Interaction of Ritonavir on Tissue Distribution of a [c]l-valinamide, a Potent Human Immunodeficiency Virus-1 Protease Inhibitor, in Rats Using Quantitative Whole-body Autoradiography

N-[(3-fluorophenyl)methyl]glycyl-N-{3-[((3-aminophenyl)sulfonyl)2-(aminophenyl)amino]-(1S,2S)-2-hydroxy-1-(phenylmethyl)propyl}3-methyl-L-valinamide (DPC 681, DPC) on oral coadministration with ritonavir (RTV) in rats caused a significant increase in systemic exposure to DPC. Following a single oral dose of [C]DPC with and without RTV pretreatment in rats, and subsequent analysis of whole-body sections, prepared at 1 and 7 or 8 h postdose, using whole-body autoradiography showed an increase in radioactivity in tissues (e.g., brain, and testes) upon coadministration. The distribution of radioactivity in the brain parenchyma and ventricles was different, such that the concentration of radioactivity was greater in cerebrospinal fluid (CSF) than in central nervous system. Thus, the use of CSF concentration of the total radioactivity as a surrogate for brain penetration would result in an overestimation. DPC was determined to be metabolized prominently by rCYP3A4. The increased tissue exposure to DPC in rats could largely be attributed to inhibition of CYP3A1/2 by RTV. DPC was also a good substrate for P-glycoprotein (Pgp), with Km of 4 M and Vmax of 13 pmol/min. The Pgp-mediated transport of DPC across Caco-2 cells was readily saturated at >10 M and was inhibited significantly by RTV at 5 to 10 M. The data above and the reported RTV concentrations suggested that both the Pgp and CYP3A4 inhibition by RTV may play a significant role in enhancing the systemic and tissue exposure to DPC in humans. Multidrug and/or multitarget, highly active antiretroviral therapy of HIV-1, to overcome progression to acquired immunodeficiency syndrome, is still a method of choice for viral suppression. There is also currently an inclusion of pharmacoenhancers in such treatments, in particular the use of RTV. RTV has been previously demonstrated to boost and maintain exposure levels for various HIV protease inhibitors, such as indinavir, amprenavir, saquinavir, lopinavir, and nalfinavir in the clinic, mainly because of inhibition of the metabolizing enzyme CYP3A4 and the transporter Pgp (Casada et al., 2000; Moyle and Back, 2001). Apart from measuring the systemic exposure to various drugs, it is imperative to determine drug distribution into various organs in which the virus can find a sanctuary (e.g., brain and testes) and escape the onslaught of drugs, allowing the virus to replicate, causing CNS damage and viral mutations. The conventional method for such measurement has been excising each organ separately, homogenizing it, followed by oxidizing and liquid scintillation counting of the trapped radioactivity. Currently, quantitative whole-body autoradiography (QWBA) has provided the means for quantitating radioactivity in whole-body sections of animals, which can determine distribution of radioactivity in many more tissue and fluid compartments (Shigematsu et al., 1995, 1999; Zane et al., 1997; Potchoiba et al., 1998). With the continued improvement and standardization of the process, QWBA is rapidly becoming the method of choice for the study of tissue distribution of radiolabeled compounds in animals. The technique has been shown to yield results similar to those obtained by excision of tissue followed by combustion and liquid scintillation counting (Chay and Pohland, 1994). The technique, apart from its major use for human dosimetry estimations (Dain et al., 1994), has also been used for studies like placental transfer (Chay and Herman, 1998), brain penetration (Polli et al., 1999), and in general, assessing accumulation of drugrelated materials in organs for toxicological evaluations. DPC is a potent and selective inhibitor of HIV-1 (Ki, 0.012 nM; IC90, 4 nM), and also of various mutant strains (IC90 of 20 nM for various triple to quintuple mutant variants) (Kaltenbach et al., 2001). In the present studies, QWBA was used to determine the effect of RTV on the disposition of DPC in rats. Possible mechanisms for increases in exposure to [C]DPC in rats and extrapolation of these results to humans are also discussed. Materials and Methods DPC (Scheme 1), [C]DPC, GF120918 were prepared at Bristol-Myers Squibb Company (Wilmington, DE). RTV was obtained from Moravek Biochemicals (Brea, CA). All the other chemicals were of analytical or HPLC grade. 