Short communication

Time and dose dependence of 3-methylcholanthrene-induced metabolism in rat intestinal mucosal cells and microsomes

A Physiologically-based pharmacokinetic (PB-PK) model was developed to describe the aspects of pharmacokinetic interactions between five HIV protease inhibitors (ritonavir, amprenavir, nelfinavir, saquinavir, indinavir) in rats. To increase usefulness of this BP-PK model, liver, intestinal tissue and other organ were assumed as compartments in this model. Each compartment was linked with the blood flow and the blood-to-plasma concentration ratios of those drugs, and the absorption process in the intestinal tract was presumed as a first-order kinetics. In addition, this PB-PK model incorporates two elimination processes due to hepatic and intestinal metabolism constructed by in vitro metabolic clearance rates and inhibition constants between HIV protease inhibitors. Excellent agreements were obtained between the predicted and observed concentrations of HIV protease inhibitors in rat plasma after a 20 mg/kg oral dose or co-administration of two kinds of HIV protease inhibitors (amprenavir/indinavir, nelfinavir/amprenavir, saquinavir/amprenavir, amprenavir/ritonavir, indinavir/ritonavir, nelfinavir/ritonavir, and saquinavir/ritonavir) with each 20 mg/kg oral dose. However, underestimates in the predicted plasma concentrations of saquinavir, indinavir and amprenavir were observed during the terminal phase after co-administration with ritonavir or amprenavir, suggesting that a term of other inhibitory process, such as a mechanism-based inhibition, might be incorporated into this PB-PK model. This BP-PK model enables us to know useful information about pharmacokinetic interaction when HIV infected patients would receive double protease therapy. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:680–689, 2002

The discovery of indinavir is a successful example in which pharmacokinetic and metabolic information were incorporated into drug design. The use of animal and in vitro human metabolic data in predicting the oral bioavailability and hepatic clearance in humans was critical in selecting indinavir as a drug candidate for development. In its development stage, pharmacokinetics continued to play an important role in identifying the key properties of indinavir in vivo, which allowed the characterization and prediction of the time course of drug action under physiological and pathological conditions. This review describes the role of pharmacokinetics and drug metabolism in the discovery and development of indinavir.

The discovery of human immunodeficiency virus (HIV) protease inhibitors is an example in which pharmacokinetic evaluation was implemented early in the discovery phase to obtain optimal pharmacological and pharmacokinetic properties. Currently, three HIV protease inhibitors, saquinavir, indinavir and ritonavir are clinically available. As a family, these HIV protease inhibitors are characterized pharmacologically by their ability to inhibit the viral protease enzyme. Pharmacokinetically, they are quite different due to their dissimilarity in physicochemical properties. Bioavailability appears to be limited with saquinavir, but not with indinavir and ritonavir. Although all three drugs are metabolized extensively by cytochrome P-450, saquinavir and indinavir are high clearance drugs while ritonavir is a low clearance drug. Despite their significant differences in elimination clearance, all three HIV proteases are given at high oral doses (600–800 mg) either twice or three times daily. These HIV protease inhibitors show superior therapeutic activity and a more favorable safety profile than those reported for the established reverse transcriptase inhibitors. However, the potential for interactions with other drugs metabolized by the CYP 3A4 isoform appears to be considerable. In addition, repeated administration of enzyme inducers results in a substantial decrease of plasma concentrations of protease inhibitors. Therefore, co-administration of drugs, such as rifampicin and rifabutin, must be avoided. HIV protease inhibitors are promising in the treatment of AIDS. Although they are not a cure, they can significantly inhibit that viral replication and improve the quality of life for people who have HIV infection.

