Short Communication Pharmacokinetics of Aminolevulinic Acid after Oral and Intravenous Administration in Dogs

The purpose of these studies was to examine the pharmacokinetics, oral bioavailability, and systemic side effects of aminolevulinic acid (ALA) in beagle dogs after oral and i.v. administration. Oral and i.v. doses of ALA (128 mg of ALA hydrochloride, equivalent to 100 mg of ALA) were administered to four animals using a crossover design. Animals were allowed a 2-week washout period between doses. Plasma ALA concentrations were determined using precolumn fluorescent derivatization and reversed-phase HPLC. Plasma concentrations after i.v. administration declined rapidly with a terminal half-life of 19.5 6 2.5 min (mean 6 S.D.). Total body clearance and volume of distribution at steady state averaged 6.79 6 1.77 ml/min/kg and 259 6 128 ml/kg, respectively. Peak plasma concentrations of ALA after oral administration ranged from 1.27 to 9.42 mg/ml. Oral bioavailability in these animals averaged 41.2 6 14.8% (range, 23.5–58.5%). These studies demonstrate that oral administration may provide a convenient and efficient route of delivery of ALA for photodynamic therapy in patients. Photodynamic therapy (PDT) is a promising form of regional chemotherapy in which photosensitized cells can be selectively eradicated by exposure to light. A variety of porphyrins have been used for PDT (Benson, 1988; Harty et al., 1989). However, these agents require i.v. administration and cause prolonged photosensitization. These attributes have limited their clinical acceptance and intensified the search for orally bioavailable photosensitizing agents with improved safety profiles. Aminolevulinic acid (ALA) is an endogenous metabolite present in virtually all mammalian cells as the first committed step in heme biosynthesis. The conversion of glycine and succinyl CoA into ALA is rate-limiting and is tightly regulated by feedback inhibition of ALA synthetase by heme. The next rate-limiting step in the heme biosynthetic pathway occurs at the conversion of protoporphyrin IX (PpIX) to heme. PpIX is a potent photosensitizer that accumulates in both normal and neoplastic tissues after exogenous administration of ALA (Loh et al., 1992; Bedwell et al., 1992; Loh et al., 1993a). Topical, oral, and i.v. administration of ALA followed by light treatment have been used in clinical trials to examine the utility of ALA PDT for the treatment of a variety of neoplasms, including superficial basal cell carcinoma, squamous cell carcinoma of the mouth, and colorectal adenocarcinoma (Marcus et al., 1996). Loh et al. (1993b) examined the tissue accumulation of PpIX-associated fluorescence after oral and i.v. administration of ALA and found that higher oral doses of ALA were required to achieve equal PpIX tissue concentrations. The pharmacokinetics and bioavailability of ALA have not been reported, but are needed to define appropriate dosing guidelines in patients. The goal of these studies was to examine the pharmacokinetics, bioavailability, and safety of ALA after single doses in dogs to guide oral and i.v. dosing studies in human volunteers at our institution. Materials and Methods Chemicals. ALA hydrochloride (Levulan) was provided by DUSA Pharmaceuticals, Inc. (Valhalla, NY). All other chemicals and solvents were purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ) and were used as received from the manufacturer. Animal Protocol. All animal procedures used in this research adhered to Principles of Laboratory Animal Care (National Institutes of Health publication no. 85–23, revised 1985) and were approved by the University of Tennessee Animal Care and Use Committee. Four male beagle dogs weighing 10.2 to 12.6 kg were purchased from Antech, Inc. (Dallas, TX). Animals were given two doses of ALA separated by a 2-week washout period. Two animals received an i.v. ALA dose on the first occasion, followed 2 weeks later by an oral ALA dose. The remaining two animals received an oral ALA dose on the first occasion, followed 2 weeks later by an i.v. ALA dose. The dosage form used for these studies, consisting of lyophilized ALA and sodium acetate/ mannitol solution in separate vials, was provided by the University of Tennessee Parenterals Medication Laboratory. The vials contained 128 mg of sterile, lyophilized ALA-hydrochloride (equivalent to 100 mg of ALA). Diluent vials contained 10 ml of sodium acetate (9 mg/ml) with mannitol (12.5 mg/ml) solution. ALA was reconstituted with the diluent less than 30 min before administration to provide an aqueous dosing solution of pH 5.0. Oral (7.29 6 1.15 mg/kg ALA) and i.v. doses (8.28 6 1.05 mg/kg ALA) were administered by oral gavage and a jugular vein angiocatheter, respectively. This dose was approximately 6 times greater than the dose chosen (i.e., 1.34 mg/kg) for initial patient studies at our institution and resulted in similar plasma concentrations in dogs and humans (unpublished data). A smaller i.v. dose (1.77 6 0.19 mg/kg) was used to examine the emetic side effects of ALA. Angiocatheters with a heparin well were placed in the jugular and a These studies were supported by a grant from DUSA Pharmaceuticals, Inc. (Valhalla, NY). 