Application of chimeric mice with humanized liver for study of human-specific drug metabolism.

Human-specific or disproportionately abundant human metabolites of drug candidates that are not adequately formed and qualified in preclinical safety assessment species pose an important drug development challenge. Furthermore, the overall metabolic profile of drug candidates in humans is an important determinant of their drug-drug interaction susceptibility. These risks can be effectively assessed and/or mitigated if human metabolic profile of the drug candidate could reliably be determined in early development. However, currently available in vitro human models (e.g., liver microsomes, hepatocytes) are often inadequate in this regard. Furthermore, the conduct of definitive radiolabeled human ADME studies is an expensive and time-consuming endeavor that is more suited for later in development when the risk of failure has been reduced. We evaluated a recently developed chimeric mouse model with humanized liver on uPA/SCID background for its ability to predict human disposition of four model drugs (lamotrigine, diclofenac, MRK-A, and propafenone) that are known to exhibit human-specific metabolism. The results from these studies demonstrate that chimeric mice were able to reproduce the human-specific metabolite profile for lamotrigine, diclofenac, and MRK-A. In the case of propafenone, however, the human-specific metabolism was not detected as a predominant pathway, and the metabolite profiles in native and humanized mice were similar; this was attributed to the presence of residual highly active propafenone-metabolizing mouse enzymes in chimeric mice. Overall, the data indicate that the chimeric mice with humanized liver have the potential to be a useful tool for the prediction of human-specific metabolism of xenobiotics and warrant further investigation.

Species differential stereoselective oxidation of a methylsulfide metabolite of MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide], a peroxisome proliferator-activated receptor dual agonist.

MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide], a thiazolidinedione (TZD)-containing peroxisome proliferator-activated receptor agonist, is a rapidly interconverting racemate that possesses a chiral center at the five position of the TZD ring. M25 is a methyl sulfide metabolite generated from MK-0767 following CYP3A4-mediated TZD ring opening and subsequent methylation of the sulfide intermediate M22. M25, a major in vitro and in vivo metabolite, was further metabolized in liver microsomes to the methyl sulfoxide amide (M16) with two chiral centers and the methyl sulfone amide (M20) with one chiral center. Previous studies demonstrated that both CYP3A4 and flavin monooxygenase-3 (FMO3) catalyzed the formation of M16, whereas M20 was formed exclusively by CYP3A4. The relative contribution of CYP3A4 and FMO3 in the formation of M16 in human and preclinical species was evaluated by chiral analysis using supercritical fluid chromatography. No stereoselectivity was observed in incubations of M25 with human and rhesus liver and recombinant CYP3A4 microsomes, whereas a high degree of stereoselectivity (63 to >99% enantiomeric excess) was observed in rat and dog liver and human recombinant FMO3 microsomes. Also, polyclonal anti-rat CYP3A2 antibody and cytochrome P450 (P450) chemical inhibitors did not inhibit the oxidation of M25 in rat liver microsomes. Furthermore, M25 oxidation was more sensitive to heat inactivation at pH 8 and 8.7 in rat and dog liver microsomes than in human and monkey liver microsomes, consistent with the species difference in involvement of FMOs. Collectively, these results indicated that S-oxidation of M25 was catalyzed primarily by P450 enzymes in human and monkey liver microsomes and by FMO enzymes in rat and dog liver microsomes.

Telomerase--a potential molecular marker of lung and cervical cancer.

Telomerase, a ribonucleoprotein enzyme that adds hexameric TTAGGG nucleotide repeats onto telomeres is reactivated in most malignancies. Lung cancer is a common malignant disease worldwide as well as in India. Most patients present in advanced stages. As noninvasive diagnostic techniques are preferred, we assayed the telomerase activity in pre-bronchoscopy sputum and compared it with that of bronchial washings and bronchoscopic biopsies by telomeric repeat amplification protocol (TRAP) in 53 cases of lung cancer. These were corroborated with cytopathological/histopathological examinations. Telomerase activity was detected in 58.5% of sputum samples, 70% of bronchial washings and 74% of bronchoscopic biopsies thereby making it a good noninvasive diagnostic marker of lung cancer. Cervical cancer is the 7th most common cancer worldwide, with 100,000 new cases being reported annually in India. It is routinely screened by Papanicolaou's (Pap) smear. Human papilloma virus (HPV) is one of its etiological agents. We have assayed telomerase activity in relation to HPV-16/18 in cervical samples from 93 subjects ranging from normal to precancerous to frank cancers in tissue biopsies and cervical scrapings. HPV infection was detected by polymerase chain reaction (PCR) in 81% of tumor samples, in 6% of control hysterectomy samples and in 2% of cervical scrapings of normal healthy controls with HPV-16 being the predominant type. Telomerase activity was detected in 96.5% of cervical tumor samples, in 68.7% of premalignant cervical scrapings but was not detected in control hysterectomy samples, or in cervical scrapings of normal healthy controls. There was 71% correlation between telomerase activity and HPV-16/18 infection.

Potentiation of the effect of paclitaxel and carboplatin by antioxidant mixture on human lung cancer h520 cells.

OBJECTIVE: Antioxidants have been shown to enhance the effect of certain chemotherapeutic agents on tumor cells in culture. However, this effect differs depending upon the type of tumor and the drugs. In this study, the objective was to see whether pretreatment with antioxidant mixture could enhance the cytotoxic and apoptotic effect of commonly used chemotherapeutic agents, paclitaxel and carboplatin for the treatment of NSCLC. METHODS: Human lung squamous cell carcinoma cell line, H520, was treated with antioxidant mixture (vitamin C, vitamin E and beta carotene), paclitaxel and carboplatin, individually and in combination of different doses in different sequences. Growth inhibition and induction of apoptosis was studied by morphological changes, MTT assay and flow-cytometric analysis. RESULTS: The antioxidant mixture by itself led to 15% apoptosis in H520 cells. Paclitaxel treatment 24 hours prior to carboplatin caused 54% apoptosis, more than that produced by simultaneous treatment with both agents (40%). A statistically significant improvement in the degree of apoptosis, induced by paclitaxel and carboplatin combination, was seen when the cells were pretreated with antioxidant mixture immediately before paclitaxel exposure (70%) or 24 hours before paclitaxel exposure (89%). CONCLUSION: The data suggests that the apoptotic effects of paclitaxel and carboplatin are enhanced by pretreatment with the antioxidant mixture. Thus, the most promising sequence of these agents, which emerged in this study, was pretreatment with antioxidant mixture for 24 hours followed by paclitaxel treatment for 24 hours followed by carboplatin exposure for 24 hours.