1 Current Address: E. G. Solon, Quest Pharmaceuticals Services, Inc., Delaware Technology Park, 3 Innovation Way, Suite 240, Newark, DE 19711. E-mail: [email protected] 2 Abbreviations used are: HIV, human immunodeficiency virus; RTV, ritonavir; Pgp, P-glycoprotein; CNS, central nervous system; QWBA, quantitative wholebody autoradiography; DPC, N-[(3-fluorophenyl)methyl]glycyl-N-{3-[((3aminophenyl)sulfonyl)-2-(aminophenyl)amino]-(1S,2S)-2-hydroxy-1-(phenylmethyl)propyl}-3-methyl-L-valinamide; GF120918, N-(4-[2-(1,2,3,4-tetrahydro-6,7dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4acridine carboxamide; HPLC, high performance liquid chromatography; IP, imaging plates; AUC, area under the curve. Address correspondence to: Suresh K Balani, Ph.D. Millennium Pharmaceuticals, Inc., 75 Sidney Street, Cambridge, MA 02139. E-mail: [email protected] 0090-9556/02/3011-1164–1169$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 11 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 715/1020356 DMD 30:1164–1169, 2002 Printed in U.S.A. 1164 at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from Rat Pharmacokinetics. DPC was dissolved in a vehicle of 0.1% methanesulfonic acid and 1% Tween 80 at a concentration of 5 mg/ml. RTV was formulated at a concentration of 1 mg/ml in a vehicle of 5% aqueous methylcellulose. The dosing formulations of either DPC or RTV were prepared and stored refrigerated on the day before dosing. Eight, male, Sprague-Dawley rats (ca. 250 g), which were obtained with surgically implanted jugular vein cannuli from Charles River Laboratories Inc. (Wilmington, MA) were acclimated for 5 days prior to administration of test compound. One group of 4 rats was pretreated with oral doses of RTV (10 mg/kg) at approximately 12 and 2 h prior to dosing with a single oral dose of DPC (20 mg/kg). Another group of four rats received only a single oral dose of DPC (20 mg/kg). Following administration of the test material, each animal in both groups was placed into an individual cage and blood samples (ca. 0.5 ml) were collected from each rat, via jugular vein, at 5, 15, and 30 min and at 1, 2, 3, 4, 6, 8, 10 and 24 h postdose. Whole blood from donor rats was infused into each study rat to maintain blood volume lost due to sample collection. The plasma was separated by centrifugation and was stored frozen until analysis. The animals were sacrificed by CO2 asphyxiation after the last blood collection at 24 h postdose. Plasma Sample Assay. Plasma samples were pooled from each rat in each group at each time point to create one sample per time point per group. The plasma concentrations were determined using a standard assay method, in the concentration range of 50 to 10,000 nM, using a structural analog as an internal standard. A 200 l of aliquot of pooled plasma samples was extracted with 5 ml of methyl t-butyl ether, after the addition of 2 mM ammonium acetate solution, followed by the addition of 0.2 ml of dilute sulfuric acid (pH 1) to the organic layer. HPLC-fluorescence (excitation 254 nm; emission 377 nm) was performed using a MetaChem Basic C18 HPLC column (3 150 mm; ANSYS, Lake Forest, CA) heated at 30°C and an acetonitrile/water mobile phase that was run over 10 min at a flow rate of 0.7 ml/min. Retention times of DPC and the internal standard were 6.6 and 7.6 min, respectively. Pharmacokinetic Analysis. The noncompartmental pharmacokinetic parameters were calculated using the WATSON program (WATSON version 5.4 v2, 1998; Pharmaceutical Software Systems, Inc. Wayne, PA). Substrate Selectivity to P450 Isoforms. DPC (2 M) was incubated individually with microsomes prepared from baculovirus-infected insect cells transfected with cDNAs encoding CYP1A1, CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, or CYP2D6. Each incubation contained 100 pmols of the individual P450 isozyme, potassium phosphate buffer (0.05 M or 0.1 M, pH 7.4) or 0.05 M Tris buffer (pH 7.4), 2 mM NADPH, and 3 mM magnesium chloride, at 37°C. Aliquots were taken at 0 and 30 min, deproteinized with acetonitrile, and analyzed by a specific liquid chromatography/tandem mass spectrometry method. Rat Tissue Distribution by QWBA. Study design. Specific activity of the final dosing formulation of [C]DPC was 14.12 Ci/mg in a vehicle of 0.1% methanesulfonic acid and 1% Tween 80 at a concentration of 1.667 mg/ml. RTV was formulated at a concentration of 2 mg/ml in a vehicle of 0.5% aqueous methylcellulose. [C]DPC (17 mg/kg) was administered to male Sprague-Dawley rats (n 1/time point) via oral gavage either alone or approximately 2 h after the last of two oral doses of RTV (10 mg/kg oral gavage, ca. 12 h b.i.d.). Rats were individually housed with free access to water and were fasted overnight before dosing and during the in-life phase of the study. Rats dosed with [C]DPC alone were euthanized 1 and 8 h postdose and rats given RTV and [C]DPC were euthanized 1 and 7 h postdose. All rats were processed for whole-body autoradiography, and tissue concentrations of [C]DPC were quantified using phosphor image analysis as described below. QWBA method. Rats were prepared for QWBA as described by Ullberg (1977). Briefly, one rat each was euthanized by CO2 inhalation at the appropriate time after dosing, and each rat carcass was immediately frozen by immersion in a hexane/dry ice bath ( 70°C) for approximately 10 min. Carcasses were drained, blotted dry, and placed on dry ice for at least 2 h to complete the freezing process. Each carcass was removed from the dry ice and placed into appropriately labeled bags with an identification card and stored at approximately 20°C until embedding. Frozen rat carcasses were individually embedded along with