Aldophosphamide-thiazolidine (NSC-613060) an oxazaphosphorine cytostatic that crosses the blood brain barrier.

The pharmacologically active metabolite of cyclophosphamide is aldophosphamide. With cysteine, aldophosphamide forms stable aldophosphamide-thiazolidine which under physiological pH and temperature conditions hydrolyzes to aldophosphamide and cysteine. Aldophosphamide-thiazolidine was synthesized and tested for its ability as a cytostatic.

Causes and possibilities to circumvent cyclophosphamide toxicity.

Cyclophosphamide is an inert prodrug converted into 4-hydroxycyclophosphamide (OHCP) by hepatic hydroxylation. OHCP is in equilibrium with its tautomeric aldophosphamide (ALDO). From ALDO, the cytotoxic active metabolites are formed enzymatically by phosphodiesterases; these are the alkylating metabolite phosphoramide mustard (PAM) and the proapoptotic aldehyde 3-hydroxypropanal (HPA).

Oxazaphosphorine cytostatics: from serendipity to rational drug design.

On the basis of the discovery that the proapoptotic aldehyde 3-hydroxypropanal is a cyclophosphamide metabolite, a novel mechanism of action of oxazaphosphorine cytostatics is presented and confirmed by animal experiments. Furthermore, it is shown that new oxazaphosphorine cytostatics, which are on orders of magnitude more effective than already existing, can be developed on the basis of the new model for the mechanism of action.

Immunostimulating and cancer-reductive experimental therapy with the oxazaphosphorine cytostatic SUM-IAP.

SUM-IAP is an aldo-ifosfamide-perhydrothiazine derivative that, under physiological conditions, spontaneously hydrolyzes to SUM-aldo-ifosfamide. In SUM-IAP, one 2-chloroethyl group of the alkylating function of aldo-ifosfamide-perhydrothiazine is substituted by a mesyl-ethyl group. The compound was synthesized to investigate the influence of the alkylating function of aldo-ifosfamide on the antitumor activity of oxazaphosphorine cytostatics.

Influence of the alkylating function of aldo-Ifosfamide on the anti-tumor activity.

The present work investigates the influence of different DNA damages caused by different isophosphoramide mustards on the 3-hydroxypropanal-assisted apoptotic antitumor activity of oxazaphosphorine cytostatics using I-aldophosphamide-perhydrothiazine (IAP) and mesyl-I-aldophosphamide-perhydrothiazine (SUM-IAP) for in-vitro and in-vivo experiments. IAP and SUM-IAP hydrolyze spontaneously to the corresponding I-aldophosphamide derivatives. They differ in the chemical structure of the alkylating moiety, whereas IAP has two chlorethyl groups in the SUM-IAP molecule, one chlorethyl group is substituted by a mesylethyl group.

Enhancement of antitumor activity of the oxazaphosphorine cytostatic SUM-IAP by N-methylformamide.

Purpose: SUM-IAP has been developed with the aim to optimize therapeutic response and minimize toxic reactions of oxazaphosphorine cytostatics. In therapy tests in mice, the primary tumor was successfully eradicated, but animals died due to formation of lethal metastases. We supposed that high activities of SUM-IAP detoxifying enzymes caused metastasis formation in the liver.

Synthesis of 1-aldofosfamide-perhydrothiazines.

Aldofosfamide-perhydrothiazine derivatives are a new class of prodrugs which spontaneously, with half-life times of 2 to > 12 h hydrolyse to the corresponding aldophosphamide in aquous solution. Synthesis of 1-aldofosfamide-perhydrothiazine (N,N'-(2-chloroethyl)-phosphorodiamide-2-(2'-[4'-carboxy-1',3'- perhydrothiazinyl])-ethylester) and a derivative, in which one 2-chlorethyl group of the alkylating function is substituted by a mesyl-ethyl-group (N-(2-Chloroethyl)-N'-(methanesulphonylethyl)- phosphorodiamide-2-(2'-[4'-carboxy-1',3'-perhydro-thiazinyl] )-ethylester), is described.

Increased antitumour activity of mesyl-I-aldophosphamide-perhydrothiazine, in vivo but not in vitro, compared to I-aldophosphamide-perhydrothiazine.

