Highly dynamic mitotic-spindle microtubules are among the most successful targets for anticancer therapy. Microtubule-targeted drugs, including paclitaxel and Vinca alkaloids, were previously considered to work primarily by increasing or decreasing the cellular microtubule mass. Although these effects might have a role in their chemotherapeutic actions, we now know that at lower concentrations, microtubule-targeted drugs can suppress microtubule dynamics without changing microtubule mass; this action leads to mitotic block and apoptosis. In addition to the expanding array of chemically diverse antimitotic agents, some microtubule-targeted drugs can act as vascular-targeting agents, rapidly depolymerizing microtubules of newly formed vasculature to shut down the blood supply to tumours.

Microtubules as a target for anticancer drugs.

Intranasal delivery to the central nervous system: Mechanisms and experimental considerations

This article reviews the information available to assist pharmacokineticists in the prediction of metabolic drug interactions. Significant advances in this area have been made in the last decade, permitting the identification in early drug development of dominant cytochrome P450 (CYP) isoform(s) metabolising a particular drug as well as the ability of a drug to inhibit a specific CYP isoform. The major isoforms involved in human drug metabolism are CYP3A, CYP2D6, CYP2C, CYP1A2 and CYP2E1. Often patients are taking multiple concurrent medications, and thus an assessment of potential drug-drug interactions is imperative. A database containing information about the clearance routes for over 300 drugs from multiple therapeutic classes, including analgesics, anti-infectives, psychotropics, anticonvulsants, cancer chemotherapeutics, gastrointestinal agents, cardiovascular agents and others, was constructed to assist in the semiquantitative prediction of the magnitude of potential interactions with drugs under development. With knowledge of the in vitro inhibition constant of a drug (Ki) for a particular CYP isoform, it is theoretically possible to assess the likelihood of interactions for a drug cleared through CYP-mediated metabolism. For many agents, the CYP isoform involved in metabolism has not been identified and there is substantial uncertainty given the current knowledge base. The mathematical concepts for prediction based on competitive enzyme inhibition are reviewed in this article. These relationships become more complex if the inhibition is of a mixed competitive/noncompetitive nature. Sources of uncertainty and inaccuracy in predicting the magnitude of in vivo inhibition includes the nature and design of in vitro experiments to determine Ki, inhibitor concentration in the hepatic cytosol compared with that in plasma, prehepatic metabolism, presence of active metabolites and enzyme induction. The accurate prospective prediction of drug interactions requires rigorous attention to the details of the in vitro results, and detailed information about the pharmacokinetics and metabolism of the inhibitor and inhibited drug. With the discussion of principles and accompanying tabulation of literature data concerning the clearance of various drugs, a framework for reasonable semiquantitative predictions is offered in this article.

Use of In Vitro and In Vivo Data to Estimate the Likelihood of Metabolic Pharmacokinetic Interactions

Pharmacogenetics is the study of how interindividual variations in the DNA sequence of specific genes affect drug response. This article highlights current pharmacogenetic knowledge on important human drug-metabolizing cytochrome P450s (CYPs) to understand the large interindividual variability in drug clearance and responses in clinical practice. The human CYP superfamily contains 57 functional genes and 58 pseudogenes, with members of the 1, 2, and 3 families playing an important role in the metabolism of therapeutic drugs, other xenobiotics, and some endogenous compounds. Polymorphisms in the CYP family may have had the most impact on the fate of therapeutic drugs. CYP2D6, 2C19, and 2C9 polymorphisms account for the most frequent variations in phase I metabolism of drugs, since almost 80% of drugs in use today are metabolized by these enzymes. Approximately 5-14% of Caucasians, 0-5% Africans, and 0-1% of Asians lack CYP2D6 activity, and these individuals are known as poor metabolizers. CYP2C9 is another clinically significant enzyme that demonstrates multiple genetic variants with a potentially functional impact on the efficacy and adverse effects of drugs that are mainly eliminated by this enzyme. Studies into the CYP2C9 polymorphism have highlighted the importance of the CYP2C9*2 and *3 alleles. Extensive polymorphism also occurs in other CYP genes, such as CYP1A1, 2A6, 2A13, 2C8, 3A4, and 3A5. Since several of these CYPs (e.g., CYP1A1 and 1A2) play a role in the bioactivation of many procarcinogens, polymorphisms of these enzymes may contribute to the variable susceptibility to carcinogenesis. The distribution of the common variant alleles of CYP genes varies among different ethnic populations. Pharmacogenetics has the potential to achieve optimal quality use of medicines, and to improve the efficacy and safety of both prospective and currently available drugs. Further studies are warranted to explore the gene-dose, gene-concentration, and gene-response relationships for these important drug-metabolizing CYPs.

