In a drug discovery environment, reasonable go/no-go human in-vivo pharmacokinetic (PK) decisions must be made in a timely manner with a minimum amount of animal in-vivo or in-vitro data. We have investigated the accuracy of the in-vivo correlation between rat and human for the prediction of the total systemic clearance (CL), the volume of distribution at steady state (Vss), and the half-life (t1/2) using simple allometric scaling techniques. We have shown, using a large diverse set of drugs, that a fixed exponent allometric scaling approach can be used to predict human in-vivo PK parameters CL, Vss and t(1/2) solely from rat in-vivo PK data with acceptable accuracy for making go/no-go decisions in drug discovery. Human in-vivo PK predictions can be obtained using the simple allometric scaling relationships CL(Human) approximately = 40 CL(Rat) (L/hr), Vss(Human) approximately = 200 Vss(Rat) (L), and t1/2(Human) approximately = 4 t1/2(Rat) (hr). The average fold error for human CL predictions for N = 176 drugs was 2.25 with 79% of the drugs having a fold error less than 3. The average fold error for human Vss predictions for N = 144 drugs was 1.85 with 84% of the drugs having a fold error less than 3. The average fold error for human t1/2 predictions for N = 145 drugs was 2.05 with 76% of the drugs having a fold error less than 3. Using these simple allometric relationships, the sorting of drug candidates into a low/medium/high/very high human classification scheme was also possible from rat data. Since these simple allometric relationships between rat and human CL, Vss, and t1/2 are reasonably accurate, easy to remember and simple to calculate, these equations should be useful for making early go/no-go in-vivo human PK decisions for drug discovery candidates.

Heat shock protein 90 (Hsp90) is critical for the maturation of numerous client proteins, many of which are involved in cellular transformation and oncogenesis. The ansamycins, geldanamycin (GA) and its derivative, 17-allylaminogeldanamycin (17-AAG), inhibit Hsp90. As such, the prototypical Hsp90 inhibitor, 17-AAG, has advanced into clinical oncology trials. GA and 17-AAG potently inhibit tumor cell proliferation and survival but have been reported to bind weakly to Hsp90 in vitro. Recent studies have suggested that the in vitro potency of ansamycins against Hsp90 may be enhanced in the presence of cochaperones. Here, we present evidence of an alternative explanation. Ansamycins reduced to their dihydroquinones in the presence of common reducing agents in vitro have approximately 40-fold greater affinity than the corresponding oxidized quinones. The dihydroquinone of 17-AAG is not generated in an aqueous environment in the absence of reducing agents but is produced in both tumor and normal quiescent epithelial cells. The reduced form of 17-AAG is differentiated from its oxidized form not only by the higher affinity for Hsp90 but also by a protracted K(off) rate. Therefore, the in vivo accumulation of the high-affinity dihydroquinone ansamycins in tumor cells contributes to the antitumor activity of these compounds and alters our understanding of the active species driving the efficacy of this class of compounds.

Glucuronidation is a common mechanism in drug metabolism. In-source dissociation of glucuronides in electrospray generates fragment ions identical to those of the precursor ions of the original drugs. The effect of experimental parameters on the process was investigated in the present study using both commercially available compounds and glucuronides generated from microsomal glucuronidation incubations. It was found that cone voltage was the most critical parameter contributing to in-source fragmentation of both O- and N-glucuronides, whereas both the desolvation temperature and the source temperature had little effect. Additionally, the extent of in-source dissociation varied for different glucuronides and could be minimized by lowering cone voltage. As demonstrated in real examples, minimizing in-source dissociation can lead to higher sensitivity in detecting glucuronides in biological samples. In addition, product ions resulting from in-source dissociation of glucuronides potentially interfere with accurate determinations of corresponding drug levels if chromatographic separation is not adequate. For cases in which chromatographic separation of glucuronides from the original drugs is not readily achieved or high-throughput analyses are desired, interference caused by in-source dissociation can usually be eliminated simply by using lower cone voltage. This approach has been proven to be effective in the analysis of more than 100 glucuronides generated from in vitro microsomal incubations.

We have used capillary electrophoresis in the frontal analysis mode (CE/FA) to determine the binding capacity of beta-adrenoceptor blocking drugs to individual serum proteins, serum protein mixtures and human serum. The free drug concentration was directly measured from the height of the frontal peak and used to calculate the bound drug concentration. From the bound drug concentration, the percentage of drug bound to the serum proteins alpha1-acid glycoprotein (AGP) and human serum albumin (HSA) was then determined. In addition to determining the percent of a drug bound to a protein, the drug-protein association constant (Ka) was determined for AGP binding to beta-blockers. The data-estimated association constants were consistent with literature values. The CE/FA studies on the beta-adrenoceptor blocking drugs and the serum proteins indicated that HSA, AGP, high density lipoprotein (HDL), and low density lipoprotein (LDL) were the main contributors to serum binding for this series of compounds. The serum-drug binding data sorted the beta-adrenoceptor blocking drugs into high and low binding categories. The protein mixture (AGP + HSA + HDL + LDL) resulted in dividing the beta-blockers into the same high/low rankings. The protein mixture (AGP + HSA + HDL + LDL) was amenable to automation, did not autoaggregate, and had constant concentrations for the proteins.

Attenuation of fructose metabolism by the inhibition of ketohexokinase (KHK; fructokinase) should reduce body weight, free fatty acids, and triglycerides, thereby offering a novel approach to treat diabetes and obesity in response to modern diets. We have identified potent, selective inhibitors of human hepatic KHK within a series of pyrimidinopyrimidines (1). For example, 8, 38, and 47 exhibited KHK IC 50 values of 12, 7, and 8 nM, respectively, and also showed potent cellular KHK inhibition (IC 50 < 500 nM), which relates to their intrinsic potency vs KHK and their ability to penetrate cells. X-ray cocrystal structures of KHK complexes of 3, 8, and 47 revealed the important interactions within the enzyme's adenosine 5'-triphosphate (ATP)-binding pocket.