Bioactivation of drugs: risk and drug design.

Bioactivation through drug metabolism is frequently suspected as an initiating event in many drug toxicities. The CYP450 and peroxidase enzyme systems are generally considered the most important groups of enzymes involved in bioactivation, producing either electrophilic or radical metabolites. Drug design efforts routinely consider these factors, and a number of structural alerts for bioactivation have been identified. Among the most frequently encountered structural alerts are aromatic systems with electron-donating substituents and some five-membered heterocycles. Metabolism of these groups can lead to chemically reactive electrophiles. Strategies that have been used to minimize the associated risk involve replacing the structural-alert moiety, blocking or making metabolism less favorable, and incorporating metabolic soft spots to facilitate metabolism away from the structural-alert substituent. The metabolism of drugs to radicals usually leads to cellular oxidative stress. The formation of radical metabolites can be minimized through the use of similar approaches but remains an area less frequently considered in drug design.

Bis(monoacylglycerol)phosphate as a non-invasive biomarker to monitor the onset and time-course of phospholipidosis with drug-induced toxicities.

IMPORTANCE TO THE FIELD: Drug-induced phospholipidosis (PL) is a phospholipid storage disorder characterized by the accumulation of multi-lamellar bodies (myeloid bodies) in tissues. A major unanswered question is whether PL represents a benign adaptive response, symptom or early event in drug toxicity. The absence of a non-invasive biomarker to monitor tissue PL has made it difficult to determine the prevalence and implications of PL in the clinic. As a result, the interpretation of PL in risk assessment remains uncertain in preclinical and clinical drug development. AREAS COVERED IN THIS REVIEW: This review describes the rationale for bis(monoacylglycerol)phosphate (BMP) as a biomarker of PL and explores the potential links between PL and the toxicities of drugs. WHAT THE READER WILL GAIN: The similarities between the hypothesized roles of BMP in PL and Niemann-Pick type C disease are discussed. The potential implications of PL for cellular function are described in the context of drug-induced QT prolongation, myopathy and renal toxicity. TAKE HOME MESSAGE: A specific species of BMP, di-docosahexaenoyl-BMP, should be investigated further as a non-invasive biomarker to monitor the onset and time course of PL and to better understand the functional consequences which could contribute to the toxicities of drugs.

Interactions of cyclosporin a with breast cancer resistance protein.

The objective of this study was to investigate whether cyclosporin A (CsA) is a modulator for breast cancer resistance protein (BCRP). The interactions between CsA and BCRP were evaluated by using both membrane- and cell-based assays. CsA inhibited BCRP or BCRP R482T mutant-associated ATPase with an IC(50) of 26.1 and 7.3 microM (31,388 and 8779 ng/ml), respectively, indicating that CsA is a modulator for BCRP and its R482T mutant. The apparent permeability (P(app)) of CsA was not affected by the BCRP-specific inhibitor Ko143 in both apical-to-basolateral (A-to-B) and basolateral-to-apical (B-to-A) directions in hBCRP- or mBcrp-transfected MDCKII cells, whereas CsA at 50 microM significantly increased the A-to-B transport and decreased B-to-A transport of BCRP substrates, [(3)H]estrone-3-sulfate ([(3)H]E3S) and [(3)H]methotrexate ([(3)H]MTX), in hBCRP- and mBcrp1-trasfected MDCKII cells. Similar to cellular transport studies, CsA did not exhibit ATP-dependent uptake in BCRP-expressed membrane vesicles but inhibited the ATP-mediated E3S and MTX uptake in the same vesicles. The inhibitory constant (K(i)) of CsA toward BCRP was 6.7 microM (8507 ng/ml) and 7.8 microM (9380 ng/ml) when using E3S or MTX, respectively, as a BCRP substrate. The inhibitory potency of CsA on BCRP wild type or its R482T mutant was lower than that on P-glycoprotein. The present studies demonstrate that CsA is an inhibitor but not a substrate for BCRP, and has low potential to cause drug-drug interactions with BCRP substrate drugs due to its weak inhibitory effect on BCRP and BCRP R482T mutant at its normal therapeutic blood concentrations (200-400 ng/ml) (Blood 91:362-363, 1998).

A novel model for the prediction of drug-drug interactions in humans based on in vitro cytochrome p450 phenotypic data.