Polymorphism of human cytochrome P450 enzymes and its clinical impact

http://www.cornellpharmacology.org/faculty/labs/giannakakou/documents/MabjeeshNJetal.CancerCell2003.pdf

2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF.

Estrogens occurring naturally in the body are metabolized to catecholestrogens (2- and 4-hydroxyestradiol) by the cytochrome P450 enzymes. 2-Hydroxy catecholestrogens are further metabolized by catechol-O-methyltransferase to 2-methoxyestradiol, which is known to be protective against tumor formation. 2-Methoxyestradiol exhibits potent apoptotic activity against rapidly growing tumor cells. It also possesses antiangiogenic activity through a direct apoptotic effect on endothelial cells. Other molecular mechanisms, including microtubule stabilization by inhibition of the colchicine-binding site, have been reported. The exact mechanism of action of 2-methoxyestradiol is still unclear, but it has been shown to be effective in preventing tumor growth in a variety of cell lines. 2-Methoxyestradiol also possesses cardioprotective activity by inhibiting vascular smooth muscle cell growth in arteries. It has a lower binding affinity for estrogen receptor alpha compared with that of estradiol, and its affinity for estrogen receptor beta is even lower than that of estrogen receptor alpha, thus it has minimal estrogenic activity. 2-Methoxyestradiol is distinct because of its inability to engage estrogen receptors as an agonist, and its unique antiproliferative and apoptotic activities are mediated independently of estrogen receptors alpha and beta. A phase I clinical trial of 2-methoxyestradiol 200, 400, 600, 800, and 1,000 mg/day in 15 patients with breast cancer showed significant reduction in bone pain and analgesic intake in some patients, with no significant adverse effects. Another phase I study of 2-methoxyestradiol 200-1,000 mg/day in combination with docetaxel 35 mg/m2/week for 4-6 weeks performed in 15 patients with advanced refractory metastatic breast cancer showed no serious drug-related adverse effects. A phase II randomized, double-blind trial of 2-methoxyestradiol 400 and 1,200 mg/day in 33 patients with hormone-refractory prostate cancer showed that it was well tolerated and showed prostate specific antigen stabilizations and declines. We have started a phase I clinical trial to explore dosages greater than 1,000 mg/day.

/pdf/2-methoxyestradiol-a-promising-anticancer-agent-2qpdgtz4o2.pdf

2-Methoxyestradiol, a promising anticancer agent.

Nasal delivery is a potential alternative for systemic availability of drugs restricted to intravenous administration, such as peptide and protein drugs. Although nasal delivery avoids the hepatic first-pass effect, the enzymatic barrier of the nasal mucosa creates a pseudo-first-pass effect. The xenobiotic metabolic activity in the nasal epithelium has been investigated in several species including humans. The Phase I, cytochrome P-450 enzymes have been studied extensively for their toxicological significance, since these enzymes metabolize inhaled pollutants into reactive metabolites which may induce nasal tumors. The cytochrome P-450 activity in the olfactory region of the nasal epithelium is higher even than in the liver, mainly because of a three- to fourfold higher NADPH-cytochrome P-450 reductase content. Phase II activity has also been found in the nasal epithelium. The delivery of peptides and proteins has been hindered by the peptidase and protease activity in the nasal mucosa. The predominant enzyme appears to be aminopeptidase among other exopeptidases and endopeptidases. The absorption of peptide drugs can be improved by using aminoboronic acid derivatives, amastatin, and other enzyme inhibitors as absorption enhancers. It is possible that some of the surfactants, e.g., bile salts, increase absorption by inhibiting the proteolytic enzymes. Thus, in addition to the permeation barriers, there also exists an enzymatic barrier to nasal drug delivery, which is created by metabolic enzymes in the nasal epithelium.

