 in rhesus monkeys and DBA/2 mice.

The time course of plasma drug levels and urinary recovery for two lipopeptide antifungal antibiotics, L-671,329 and cilofungin, were measured in male rhesus monkeys (Macaca mulatta) and in female DBA/2 mice. The antibiotics were administered intravenously at 10 mg/kg of body weight in phosphate-buffered saline-26% polyethylene glycol for the rhesus monkeys and in 5% dimethyl sulfoxide for the mice. Plasma and urine drug concentrations were determined by high-pressure liquid chromatography and/or a microbiological assay versus Aspergillus niger, and pharmacokinetic parameters were determined for both species. In each of the two rhesus crossover tests as well as in the mouse studies, the pharmacokinetics of the two compounds were similar; however, a marked difference was evident between species. The half-lives of L-671,329 and cilofungin in plasma were 39 and 34 min in the mice and averaged 1.8 and 2 h in the rhesus monkeys, respectively. In mice and rhesus monkeys, urinary recovery was less than 4% for both compounds.

Discovery of N-[Bis(4-methoxyphenyl)methyl]-4-hydroxy-2-(pyridazin-3-yl)pyrimidine-5-carboxamide (MK-8617), an Orally Active Pan-Inhibitor of Hypoxia-Inducible Factor Prolyl Hydroxylase 1-3 (HIF PHD1-3) for the Treatment of Anemia.

The discovery of novel 4-hydroxy-2-(heterocyclic)pyrimidine-5-carboxamide inhibitors of hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD) is described. These are potent, selective, orally bioavailable across several species, and active in stimulating erythropoiesis. Mouse and rat studies showed hematological changes with elevations of plasma EPO and circulating reticulocytes following single oral dose administration, while 4-week q.d. po administration in rat elevated hemoglobin levels. A major focus of the optimization process was to decrease the long half-life observed in higher species with early compounds. These efforts led to the identification of 28 (MK-8617), which has advanced to human clinical trials for anemia.