Ketoconazole has generally been used as a standard inhibitor for studying clinical pharmacokinetic drug-drug interactions (DDIs) of drugs that are primarily metabolized by CYP3A4/5. However, ketoconazole at therapeutic, high concentrations also inhibits cytochromes P450 (P450) other than CYP3A4/5, which has made the predictions of DDIs less accurate. Determining the in vivo inhibitor concentration at the enzymatic site is critical for predicting the clinical DDI, but it remains a technical challenge. Various approaches have been used in the literature to estimate the human hepatic free concentrations of this inhibitor, and application of those to predict DDIs has shown some success. In the present study, a novel approach using cryopreserved human hepatocytes suspended in human plasma was applied to mimic the in vivo concentration of ketoconazole at the enzymatic site. The involvement of various P450s in the metabolism of compounds of interest was quantitatively determined (reactive phenotyping). Likewise, the effect of ketoconazole on various P450s was quantitated. Using this information, P450-mediated change in the area under the curve has been predicted without the need of estimating the inhibitor concentrations at the enzyme active site or the K(i). This approach successfully estimated the magnitude of the clinical DDI of an investigational compound, MLX, which is cleared by multiple P450-mediated metabolism. It also successfully predicted the pharmacokinetic DDIs for several marketed drugs (theophylline, tolbutamide, omeprazole, desipramine, midazolam, alprazolam, cyclosporine, and loratadine) with a correlation coefficient (r(2)) of 0.992. Thus, this approach provides a simple method to more precisely predict the DDIs for P450 substrates when coadministered with ketoconazole or any other competitive P450 inhibitors in humans.

Determination of drug plasma protein binding by solid phase microextraction.

The plasma protein binding of drugs has been shown to have significant effects on the quantitative relationship between clinical pharmacokinetics and pharmacodynamics. In many clinical situations, measurement of the total drug concentration does not provide the needed information concerning the unbound fraction of drug in plasma, which is available for pharmacodynamic action. Therefore, the accurate determination of unbound plasma drug concentrations is important in understanding drug action. Many methodologies exist for determining the extent of plasma protein binding, but different methods produce a rather wide range of results for the same compound at the same concentration level. The solid phase microextraction (SPME) method reported in the present study attempts to eliminate many experimental variables that could lead to the lack of reproducibility, such as the variable content of organic solvent or ionic strength in plasma, pH shifts, and volume shifts. Five well-known drugs were chosen to study plasma protein binding: ibuprofen, warfarin, verapamil, propranolol, and caffeine, with high, intermediate and low binding properties. Dilution of plasma with isotonic PBS or incubation with 10% CO(2) in the atmosphere was found to compensate for changes in pH during incubation. The data obtained using these pH-controlled methods correlate well with the average values of plasma protein binding found in the literature. SPME, which uses an extraction phase that dissolves or adsorbs the drug of interest and rejects proteins, overcomes several limitations of currently available techniques and is a thermodynamically sound method, since the measurements are always performed at equilibrium. Compared to other methods, SPME offers several advantages: small sample size, short analysis time, possibility to automate, and ability to directly study complex samples.

Comparison of intrinsic clearance in liver microsomes and hepatocytes from rats and humans: evaluation of free fraction and uptake in hepatocytes.

Apparent intrinsic clearance (CL(int,app)) of 7-ethoxycoumarin, phenacetin, propranolol, and midazolam was measured using rat and human liver microsomes and freshly isolated and cryopreserved hepatocytes to determine factors responsible for differences in rates of metabolism in these systems. The cryopreserved and freshly isolated hepatocytes generally provided similar results, although there was greater variability using the latter system. The CL(int,app) values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CL(int,app). This could partly be attributed to the differences in the free fraction (fu). The fu in hepatocyte incubations (fu,hep-inc) was influenced not only by the free fraction of compounds in the incubation buffer (fu,buffer) but also by the rate constants of uptake (k(up)) and metabolism (k(met)). This report provides a new derivation for fu,hep-inc, which can be expressed as fu,hep-inc = [k(up)/(k(met) + k(up))]/[1 + (C(hep)/C(buffer)) x (V(hep)/V(buffer))], where the C(hep), C(buffer), V(hep), and V(buffer) represent the concentrations of a compound in hepatocytes and buffer and volumes of hepatocytes and buffer, respectively. For midazolam, the fu,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.

Investigation of drug-drug interaction potential of bortezomib in vivo in female Sprague-Dawley rats and in vitro in human liver microsomes.

