Determination of in Vivo Enzyme Occupancy Utilizing Inhibitor Dissociation Kinetics.

During drug discovery, assessment of in vivo target occupancy by therapeutic candidates is often required for predicting clinical efficacy. Current strategies for determining target occupancy include using radiolabeled or irreversible surrogates, which can be technically challenging, and the results are often not sufficiently quantitative. We developed a straightforward method by applying slow-dissociation kinetics to quantitatively determine enzyme occupancy without using specialized reagents. We applied this method to determine occupancy of Cathepsin K inhibitors in bone tissues harvested from rabbit femurs. Tissues from dosed animals were harvested, flash frozen, lysed, then analyzed by a jump-dilution assay with substrate. The rate of substrate turnover was monitored continuously until reaching steady state and progress curves were fit with the equation [product] = vst + ((vi - vs)/kobs)(1 - exp(-kobst)). The initial rate vi represents the residual activity of the enzyme before inhibitor dissociation; vs is the reaction rate after dissociation of the inhibitor. Occupancy is derived from the ratio of vi/vs. A significant benefit of the method is that data from both the occupied and unoccupied states are obtained in the same assay under identical conditions, which provides greater consistency between studies. The Cat K inhibitor MK-0674 (in vitro IC50 1 nM) was tested in young rabbits (<6 month old) and showed a dose-dependent increase in occupancy, reaching essentially complete occupancy at 1.0 mg/kg. In addition the method enables measurement of the total Cat K in the target tissue. Results confirmed complete occupancy even as the osteoclasts responded to higher doses with increased enzyme production.

Discovery of selective glucocorticoid receptor modulator MK-5932.

A series of partial agonists of the Glucocorticoid Receptor were prepared targeting reduced transactivation activity, while maintaining significant transrepression activity. Incorporation of an ortho-aryl amide produced compounds with the desired in vitro profile. Bioreactors consisting of Suspension cultures of Sf21 cells co expressing a CYP3A4 and NADPH-cytochrome P450 oxireductase were used to prepare the major metabolites of these compounds and revealed that oxidative N-dealkylation provided a pathway for formation of metabolites that were more agonistic than the parent partial agonists. Oxidative N-dealkylation was blocked in a new series of compounds, however oxidation alone was capable of producing full agonist metabolites. Incorporation of an ortho-primary amide and utilization of fluorine to modulate agonism afforded partial agonist MK-5932. Synthesis of the major metabolites of MK-5932 using bioreactor technology revealed that no significant GR-active metabolites were formed. Orally administered MK-5932 displayed anti-inflammatory efficacy in a Rat Oxazolone-induced chronic dermatitis model, while sparing plasma insulin.

Development of a pharmaceutical cocrystal of a monophosphate salt with phosphoric acid.

We report the first case of a pharmaceutical cocrystal formed between an inorganic acid and an active pharmaceutical ingredient (API), which enabled us to develop a stable crystalline and bioavailable solid dosage form for pharmaceutical development where otherwise only unstable amorphous free form or salts could have been used.

Applicability of in vitro-in vivo translation of cathepsin K inhibition from animal species to human with the use of free-drug hypothesis.

The correlation of in vitro inhibition of cathepsin K (CatK) activity and in vivo suppression of collagen I biomarkers was examined with three selective CatK inhibitors to explore the potential translatability from animal species to human. These inhibitors exhibited good in vitro potencies toward recombinant CatK enzymes across species, with IC50 values ranging from 0.20 to 6.1 nM. In vivo studies were conducted in animal species following multiple-day dosing of the CatK inhibitors to achieve steady-state plasma drug concentration-time profiles. Measurement of urinary bone resorption biomarkers (cross-linked N-terminal telopeptide and helical peptide of type I collagen) revealed drug concentration-dependent suppression of biomarkers, with EC50 values estimated to be 12 to 160 nM. Marked improvement in the correlation between in vitro and in vivo CatK activities was observed with the application of unbound (free) fraction in plasma, consistent with the conditions stipulated by the free-drug hypothesis. These results indicate that the in vitro-in vivo translation of CatK inhibition observed in animal species can translate to humans when the unbound fraction of the inhibitor is considered. Interestingly, residual levels of urinary bone resorption marker were detected as the suppression reached saturation (at an average of 82% inhibition), an apparent phenomenon observed regardless of the species, biomarker, or compound examined. Since cathepsin enzymes other than CatK were reported to catalyze cleavage of collagen I, it is hypothesized that CatK-mediated degradation of collagen I in bone represents ~82% of overall collagen I turnover in the body.

Comparative bioavailability study in dogs of a self-emulsifying formulation of progesterone presented in a pellet and liquid form compared with an aqueous suspension of progesterone.