section thickness quality control standards (C-spiked rat blood) in carboxymethylcellulose (Chay and Pohland, 1994) (frozen at 70°C). Appropriate sections ( 30thick) were collected on adhesive tape (Scotch Brand 810; 3M, St. Paul, MN) using a Leica CM3600 Cryomicrotome (Leica Microsystems, Deerfield, IL) with temperature at approximately 20°C. Sections were collected at eight levels of interest in the sagittal plane, and all major tissues, organs, and fluids were included in these levels. Sections were lyophilized, mounted on a black cardboard support along with C-autoradiographic calibration standards (Code RPA 511; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK), wrapped with Mylar film and exposed to phosphor imaging plates (IPs) (BASIII; Fuji Photo Film Co., Ltd., Tokyo, Japan) for 4 days. Exposed IPs were scanned into the WBA imaging system via a FLA 3000 BioImaging Analyzer (Fuji Biomedical Products; Fuji Photo Film Co., Ltd) and digital images of the radioactivity in each section were obtained using M5 MCID software (Imaging Research Inc., St. Catharine’s, ON). Tissue concentrations were interpolated from each standard curve as nanocuries per gram and converted to g-equivalents of DPC/g of tissue, and then to M, assuming 1 g of tissue weight was equivalent to 1 ml, and based on molecular weight of DPC. The concentrations of radioactivity in the calibration standards ranged from 0 to approximately 9400 nCi/g tissue and the r values obtained for the calibration curves used ranged from 0.9994 to 0.9999, which demonstrated the linearity of IPs. Tissue concentrations were obtained from tissues and fluids that could be visually identified on the autoradiograph. The limit of quantitation was determined as the mean background radioactivity concentration value for background plus three times the standard deviation (mean of 10 measurements/IP using sampling tools provided by the image analysis software, in which small tool area 1 1 mm; large sampling tool area 5 5 mm). This was determined for small and large sampling tool areas on each IP (n 7) used for study. Small tissues included the pituitary gland, adrenal gland, thyroid gland, skin, and bone marrow, and remaining tissues were considered as large tissues. Images were printed in pseudocolor using the image analysis software. Caco-2 Cell RTV-DPC Interaction. Cell culture. Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA). Cell stocks were maintained in T-75 cm flasks (Corning, Acton, MA) at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The culture consisted of a high glucose (4.5 g/liter) Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1% nonessential amino acids, 100 U/ml penicillin, and 100 g/ml streptomycin (Invitrogen). The culture media were replaced every other day. Monolayers were subcultured using 0.05% trypsin-0.02% EDTA when they reached 75 to 85% confluency at a split ratio of approximately 1:5. Single-cell suspensions of Caco-2 cells were plated onto the 12-mm diameter Transwell polycarbonate membranes (0.4m pore size) at a density of 6 10 cells/cm. The Transwell (Corning) inserts were placed in 12-well culture plates with 0.5 ml of media in the apical compartment and 1.5 ml of media in the basolateral compartment. The media at both compartments were replaced every other day for 21 days before the cells were used for the transport studies. Transport Studies. Prior to the transport experiments, the integrity of Caco-2 cell monolayers was assessed by determining transepithelial electrical resistances using an Evon Epithelial Voltohm meter (World Precision Instruments, New Haven, CT). The transepithelial electrical resistances values were in the range of 400 to 800 ohms cm. The culture medium in the transwell was aspirated and the wells washed twice with the transport buffer (Hank’s Balance salt solution containing 25 mM glucose and 10 mM HEPES, pH 7.4). The 1.5 ml of transport buffer containing [C]DPC (23.43 mCi/mmol) was added to SCHEME 1. Structure of DPC 681. 1165 EFFECT OF RITONAVIR ON TISSUE DISTRIBUTION OF HIV PI at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from the basolateral side, and 0.5 ml of transport buffer was added to the apical side. To determine Km and Vmax of DPC transported by Pgp, concentration of 1, 2.5, 5, 10, 25, and 50 M [C]DPC in the presence and absence of GF120918, a Pgp inhibitor (Evers et al., 2000) at 2 M added to the basolateral side were studied. To determine inhibitory effect of RTV on transport of [C]DPC mediated by Pgp, 2 M [C]DPC in the presence of 0, 5, 10, 25, and 50 M RTV or GF120918 (2 M) added to the basolateral side was studied. The transport was initiated by incubating plates at 37°C. After 1 h, the radioactivity in the transport buffer at the apical side was determined by a Packard liquid scintillation analyzer (PerkinElmer Life Sciences, Boston, MA). The Caco-2 cell model was validated for the functional viability of Pgp by testing standard substrates like digoxin, taxol, or vinblastin, for A to B and B to A transport, in the presence or absence of Pgp inhibitor GF120918.