Bortezomib (Velcade, PS-341), a dipeptidyl boronic acid, is a first-in-class proteasome inhibitor approved in 2003 for the treatment of multiple myeloma. In a preclinical toxicology study, bortezomib-treated rats resulted in liver enlargement (35%). Ex vivo analyses of the liver samples showed an 18% decrease in cytochrome P450 (P450) content, a 60% increase in palmitoyl coenzyme A beta-oxidation activity, and a 41 and 23% decrease in CYP3A protein expression and activity, respectively. Furthermore, liver samples of bortezomib-treated rats had little change in CYP2B and CYP4A protein levels and activities. To address the likelihood of clinical drug-drug interactions, the P450 inhibition potential of bortezomib and its major deboronated metabolites M1 and M2 and their dealkylated metabolites M3 and M4 was evaluated in human liver microsomes for the major P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4/5. Bortezomib, M1, and M2 were found to be mild inhibitors of CYP2C19 (IC(50) approximately 18.0, 10.0, and 13.2 microM, respectively), and M1 was also a mild inhibitor of CYP2C9 (IC(50) approximately 11.5 microM). However, bortezomib, M1, M2, M3, and M4 did not inhibit other P450s (IC(50) values > 30 microM). There also was no time-dependent inhibition of CYP3A4/5 by bortezomib or its major metabolites. Based on these results, no major P450-mediated clinical drug-drug interactions are anticipated for bortezomib or its major metabolites. To our knowledge, this is the first report on P450-mediated drug-drug interaction potential of proteasome inhibitors or boronic acid containing therapeutics.

Evaluation of microdosing to assess pharmacokinetic linearity in rats using liquid chromatography-tandem mass spectrometry.

The microdosing strategy allows for early assessment of human pharmacokinetics of new chemical entities using more limited safety assessment requirements than those requisite for a conventional phase I program. The current choice for evaluating microdosing is accelerator mass spectrometry (AMS) due to its ultrasensitivity for detecting radiotracers. However, the AMS technique is still expensive to be used routinely and requires the preparation of radiolabeled compounds. This report describes a feasibility study with conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology for oral microdosing assessment in rats, a commonly used preclinical species. The nonlabeled drugs fluconazole and tolbutamide were studied because of their similar pharmacokinetics characteristics in rats and humans. We demonstrate that pharmacokinetics can be readily characterized by LC-MS/MS at a microdose of 1 microg/kg for these molecules in rats, and, hence, LC-MS/MS should be adequate in human microdosing studies. The studies also exhibit linearity in exposure between the microdose and >or=1000-fold higher doses in rats for these drugs, which are known to show a linear dose-exposure relationship in the clinic, further substantiating the potential utility of LC-MS/MS in defining pharmacokinetics from the microdose of drugs. These data should increase confidence in the use of LC-MS/MS in microdose pharmacokinetics studies of new chemical entities in humans. Application of this approach is also described for an investigational compound, MLNX, in which the pharmacokinetics in rats were determined to be nonlinear, suggesting that MLNX pharmacokinetics at microdoses in humans also might not reflect those at the therapeutic doses. These preclinical studies demonstrate the potential applicability of using traditional LC-MS/MS for microdose pharmacokinetic assessment in humans.

Strategy of utilizing in vitro and in vivo ADME tools for lead optimization and drug candidate selection.

The high-throughput screening in drug discovery for absorption, distribution, metabolism and excretion (ADME) properties has become the norm in the industry. Only a few years ago it was ADME properties that were attributed to more failure of drugs than efficacy or safety in the clinic trials. With the realization of new techniques and refinement of existing techniques better projections for the pharmacokinetic properties of compounds in humans are being made, shifting the drug failure attributes more to the safety and efficacy properties of drug candidates. There are a tremendous number of tools available to discovery scientists to screen compounds for optimization of ADME properties and selection of better candidates. However, the use of these tools has generally been to characterize these compounds rather than to select among them. This report discusses applications of the available ADME tools to better understand the clinical implication of these properties, and to optimize these properties. It also provides tracts for timing of studies with respect to the stage of the compound during discovery, by means of a discovery assay by stage (DABS) paradigm. The DABS provide the team with a rationale for the types of studies to be done during hit-to-lead, early and late lead optimization stages of discovery, as well as outlining the deliverables (objectives) at those stages. DABS has proven to be optimal for efficient utilization of resources and helped the discovery team to track the progress of compounds and projects.