When equitoxic dosages of ifosfamide and I-aldophosphamide-perhydrothiazine (IAP) were tested for antitumour activity in the P388 mouse leukaemia model, 80% long-term survival (for more than 100 days) was achieved with IAP whereas, in the ifosfamide group, all mice died from tumour growth within 60 days. Even better antitumour activity, compared with ifosfamide, was observed with an IAP derivative (SUM-IAP) in which one 2-chloroethyl group of the alkylating function is substituted by a mesylethyl group. Evaluation of tumour growth curves following treatment with IAP and SUM-IAP revealed the antitumour activity of SUM-IAP to be 10(4)-10(5) times higher than that of IAP.

Structure/activity studies with thiazolidinyl- and perhydrothiazinylphosphamide ester.

Structure/activity studies were carried out with thiazolidinyl- and perhydrothiazinylphosphamide ester, which differ in the structure of the phosphamide moiety and in the rate of spontaneous hydrolysis to activated oxazaphosphorines. Antitumour activity in mice with advanced P388 tumours growing subcutaneously was increased 30- to 40-fold when one 2-chloroethyl group in l-aldophosphamide-perhydrothiazine was substituted by a mesylethyl group.

Synthesis and characterization of 2-mercaptoethanesulfonic acid albumin.

Autoimmune patients treated with ifosfamide (CAS 3778-73-2) and mesna (2-mercaptoethanesulfonic acid, CAS 3375-50-6) in some cases suffered from severe allergic reactions that were proposed to be due to mesna linked to serum albumin by a disulfide bond. To prove the existence of the hypothetic mesna albumin adduct in vivo it was synthesized: The free thiol group of albumin (molecular mass determined by MALDI spectroscopy: 67009 Da) was converted to S-phenylsulfonyl albumin and reacted with mesna to albumin mesna (molecular mass: 67159 Da). In an alternative synthesis albumin was incubated with mesna at pH 8, 40 degrees C (molecular mass of the adduct: 67166 Da).

Thiazolidinyl- and perhydrothiazinylphosphamidesters: toxicity and preliminary antitumour evaluation.

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.

The enzymatic basis of cyclophosphamide specificity.

Metabolic activation of cyclophosphamide (CP) by microsomal mixed function hydroxylases yields 4-hydroxycyclophosphamide and aldophosphamide defined as activated CP. Activated CP shows relatively high cancerotoxic selectivity in vivo and cytotoxic specificity in vitro and can be trapped rapidly by reversible reaction of hemiaminal group of the oxazaphosphorine ring with protein thiols to form protein bound activated CP (protein-S-CP). Protein-S-CP stores activated CP in a highly stable form.

The embryotoxicity of cyclophosphamide in rabbits during the histiotrophic phase of nutrition.

After iv injection of cyclophosphamide (CP; 80 mg/kg) and dechlor-CP (60 mg/kg) on the 9th day of gestation (histiotrophic phase of nutrition) in rabbits, the concentrations of activated CP and activated dechlor-CP were determined fluorometrically in the maternal blood and in the yolk sac fluid. Activated CP and dechlor-CP could be measured in the maternal blood but not in the yolk sac fluid. This holds true for both the free as well as the protein bound form.

Pharmacokinetics of "activated" cyclophosphamide and therapeutic efficacies.

Estimation of "activated" cyclophosphamide (4-OH-CP) in blood of cancer patients and laboratory animals has revealed significant differences between pharmacokinetics of cyclophosphamide (CP) in man and laboratory animals after CP treatment. Whereas in blood of mice and rats relatively high concentrations of 4-OH-CP were found to exist for a relatively short time, in blood of humans only low, but longer-lasting, blood levels were detected after administration of comparable CP doses. In order to examine whether these different pharmacokinetic behaviors might account at least in part for the known differences of antitumor activity and toxicity of CP between humans and laboratory animals, the authors studied the influence of pharmacokinetics of activated CP on therapeutic efficacy and toxicity after injection of 4-(S-ethanol)-sulfido-cyclophosphamide (P1), a pro drug of activated CP, into nude mice bearing heterotransplanted human bladder sarcoma.

