No abstract

Aldophosphamide thiazolidine (NSC 613060) and aldophosphamide perhydrothiazine (NSC 612567), which hydrolyse spontaneously to 4-hydroxycyclophosphamide (4-OH-CP) in aqueous solution, were synthesised. These substances are prototypes of a new class of prodrugs for activated oxazaphosphorines. They were developed according to our hypothesis on the mechanism of action of oxazaphosphorine cytostatics. According to this hypothesis, toxicity and canceroselectivity are the results of phosphoramide mustard (PAM) release from 4-OH-CP catalysed by two classes of phosphodiesterase. 4-OH-CP toxicity results (a) from oxazaphosphorine-specific toxicity due to reactivity of the hemiaminal group with thiol groups of membrane proteins and (b) from PAM release catalysed by ubiquitous phosphodiesterases present in blood and tissues. Specific cytotoxicity suitable for antitumour therapy is based on specific PAM release in the vicinity of the target molecule DNA by the exonuclease subsites of DNA polymerases delta and epsilon. To unfold this specific core, which, we assume, improves efficacy in cancer treatment, low, long-lasting concentrations of OH-CP have to be guaranteed beneath the affinity range of the ubiquitous phosphodiesterase. This goal is facilitated by the rapid transfer of 4-OH-CP released from the perhyrothiazine derivative NSC 612567 to protein SH groups, as shown by protein-binding studies. Half-lives of hydrolysis and dissociation constants of the thiazolidine and perhydrothiazine derivatives, in which the reactivity of the hemiaminal group is inactivated by inclusion into the thiazolidine or perhydrothiazine ring, were determined to be 23 h and 6.0 x 10(-6) mol/l for NSC 613060 and 1.5 h and 1.1 x 10(-4) mol/l for NSC 312567. Accordingly the compounds guarantee low but long-lasting steady-state concentrations of 4-OH-CP. The acute toxicity determined in mice was 2400 mg/kg for NSC 613060 and 1900 mg/kg for NSC 612567. Except for a 30% decrease in leucocytes, daily i.p. injections of 260 mg/kg NSC 612567 (15% of LD50) were tolerated without signs of toxicity over a period of 4 weeks. In contrast, equitoxic doses of cyclophosphamide caused severe signs of toxicity, only five daily applications were tolerated. In mice treated repeatedly with NSC 613060, oxazaphosphorine toxicity was overlapped by thiazolidine toxicity. Scheduled activity tests in mice bearing P815 ascites tumour showed optimal therapeutic response when mice were treated daily. Repeated applications of 4% LD50 of NSC 613060 and 13% LD50 of NSC 612567 prevented tumour growth in mice with advanced, P388 lymphomas, implanted subcutaneously, without signs of overall toxicity to the host.

DNA polymerase I from E. coli can toxify activated cyclophosphamide (CP) by means of the 3'-5' exonuclease activity associated with the enzyme. Acrolein and an alkylating moiety are released in the process. Preincubation of DNA polymerase I with activated CP for 15-60 min leads to an increasing inhibition of DNA polymerase activity, which can be prevented when preincubation of DNA polymerase I with activated CP is carried out in the presence of 5' AMP, a competitive inhibitor of the 3'-5' exonuclease subsite of the enzyme. This demonstrates that toxification of activated CP by the 3'-5' exonuclease subsite of DNA polymerase is a prerequisite for the inhibition of DNA polymerase activity. The kinetics and the degree of DNA polymerase inhibition suggest that the alkylating moiety rather than acrolein released from activated CP during toxification is responsible for the inhibition of DNA polymerase. DNA polymerase with associated 3'-5' exonuclease activity has also been isolated from eukaryotic cells, and toxification of activated CP by such an enzyme (DNA polymerase delta from rabbit bone marrow) has been shown previously. Thus we suggest that toxification of activated CP by DNA polymerases/3'-5' exonucleases present mainly in proliferating cells might lead to the specific alkylation of macromolecules involved in the cell proliferation process, such as the DNA polymerase subsite of these enzymes and probably also the DNA bound to the enzymes. The relatively high cancerotoxic selectivity and cytotoxic specificity of activated CP could be based on this specific enzyme-mediated alkylation.

3'-5' Exonucleases from various sources were found to toxicogenate 4-hydroxycyclophosphamide ("activated" cyclophosphamide) by splitting the oxazaphosphorinane ring and releasing an alkylating moiety and acrolein. Neither cyclophosphamide (CP) nor the deactivated metabolites of CP, 4-keto-CP and carboxyphosphamide nor 4-(S-ethanol)-sulfido-CP were attacked by 3'-5' exonucleases. DNA polymerases with proofreading activity, such as DNA polymerase I from E. coli or DNA polymerase delta from rabbit bone marrow, exhibited a tenfold higher specific activity with "activated" CP than "plain" 3'-5' phosphodiesterases such as snake venom phosphodiesterase or 3',5'cyclic AMP phosphodiesterase from bovine heart tissue. High levels of toxicogenating activity were estimated in peripheric human lymphocytes and tissues of lymphatic origin, suggesting that enzymatic toxicogenation plays a key role with respect to the cytotoxic specificity of "activated" CP.

The effect of 6-aminonicotinamide (6-AN) on putative amino acid neurotransmitters, namely glutamate, GABA and aspartate was studied on brains of rats treated with this antimetabolite (35 mg/kg i.p.). After 6-AN application the following substrates and metabolites were determined: phosphocreatine, ATP, ADP, AMP, glucose, glucose 6-phosphate, fructose, glutamate, GABA, aspartate, ammonia, and 6-phosphogluconate. The alterations in the cerebral energy metabolism were found as reported in the literature (increased levels of glucose, glucose 6-phosphate, decreased levels of lactate and pyruvate) and could be interpreted as the result of a reduced glycolytic flux rate. After a prolonged period of 6-AN pretreatment (16-30 h) the GABA and glutamate concentrations were significantly reduced, whereas the level of aspartate remained unchanged. From the result presented the two following conclusions may be drawn: a) The changes in the concentration of neurotransmitters as GABA and glutamate could be responsible for some neurological symptoms produced by 6-AN. b) As 6-AN seems to affect the GABA-shunt it represents a model substance for studying this pathway in the nervous system.