Biochemical Pharmacolog). Vol 23. pp. 553-566. Pergamon Press. 1974. Printed in Great Britain. BINDING OF DIAZOXIDE AND OTHER BENZOTHIADIAZINES TO HUMAN ALBUMIN* EDWARD M. SELLERSt and JAN KOCH-WESER~ Clinical Pharmacology Unit, Departments of Medicine and Pharmacology, Massachusetts General Hospital and Harvard Medical School. Boston. Mass.. U.S.A. (Received 28 April 1973: accepted 11 July 1973) Abstract--The binding of seven benzothiadiazines to human albumin was studied by equi- librium dialysis. All these 1,2,4-benzothiadiazine-l,l-dioxide analogs are highly bound to human albumin. The unsubstituted benzothiadiazine nucleus is bound less than the substi- tuted analogs. Addition of a chlorine at C-6 and C-7 markedly increases binding, but further addition of methyl or sulfamyl groups results in some reduction of binding. Binding studies on benzothiadiazines do not demonstrate independent binding sites on albumin. Binding of drugs to albumin can be evaluated by fitting a logistic function to the experimental points with a least squares method, This empirical, objective technique allows examination of the nature of the bonds between drug and protein. Diazoxide, the 7-chloro-3-methyl analog, was used for detailed investigations into the mechanism of protein binding of the benzothia- diazines. The effects of pH, temperature, ionic strength, cations and deuterium on the bind- ing of diazoxide to human albumin indicate that the drug is bound mainly by hydrophobic interaction and to a lesser extent by hydrogen bonding. Difference spectroscopy studies show a shift in the electron distribution of diazoxide with binding. THE EXTENTand mechanism of the binding of benzothiadiazines to human plasma proteins have not been established. Chlorothiazide, hydrochlorothiazide, benzthia- zide and triflumethiazide are bound to the albumin fraction of dog plasma. 1'2 Dia- zoxide, a nondiuretic, hypotensive benzothiadiazine, is highly bound to human albu- min. 3'4 Since the drug is eliminated mainly by glomerular filtration, this binding results in a plasma half-life of 22 hr. 3'4 It may also account for the increase with intra- venous injection rate of the hypotensive response to diazoxide. 3'4 The present study was conducted to determine the extent of binding of diazoxide and of its benzothia- diazine analogs to human albumin and to define the types of bonds formed. METHODS Most of the analogs studied were obtained from the Schering Corp., Bloomfield. N.J. Analogs with sulfamyl substitution at C-7 were obtained from Merck & Co., Rah- way, N.J. The 6-chloro- 1,2,4-benzothiadiazine- 1, l-dioxide analog was synthesized by refluxing 10.0 g of 4-chloro-2-aminobenzene sulfonamide in 10~ excess 95~o formic acid 5-7 for 2 hr followed by precipitation in ice-cold water and subsequent crystalli- zation from butanone and then from methanol. A yield of 46.4 per cent was obtained. * This study was supported in part by United States Public Health Service Research Grants HE-06664 and HE-08643. t Former recipient Medical Research Council of Canada Fellowship. Requests for reprints: Head, Clini- cal Pharmacology Program, Addiction Research Foundation, 33 Russell St., Toronto, Canada. + Burroughs Wellcome Scholar in Clinical Pharmacology. 553