Drug metabolism in the nasal mucosa.

Theophylline is metabolized in the liver by one or more cytochrome P-450 enzymes. To assess the amounts and types of these human cytochromes P-450, we incubated theophylline with microsomes prepared from 22 different human livers in the presence of NADPH, and measured simultaneous rates of 1- and 3-N-demethylations to 3-methylxanthine (3-MX) and 1-methylxanthine (1-MX), respectively; and 8-hydroxylation to 1,3-dimethyluric acid (1,3-DMU). Under optimal conditions, 3-MX, 1-MX, and 1,3-DMU formation proceeded with mean Km values of 2.05, 1.93, and 5.34 mM and Vmax values of 2.28, 2.48, and 23.4 pmol/mg/min, respectively. Formation of 3-MX and 1-MX correlated best with amounts of the immunoreactive protein HLd (P-450IA2) (p less than 0.05), whereas formation of 1,3-DMU correlated with the microsomal content of HLp (P-450IIIA3) and HLj (P-450IIE1). In immunoinhibition experiments, incubations conducted with a polyclonal anti-rat P-450c/d antibody, the formation of all the three theophylline metabolites (p less than 0.05) was significantly inhibited. However, addition of isoform-specific anti-rat-P-450d antibodies to the microsomal mixture significantly inhibited 1-N-demethylation, selectively, with little (if any) inhibition of 3-N-demethylation or 8-hydroxylation. Nonspecific cytochrome P-450 inhibition was ruled out by showing that erythromycin N-demethylation, an activity catalyzed by HLp, was unaffected by either anti-P-450c/d (P-450IA1/IA2) or anti-P-450d. Anti-rat-P-450p antibodies failed to block formation of theophylline metabolism, but did inhibit erythromycin N-demethylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of human liver cytochromes P-450 involved in theophylline metabolism.

Polycyclic aromatic hydrocarbons present in cigarette smoke induce cytochromes P4501A1 and P4501A2. These isozymes are of toxicological importance because they convert several environmental pollutants to reactive intermediates that form covalent adducts with cellular DNA resulting in mutations and/or malignant transformations. The aim of our research was to investigate whether theophylline metabolites could be used as probes for P4501A1 and P4501A2. It is known that at least two human P450 isozymes are involved in theophylline metabolism. The N-demethylations of theophylline to 3-methylxanthine (3-MX) and 1-methylxanthine (1-MX) appear to be mediated by P4501A1 and/or P4501A2 and the 8-hydroxylation by different isozymes. Theophylline metabolism was measured in liver microsomes from control, benzo(a)pyrene (BP)-, and isosafrole (ISO)-induced rats. Theophylline was also incubated in microsomes prepared from cells expressing high levels of human P4501A1 and P4501A2. A plot of v vs. v/S was linear for 3-MX in the ISO-induced microsomes, but nonlinear for 1-MX, indicating that P4501A2 mainly forms 3-MX, whereas P4501A1 and 1A2 probably mediate 1-MX. A similar nonlinear relation was also obtained in the BP-induced microsomes. Incubation of theophylline in the microsomes from the cells indicated that only 1-MX could be measured in cells expressing P4501A1, and both 1-MX and 3-MX were formed in the P4501A2 cell microsomes. Therefore 1-MX seems to be mediated by P4501A1/P4501A2 and 3-MX specifically by P4501A2, and theophylline N-demethylations can be used as probes for P4501A1 and P4501A2.(ABSTRACT TRUNCATED AT 250 WORDS)

Mohamadi A. Sarkar

Papers

2-Methoxyestradiol, a promising anticancer agent.

Drug metabolism in the nasal mucosa.

Characterization of human liver cytochromes P-450 involved in theophylline metabolism.

Theophylline N-demethylations as probes for P4501A1 and P4501A2.

Regiospecific expression of cytochrome P4501A1 and 1B1 in human uterine tissue.