A pellet formulation of progesterone in a self-emulsifying system (SES) was prepared by the process of extrusion/spheronization to provide a good in vitro drug release (100% within 30 min, T(50%) at 13 min). A three-way randomized crossover study was performed in six fasted male beagle dogs with these pellets and the same SES liquid formulation, both contained in a hard shell capsule, and an aqueous suspension. The same dose of progesterone (16 mg) in pellets and in the SES liquid formulation resulted in similar AUC, C(max) and T(max) values, estimated from progesterone plasma levels by (125)I radioimmunoassay. Although the maximum absorption was slightly retarded (0.5 to 1 h) by SES (pellets and liquid), AUC and C(max) were approximately seven and nine times greater then those obtained when an aqueous suspension formulation of the same dose of progesterone was administered to the same dogs. These results showed that it was possible to improve the bioavailability of the poorly soluble, poorly permeable progesterone when administered in SES. Moreover, presenting the progesterone in the form of a pellet did not prevent the release of the drug in vivo. These data demonstrate the utility of extrusion/spheronization in delivering a nonaqueous system in a novel solid dosage form.

Influence of physicochemical properties and intestinal region on the absorption of 3-fluoro-2-pyrimidylmethyl 3-(2,2-difluoro-2-(2-pyridyl)ethylamino)-6-chloropyrazin-2-one-1-acetamide, a water insoluble thrombin inhibitor, in dogs.

In this paper, we describe the physicochemical and biopharmaceutical properties of 3-fluoro-2-pyrimidylmethyl 3-(2,2-difluoro-2-(2-pyridyl)ethylamino)-6-chloropyrazin-2-one-1-acetamide, a direct thrombin inhibitor (1, Fig. 1). Three crystalline forms were characterized and studies were planned to investigate the absorption characteristics of the three selected crystalline forms. Due to the short half-life observed in preclinical species, regional absorption studies were also conducted to support potential controlled release formulation development. Results showed that the absorption of 1 was dependent on the surface area of the particles administered as suspensions and was independent of the crystal forms. From Caco-2 cell transport studies, it was determined that the permeability of 1 was high. Based on the low aqueous solubility it would be classified as a class 2 compound in the Biopharmaceutics Classification System. Regional absorption results suggested that the compound was absorbed along the gastrointestinal tract in Beagle dogs, however colonic absorption appeared to be reduced by slower dissolution.

The preclinical efficacy, selectivity and pharmacologic profile of MK-5932, an insulin-sparing selective glucocorticoid receptor modulator.

Glucocorticoids are used widely in the treatment of inflammatory diseases, but use is accompanied by a significant burden of adverse effects. It has been hypothesized that gene- and cell-specific regulation of the glucocorticoid receptor by small molecule ligands could be translated into a therapeutic with an improved risk-benefit profile. MK-5932 is a highly selective glucocorticoid receptor modulator that is anti-inflammatory in vivo with an improved profile on glucose metabolism: Bungard et al. (2011). Bioorg. Med. Chem. 19, 7374-7386. Here we describe the full biological profile of MK-5932. Cytokine production following lipopolysaccharide (LPS) challenge was blocked by MK-5932 in both rat and human whole blood. Oral administration reduced inflammatory cytokine levels in the serum of rats challenged with LPS. MK-5932 was anti-inflammatory in a rat contact dermatitis model, but was differentiated from 6-methylprednisolone by a lack of elevation of fasting insulin or glucose levels after 7 days of dosing, even at high exposure levels. In fact, animals in the vehicle group were consistently hyperglycemic at the end of the study, and MK-5932 normalized glucose levels in a dose-dependent manner. MK-5932 was also anti-inflammatory in the rat collagen-induced arthritis and adjuvant-induced arthritis models. In healthy dogs, oral administration of MK-5932 exerted acute pharmacodynamic effects with potency comparable to prednisone, but with important differences on neutrophil counts, again suggestive of a dissociated profile. Important gaps in our understanding of mechanism of action remain, but MK-5932 will be a useful tool in dissecting the mechanisms of glucose dysregulation by therapeutic glucocortiocids.

The importance of chromatographic separation in LC/MS/MS quantitation of drugs in biological fluids: detection, characterization, and synthesis of a previously unknown low-level nitrone metabolite of a substance P antagonist.

The observation of an interference peak in plasma samples from dogs dosed with compound I led to the discovery of an unidentified metabolite. The unknown metabolite had the same molecular weight as the parent drug, and their fragmentation profiles were also quite similar. LC/MS/ MS analysis of the plasma extracts of dogs and rats dosed with I and its deuterium-labeled analogue suggested a nitrone structure for the unknown metabolite. Synthesized nitrone matched the unknown metabolite with identical retention time and nearly identical fragmentation profile. The nitrone slowly decomposed in acidic aqueous solution at ambient temperature and also underwent in-source, thermal-induced hydrolysis during electrospray ionization mass spectrometric analysis. The reaction of the nitrone with diethyl acetylenedicarboxylate readily generated a [2 + 3] cycloaddition product. The example shown here clearly demonstrates that precautions must be taken when LC/MS/MS quantitation is conducted in the selected reaction monitoring mode.