1 Abbreviations used are: PDT, photodynamic therapy; ALA, aminolevulinic acid; PpIX, protoporphyrin IX; Tmax, time to reach maximum plasma concentration; Vdss, volume of distribution at steady state; CL, total plasma clearance; AUC, area under the plasma concentration-time curve. Send reprint requests to: James T. Dalton, Ph.D., Department of Pharmaceutical Sciences, University of Tennessee, Memphis, 874 Union Avenue, Crowe Building, Room 5, Memphis, TN 38163. E-mail: [email protected] 0090-9556/99/2704-0432–435$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 27, No. 4 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 432 at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from saphenous vein before each dose. Blood samples (8 ml each) were withdrawn before each dose and at 2, 5, 10, 15, 30, 60, 120, 240, 480, and 720 min after i.v. doses and at 5, 10, 20, 30, 40, 50, 60, 70, 90, 120, 240, 480, and 720 min after oral doses. Blood pressure and heart rate were determined at various time points before and after ALA doses using a noninvasive device (Critikon Dinamap Vital Signs Monitor, model 8100; Critikon, Inc., Tampa, FL). Serum samples obtained immediately before and 12 h after ALA administration were submitted for routine clinical analysis including electrolytes, cholesterol, creatinine, bilirubin, alkaline phosphatase, serum glutamic-pyruvic transaminase, and creatine phosphokinase. Analytical Method. An HPLC method was developed to quantitate ALA in dog plasma. This method was based in part on the precolumn derivatization method used by Okayama et al. (1990) to assay ALA in urine. The limit of detection of the published method was 10 mg/ml in urine, whereas the limit of detection for the assay reported here is 0.010 mg/ml in plasma. Briefly, an aliquot of plasma (0.01–1 ml) was diluted with human plasma to a final volume of 1.0 ml. The human plasma employed for the dilution also was utilized to prepare the standard curves and contained less than 0.02 mg/ml endogenous ALA. The dilutions were necessary to quantitate plasma concentrations that were greater than the upper concentration limit of the standard curve (1.0 mg/ml). Each plasma sample was treated with 2 ml of acetonitrile to precipitate the plasma proteins. After centrifugation, the supernatant was transferred to a clean culture tube, and solutions containing acetylacetone and formaldehyde were added. The tubes then were capped and placed in a boiling water bath for 1 h. After cooling, 1 ml of 1 N hydrochloric acid and 0.2 ml of internal standard were added. The internal standard, a fluorescent derivative of 3amino-2-napthoic acid, was synthesized using the same procedures utilized to derivatize ALA. The internal standard was not prepared in situ with ALA to avoid variability associated with competition for the reaction reagents. The acidified solution containing the internal standard and derivatized ALA was then extracted with 5 ml of ethyl acetate. The organic phase was extracted further with 1 ml of 1 N sodium hydroxide. The aqueous phase was transferred to another tube, acidified with 1 ml of 1 N hydrochloric acid, and re-extracted with fresh ethyl acetate. The ethyl acetate then was transferred to another tube and evaporated to dryness, and the residue was reconstituted with HPLC mobile phase. Derivatized ALA and the internal standard were separated by isocratic reversed-phase HPLC. The stationary phase was a mBondapak C18 column (3.9 3 300 mm, 10-mm particle size; Waters Corporation, Milford, MA). The mobile phase contained 50% (v/v) methanol and 1% (v/v) glacial acetic acid in deionized water. The flow rate was 1.0 ml/min, and the analytes were monitored using a fluorescence detector (model 474; Waters Corporation) operating at an excitation wavelength of 363 nm and an emission wavelength of 470 nm. A standard curve was prepared in duplicate over a plasma concentration range of 0.01 to 1.0 mg/ml. Control samples (prepared at 0.03, 0.2, and 0.8 mg/ml) were stored frozen with the dog samples from the pharmacokinetic studies and were assayed in triplicate at the same time the dog samples were assayed. All analyses were performed at ambient temperature. The accuracy of the assay ranged from 110% of nominal at 0.01 mg/ml to 96% of nominal at 1.0 mg/ml. The precision (CV%) of the assay ranged from 20% at 0.01 mg/ml to 9% at 1.0 mg/ml. Data Analysis. Endogenous ALA plasma concentrations are low and do not demonstrate circadian variation (Gorchein and Webber, 1987). To correct for endogenous ALA, the predose plasma concentration of ALA (0.025 6 0.025 mg/ml, mean 6 S.D.) was subtracted from plasma ALA concentrations determined during the pharmacokinetic studies (Marzo and Rescigno, 1993). Thus, pharmacokinetic parameters reported herein represent only exogenous ALA concentrations (i.e., total minus endogenous). The plasma concentration-time profiles were analyzed using established noncompartmental methods. The maximum plasma concentration observed after oral dosing and the time at which it was observed (Tmax) were determined by direct inspection of the individual plasma concentration-time profiles. The terminal slope of the ln(concentration) versus time plot was calculated by linear least-squares regression and the half-life was calculated as 0.693 divided by the absolute value of slope. The area under the plasma concentration-time curve from time zero to infinity (AUC0-`) was calculated by the linear trapezoid rule. The total plasma clearance (CL) of i.v. ALA was calculated as the i.v. dose divided by the AUC0-` of i.v. ALA and the volume of distribution at steady state (Vdss) by the method of Benet and Galeazzi (1979). The relative bioavailability (F) of the oral doses was calculated by using the following equation: