Prodrugs of butyric acid. Novel derivatives possessing increased aqueous solubility and potential for treating cancer and blood diseases.

The synthesis and biological activities of acidic, basic and neutral types of butyric acid (BA) prodrugs possessing increased aqueous solubility are described. The compounds are butyroyloxyalkyl derivatives of carboxylic acids, which possess functionalities suitable for aqueous solubilization. The anticancer activity of the prodrugs in vitro was evaluated by examining their effect on the growth of human colon, breast and pancreatic carcinoma cell lines, and their solubility in aqueous media was determined. The most promising compounds, with respect to activity and solubility, were found to be the butyroyloxymethyl esters of glutaric 2a and nicotinic acids 4a and phosphoric acid as its diethyl ester 10a, which displayed IC(50) values of 100 microM or lower. These prodrugs are expected to release formaldehyde upon metabolic hydrolysis. The corresponding butyroyloxyethyl esters (2b, 4b and 10b) that release acetaldehyde upon metabolism were significantly less potent. A similar correlation was observed for growth inhibition of the human prostate carcinoma cell lines PC-3 and LnCap and for induction of differentiation and apoptosis in the human myeloid leukemia cell line HL-60. The higher biological activity of the formaldehyde-releasing prodrugs 2a and 10a was further confirmed when induction of hemoglobin (Hb) synthesis in the human erythroleukemic cell line K562 was measured. Moreover, a therapeutic index (IC(50)/ED(50)) of ca. 5 was observed. The acute i.p. toxicity LD(50) in mice for 2a, 2b, 10a and 10b was similar and in the range of 400-600 mg kg(-1). The results obtained support the potential use of the butyric acid prodrugs for the treatment of neoplastic diseases and beta-globin disorders.

Uncoupling of gonadotropin-sensitive adenylate cyclase by N,N'-dicyclohexylcarbodiimide. Evidence for modification of two sites.

Treatment of purified rat ovarian plasma membranes with N,N'-dicyclohexylcarbodiimide (DCC) abolishes the subsequent response of adenylate cyclase [EC 4.6.1.1, ATP pyrophosphate lyase (cyclizing)] to lutropin (LH) and follitropin (FSH) but not to NaF. Such treatment also inhibits binding to these membranes of iodinated [125I] human chorionic gonadotropin (125I-hCG). Preincubation for 30 min at room temperature with 70 microM DCC reduces the response of adenylate cyclase to LH by 50% whereas 4 times higher concentration is required to reduce 125I-hCG binding to the same extent. At 0.5 mM, DCC reduces both activities by 50% within 8-10 min. Preincubation of the membranes with hCG prior to treatment with DCC protects the hormone-binding site of the receptor but not the ability of the enzyme to respond to LH. Thus the enzyme becomes functionally uncoupled. It is suggested that at least two distinct sites in the enzyme system are affected by DCC. Both sites are located proximal to the regulatory step that involves GTP-binding protein. One site seems to be associated with the receptor and the second is located distal to receptor-hormone complex formation.

Photo-induced inactivation and uncoupling of gonadotropin receptors in rat ovarian plasma membrane.

Suspensions of purified rat ovarian plasma membranes were irradiated by high-intensity light in the cold. This treatment gradually reduced the ability of the membrane receptor to bind 125I-labeled human chorionic gonadotropin and the ability of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) to respond to luteinizing hormone, follicle-stimulating hormone, human chorionic gonadotropin and prostaglandin E2. In contrast adenylate cyclase activity stimulated by NaF or guanosine 5'(beta, gamma-imido)triphosphate (p(NH)ppG) was significantly more resistant to irradiation. Human chorionic gonadotropin protected the binding site from light-induced damage, but not the ability of the hormone to activate adenylate cyclase. Irradiation destroyed close to 50% of unoccupied guanosine nucleotide binding sites but apparently did not induce massive covalent binding of nucleotides to membrane components. It is suggested that high-intensity light induces damage to two separate sites in the adenylate cyclase system. One affects hormone binding and is presumably associated with the hormone receptor, the second interferes with coupling but at a step proximal to regulation of adenylate cyclase by GTP binding protein.

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. I. Inhibition by heparin of gonadotrophin-stimulated ovarian adenylate cyclase.

Heparin inhibits (I50 = 2 microgram/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5'-(beta,gamma-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by heparin (I50 = 6 microgram/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Heparin (3 microgram/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged heparin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.