[CANCER RESEARCH 47, 1571-1576, March 15, 1987] Facilitated Transport of Melphalan at the Rat Blood-Brain Barrier by the Large Neutral Amino Acid Carrier System Nigel H. Greig,1 Seiji Momma, Daniel J. Sweeney, Quentin R. Smith, and Stanley I. Rapoport Laboratory of Neurosciences, National Institute on Aging, NIH, Bethesda, Maryland 20892 ABSTRACT Melphalan has been reported to be actively transported into tumor cells by two amino acid carrier systems. As amino acids are transported across cerebral capillaries by a facilitated mechanism, studies were undertaken to assess whether or not melphalan was transported similarly, and additionally to determine melphalan's plasma and brain pharma- cokinetics. The brain uptake of [MC]melphalan was measured by an in situ brain perfusion technique in the anesthetized rat utilizing [I4C|- melphalan. The cerebrovascular permeability-surface area product of |'4C]melphalan was calculated at cold melphalan concentrations from 0 to 16.3 Aunol/iul.The permeability-surface area product was concentra tion dependent and decreased from 10.8 ±0.6 (±SE)x 10~V at 0.02 iiiiiiil/inl melphalan to 5.4 ±0.3 x 10 \ ' at 16.3 ¿imol/ml. The system became saturated at a concentration in excess of 0.1 ^mol/ml. The lYIichaelis-Menten parameters (',„„, and A',,,,determined by nonlinear regression analysis of the permeability-surface area product data, equaled 0.9 ±0.3 x 10""Mmol/s/gand 0.15 ±0.06 nmol/ml, respectively, for the saturable component of melphalan's brain uptake. The A,,of the nonsat- urable component was 5.3 ±0.03 x 10 's '. Addition of the amino acid l.-phenylalanine to the brain perfusate inhibited the saturable component of melphalan's brain uptake. The analysis of the plasma and brain concentrations of melphalan by high-performance liquid chromatography, following i.v. melphalan administration, demonstrated that approximately 15% of the drug that was present in plasma entered the brain. These data suggest that the brain uptake of melphalan is facilitated, demon strating concentration-dependent uptake, saturation, and inhibition, and that melphalan shares the large neutral amino acid carrier system at the blood-brain barrier. INTRODUCTION Antineoplastic drugs have been responsible for considerable improvements in the treatment of a variety of extracerebral malignancies. However, the mainstays for the treatment of brain tumors remain surgery and radiation therapy (1). The presence of a blood-brain barrier, although of variable integrity in brain tumors (1, 2), restricts the brain uptake of water soluble compounds (3). While stereospecific, saturable carrier-mediated transport systems exist at the level of the cerebral capillary endothelium to regulate and facilitate the brain uptake of essen tial water soluble compounds (o-glucose, L-amino acids, and certain nucleic acid precursors and ions) (3, 4), no exogenous drugs have yet been demonstrated to be similarly transported (5). Melphalan is an anticancer alkylating agent proven to be effective in the treatment of multiple myeloma (6) and of carcinoma of the breast and ovary (7, 8). It is a nitrogen mustard derivative of the large neutral amino acid L-phenylalanine (Fig. 1), and has been demonstrated to be transported into tumor cells via two amino acid carrier systems (9-18). Experiments were undertaken to assess whether melphalan was similarly transported across the blood-brain barrier. A brain perfusion technique was utilized to measure the brain uptake of melphalan Received 8/25/86; revised 12/3/86; accepted 12/5/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Laboratory of Neu- rosciences. Building 10. Room 6C 103, NIH, Bethesda, MD 20892. independent of the uptake of the endogenous amino acids, which compete for the large neutral amino acid carrier system (19). In addition, as the brain pharmacokinetics of melphalan have not previously been reported, plasma and brain concentra tions of drug were measured up to 4 h following the i.v. administration of melphalan to rats. Part of this work has been published as an abstract (20). MATERIALS AND METHODS Pharmacokinetic Study Adult male rats (Fischer 344 strain), weighing 200-250 g, were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). The left sa- phenous vein was exposed and melphalan (Sigma Chemical Co., St. Louis, MO), 10 mg/kg, was injected i.v. (1 ml/kg). At intervals from 15 min to 4 h following melphalan administration, blood was collected by cardiac puncture and the brain was removed and placed on 0.9% NaCl, ice-chilled filter paper. A minimum of five animals were killed per time point. The blood was centrifuged (7000 x g, l min), and the plasma removed and stored immediately at —70°C. Plasma and brain samples were analyzed for melphalan by high-performance liquid chro matography, as described by Sweeney and colleagues (21). Brain Perfusion Study Surgical Procedure. Adult male rats (Osborne-Mendel strain), weigh ing 250-350 g, were anesthetized with sodium pentobarbital (50 mg/ kg, i.p.). The right external carotid artery was cannulated with a polyethylene catheter for retrograde infusion. In addition, the right pterygopalatine artery was ligated, the right occipital and thyroid arter ies were coagulated and cut, and the right common carotid artery was encircled with silk thread (Fig. 2). Blood flow through the right common carotid artery was never interrupted during the surgical procedure. Following the surgery, a heat lamp connected to a feedback device (YSI Indicating Controller, Yellow Springs, OH) maintained rectal temper ature at 37°C. Brain Perfusion. The cannula to the right external carotid artery was connected to a syringe containing 0.3 iiCi/ml [l4C]melphalan, 1.0 iiCi/ ml |'M |iiiiilin, 0-16.3 iimol/ml of unlabeled melphalan (Sigma Chem ical Co., St. Louis, MO), and 0-100 ^mol/ml of L-phenylalanine (Sigma) dissolved in an HCOj-buffered physiological saline (in (¿mol/ ml, 142 NaCl, 28 NaHCO3,6.0 dextrose, 4.2 KH2PO4, 1.7 CaSO4, and 1.0 MgSO4). This perfusion fluid was prepared just prior to use to minimize any hydrolysis of the melphalan. The radiochemical purities (>98%) of the [14C] and [3H]tracers were confirmed by thin-layer chromatography and gel chromatography (22, 23), respectively. The perfusion fluid was filtered through a 10-^m polypropylene filter (Gel- man Sciences, Ann Arbor, MI), oxygenated with 95% O2:5% CO2 and warmed to 37°C. The pH and CO? tension of the perfusion fluid were 7.40 and 32 mm Hg, respectively. One second before perfusion, the right common carotid artery was ligated. Then perfusion fluid was infused retrograde into the external carotid artery at a constant rate of 8.3 x 10~2 ml/s with an infusion pump (no. 944; Harvard Apparatus, South Natick, MA). At this infusion rate, the measured carotid artery pressure was between 130 and 140 mm Hg [below 160-190 mm Hg, which has been reported to damage the blood-brain barrier (3)], and circulating blood contributed less than 5% of the net flow to the right cerebral hemisphere (19). After 60-65-s brain perfusion, the rat was decapitated. A 100-iil aliquot of perfusion fluid was transferred to a vial and stored at -70°C for radiochemical analysis by thin-layer chromatography, to determine whether any hydrolysis of melphalan 1571 on March 16, 2016. © 1987 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from