The influence of the protector thiol L-cystein on the toxic and therapeutic responses of stabilized "activated" cyclophosphamide (4-(S-ethanol)-sulfido-cyclophosphamide).

The influence of L-cystein on the toxic and therapeutic responses of 4-(S-ethanol)-sulfido-cyclophosphamide (P1), a stabilized "activated" cyclophosphamide, was investigated. Stabilized "activated" cyclophosphamides hydrolyze under physiological conditions to 4-hydroxycyclophosphamide (4-OH-CP). The antitumor activity of P1 was investigated on a heterotransplanted human bladder sarcoma in nude mice and in perfusion experiments carried out on the isolated tumor bearing limb in rats.

[Intracavitary chemotherapy of S 180 ascites sarcoma in mice with 4-(S-ethanol)-sulfido-cyclophosphamide in combination with protector thiols].

The experimental and pharmacokinetic basis for the local chemotherapy of body cavities with 4-(S-ethanol)-sulfido-cyclophosphamide (P1), a stable derivative of activated cyclophosphamide (CP), was evaluated on the S 180 ascites sarcoma in mice. The severe local toxicity of P1 observed after intraperitoneal administration was markedly reduced by increasing the injection volume (belly bath) without significant loss of cytotoxic activity on the S 180 tumor. Simultaneous application of L-cysteine as a "protector thiol" resulted in further reduction of toxicity without significantly decreasing the cytotoxic effect on tumor cells.

Enzymatic toxicogenation of "activated" cyclophosphamide by 3'-5' exonucleases.

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.

[Pharmacokinetics of cyclophosphamide and cyclophosphamide metabolites in the mouse and their influence on the therapeutic effect of "activated" cyclophosphamide (4-hydroxycyclophosphamide) (author's transl)].

Therapy tests with 2- [bis(2-chloroethyl)amino]tetrahydro (2H)-1,3,2-oxazaphosphorinane-2-oxide (cyclophosphamide (CP)) and its metabolites 4-hydroxycyclophosphamide (4-OH-CP) and phosphoramide mustard (NLDP) were carried out on heterotransplanted human breast cancer in nude mice. The results are: 1. Only 4-hydroxycyclophosphamide can imitate the therapeutic effect of cyclophosphamide.

Comparative study on human pharmacokinetics of activated ifosfamide and cyclophosphamide by a modified fluorometric test.

The activated metabolites of ifosfamide and cyclophosphamide (4-hydroxy-ifosfamide and 4-hydroxy-cyclophosphamide) were analysed fluorometrically by condensation of liberated acrolein with m-aminophenol yielding 7-hydroxyguinoline. Interfering fluorescence of blood and urine was eliminated by extraction with dichlormethane and determination of blanks in which the liberated acrolein reacted with hydrazine to non-fluorescent pyrazoline. The modified test proved effective in identifying low levels of activated metabolites in man.

[Blood level and urinary excretion of activated cyclophosphamide and its deactivation products in man (author's transl)].

Blood levels and urinary excretion of cyclophosphamide and its metabolites were determined in cancer patients receiving cyclophosphamide. Activated cyclophosphamide (4-hydroxycyclophosphamide aldophosphamide) was assayed by TLC after derivatisation to stable 4-(S-benzyl)-sulfido-cyclophosphamide. Twenty minutes after injection of 10(20) mg/kg cyclophosphamide mean peak levels of activated cyclophosphamide were found to be 1.

[Fluorometric determination of "activated" cyclophosphamide and ifosfamide in blood (author's transl)].

"Activated" N-(2-Chloroethyl)amido-oxazaphosphorines like 4-hydroxycyclophosphamide, 4-hydroperoxycyclophosphamide, 4-hydroxyifosfamide, and 4-hydroperoxyifosfamide can be determined fluorometrically by condensation of liberated acrolein with m-aminophenol yielding 7-hydroxychinolin. The method permits determination of 10(-10) mol and is specific for "activated" N-(2-Chloroethyl)amido-oxazaphosphorine metabolites which liberate acrolein under conditions of the test. Neither cyclophosphamide nor ifosfamide or other metabolites of this cytostatics interfere with the test.