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F040 STUDIES OF THE ADDUCT FORMED ON INTERACTING CISPLATIN WITH HUMAN APOTRANSFERRIN: SYNTHESIS, IN VITRO BINDING KINETICS, IN VIVO PHARMACOKINETICS AND BIODISTRIBUTION IN THE RAT. J.D. Hoeschelea L.R. Whitfield,b W.R. Leopold,b W. McNally,b J. Nawrocki,b J. Phillips,C a Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109-I 055 b Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, MI 48105 and C Cancer Therapy and Research Foundation of South Texas, San Antonio, TX 78229. Transferrins are glycoproteins which are important in the cellular transport and storage of iron and other metal ions into cells [ 11. Since many tumor cells overproduce transferrin receptors, an attractive concept is to consider using transferrin as a delivery system to target cancer drugs specifically to tumor cells. We have undertaken a test of this concept using a cisplatin(cisPt)apotransferrin(aTf) adduct which was (1) synthesized, isolated (via dialysis), and analyzed via 1g5mPt radioactivity, (2) tested in vivo for antitumor activity vs implanted M5076 ovarian sarcoma tumors in the mouse, and (3) used to study the pharmacokinetic and biodistribution properties in vivo for comparison with those of cisplatin. The results and conclusions are as follows: (1) CisPt reacts with human aTf with an apparent first-order rate constant of -2x10-5 set-l. Since this rate constant is comparable to that for the aquation reaction of cisPt, it is likely that the reaction proceeds via an aquation intermediate. (2) The isolated adduct is non-stoichiometric with an approximate empirical formula of Pt~.410_75Tf. (A Pt/Tf ratio of 1.8-2.0 would be expected if the cisPt-aTf reaction went to completion and cisPt occupied both available Fe@) binding sites.) (3) The cisPt-aTf adduct is inactive against murine M5076 ovarian sarcoma over a wide range of dose levels (799-3318 mg as Tf/Kg body weight), indicating that active cisPt is not being delivered via the adduct. (4) The cisPtaTf adduct is a unique entity which exhibits pharmacokinetic and biodistribution properties distinctly different from those of cispt.

The interaction between deoxyguanosine (dG) and cis -dichlorodiammineplatinum(II) ( cis -Pt) leads to the 2:1 and the 1:1 dG-Pt adducts. These adducts were separated on an Aminex A6 cationexchange column by use ot 0.01 M K 2 CO 3 (pH 11) as an eluent. The stoichiometry of the adducts was determined from the 195m Pt radioactivity and from the absorbance of the guanine chromophore at 280 nm. Time-course studies show that dG reacts initially with cis -Pt to form the 1:1 adduct, which then interacts with a second molecule of dG to form the 2:1 adduct. Acid hydrolysis (100°C in 88% formic acid for 5–15 min) of the 1:1 and 2:1 adducts results in their conversion to two new products, which elute differently from the column but which still contain Pt bound in the same stoichiometric ratio to dG as in the nonhydrolyzed adducts. The hydrolyzed adducts show a negative diphenylamine reaction indicative ot cleavage of the glycosidic bond. It is concluded that mild acid hydrolysis converts the 1:1 and 2:1 dG-Pt adducts into the corresponding guanine-Pt adducts, which are chromatographically distinguishable. This acid hydrolysis-high pressure liquid chromatography (HPLC) procedure has application to the identification of the Pt adducts formed in DNA.