The metabolism of indinavir, a human immune deficiency virus (HIV) protease inhibitor, has been characterized extensively in rats and humans. All oxidative metabolites found in vivo were formed when indinavir was incubated with NADPH-fortified hepatic and intestinal microsomes obtained from rats and humans. In vitro kinetic studies revealed that V_max/K_m values (μL/min/mg protein) in rat and human liver microsomes were approximately 8- and 2-fold greater than those in the intestinal microsomes of the corresponding species (55.8 and 6.7 for the liver and intestine, respectively, in rats; 16.5 and 7.7 for the liver and intestine, respectively, in humans). However, when V_max/K_m was scaled up to intrinsic clearance (mL/min/kg body weight), hepatic intrinsic clearance was much greater than the intestinal clearance by 50- to 200-fold. These results suggest that the liver plays a much greater role in first-pass metabolism of indinavir than the intestine in both species. Consistently, ketoconazole, a selective inhibitor for CYP3A, and an anti-rat CYP3A1 antibody strongly inhibited hepatic and intestinal metabolism of indinavir in both rats and humans, suggesting the involvement of CYP3 A isoforms in both organs. Oral treatment of rats with dexamethasone (50 mg/kg/day for 4 days), a potent CYP3A inducer, increased both hepatic and intestinal metabolism of indinavir by a factor of 7 and 3, respectively. Furthermore, indinavir selectively inhibited 6β-hydroxylase activity of testosterone, a CYP3A marker activity, in rat and human liver microsomes; the interactions between testosterone and indinavir were competitive with K_i values of < 1.0 μ.M.

The intestine is a crucial organ in respect of toxicity of ingested compounds, because of its extensive exposed surface area as well as its absorptive and physiological properties. Only recently has attention been paid to this organ in the investigation of pathological conditions using in vitro models. The first studies performed dealt with the metabolic capacity of isolated intestinal cells and date back to 1977. More recently, intestinal cell lines have been characterized for the presence of some activating-deactivating enzymes. Cytotoxicity studies reported in the literature predominantly concern screening of antitumoral drugs using carcinoma cell lines and investigation of the mechanisms involved. A variety of other chemicals with possible toxic effects have been studied in intestinal cells and these include food contaminants, anti-infective drugs, natural toxins and dietary compounds. However those studies are still very limited in number and represent a wide range of approaches, in spite of the fact that very interesting models of intestinal cells are now available and the technology is improving.

To investigate the drug-metabolizing potential of different sub-populations of cells along the villus-crypt surface of the small intestine, the major monooxygenase activities directed towards the substrates benzo[a]pyrene (BP), 7-ethoxycoumarin and ethylmorphine were studied. The cells were isolated in sequential fractions corresponding to the villus tip-to-crypt gradient in the small intestinal epithelium of the rat. Cells from the upper- and mid-villus regions were rich in aryl hydrocarbon (BP)hydroxylase (AHH) and 7-ethoxycoumarin deethylase (7-ECDE) activities whereas in crypt cells the activities of these enzymes were at the level of detectability. Ethylmorphine demethylase (EMD) was not detectable in the entire villus-crypt surface. The intestinal epithelial cells responded strongly to inducers. 3-Methylcholanthrene (3-MC), given to rats 24 hr previously, induced increases in AHH activity of 4- to 7-fold in the villus and of 19- to 26-fold in the crypt cells. 7-ECDE had a similar pattern. The induced level of monooxygenase activity in crypt cells was sustained for a longer time, followed in order by consecutively higher cells of the villus. Phenobarbital caused maximal expression of EMD activity in the mid-villus region whereas the activity in crypt cells was half the maximal activity. PB also significantly induced AHH and 7-ECDE in the intestinal epithelium. 7,8-Benzoflavone inhibited AHH activity to the same degree in all the cell fractions. The apparent K_m for AHH was 5 μM (BP). Treatment of rats with 3-MC, after 24 hr, enhanced the K_m and V_max differently in the cells along the villus-crypt surface. The K_m value in the villus region increased, whereas it did not change in the crypt cells; V_max increased 6-fold in the villus and about 12-fold in the crypt cells, above their basal levels.

The results suggest that the intestinal cells are capable of biotransforming various xenobiotics. The higher sensitivity of their monooxygenases, particularly of the crypt cells, might protect them directly or render the cells capable of generating metabolites that aid and abet toxicity toward target tissue in vivo.