Zur Bindung von Cyclophosphamid und Cyclophosphamid-Metaboliten an Serum-Albumin

Voelcker, Georg; Giera, H. P.; Jager, L. P.; Hohorst, H. J.

doi:10.1007/bf00284020

Cited by 15 publications

(13 citation statements)

References 6 publications

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“…The high correlation (r = 0.82) found between AUC and colony reduction emphasizes that this is a good example for a dose (concentration × time) Ϫ response relationship. The type of reaction between mafosfamide and proteins does not seem to be alkylation, because investigations of the metabolism of cyclophosphamide by Voelcker et al 28,29 showed a reversible but rather stable binding to proteins (especially albumin), which resulted in a marked influence on the drug's half-life. They demonstrated that not the parent drug, but two activated metabolites (4-OH-cyclophosphamide and phosphoramide mustard) react by a thioglycosidation with the SH-moities of proteins, generating a complex without alkylating properties.…”

Section: Discussionmentioning

confidence: 99%

The cytotoxicity of mafosfamide on G-CSF mobilized hematopoietic progenitors is reduced by SH groups of albumin – implications for further purging strategies

Schlenke¹,

Kisro

Deeken

et al. 1999

Bone Marrow Transplant

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Summary:The efficacy of mafosfamide purging depends on factors like incubation time, drug and erythrocyte concentration. To determine the influence of protein-bound SH groups in the incubation medium, the cytotoxicity of mafosfamide on G-CSF mobilized CD34 + cells was evaluated by short-term culture assays and drug concentration measurements. 100 mol/ml mafosfamide was incubated for 30 min in five buffers (PBS, PBS with 1%, 5% and 10% BSA and plasma). The mean calculated areas under the concentration-time curves (AUC) were 2489 ؎ 198, 1561 ؎ 286, 976 ؎ 201, 585 ؎ 62 and 605 ؎ 196 mol/l/min. The mean reductions of CFU-GM growth were 79.4%, 73.0%, 62.5%, 30.3%, 6.2% respectively. Similar results were obtained for BFU-E. Regression analysis showed a good correlation between cytotoxicity and AUCs (CFU-GM: r = 0.8195; BFU-E: r = 0.8207). This effect is well explained by the different concentrations of SH moieties in the incubation medium resulting in a higher drug binding capacity. The profound difference between AUCs and CFU-GMs in plasma and 10% BSA cannot be explained by the quantity of SH-groups. It is probably due to an additional enzymatic drug degeneration by the 3Ј-5Јexonuclease subsite of plasma DNA polymerase. In conclusion, the concentration of albumin-associated SH groups strongly influences the cytotoxicity of mafosfamide. It has to be considered as a new and important aspect in ex vivo bone marrow purging.

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Section: Discussionmentioning

confidence: 99%

The cytotoxicity of mafosfamide on G-CSF mobilized hematopoietic progenitors is reduced by SH groups of albumin – implications for further purging strategies

Schlenke¹,

Kisro

Deeken

et al. 1999

Bone Marrow Transplant

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“…These are approximately 100% after intraperitoneal and 55% after oral application. Extensive investigations with bovine serum albumin and blood samples from animals and patients after cyclophosphamide application revealed that 4-OH-CP is attached to serum albumin by free SH groups (Voelcker et al 1977). Whether NSC 612567 and/or NSC613060 also lead to binding of 4-OH-CP to serum albumin under comparable conditions was examined in the following tests.…”

Section: Chemotherapy Tests With Nsc 613060 and Nsc 612567mentioning

confidence: 99%

“…Enzymatic detoxi®cation to the cytotoxically ineective metabolites carboxyphosphamide and 4-ketocy- J Cancer Res Clin Oncol (1997) clophosphamide by aldehyde dehydrogenases and aldehyde oxidases (Hohorst et al 1971;Connors et al 1972) 2. Reversible reaction with thiol groups of proteins, for example mercaptalbumin, to form stable deactivated 4-(S-protein)-sulphido-oxazaphosphorines Peter et al 1976;Voelcker et al 1977) 3. Toxi®cation to the alkylating agent that is assumed to inhibit cell growth by DNA alkylation (Connors et al 1974).…”

Section: Introductionmentioning

confidence: 99%

Thiazolidinyl- and perhydrothiazinylphosphamidesters: toxicity and preliminary antitumour evaluation

Voelcker

Bielicki

Hohorst

1997

Journal of Cancer Research and Clinical Oncology

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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.

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“…All the above arguments refer to in vitro experiments. In vivo however free OHCP/ALDO is only transiently occurring before it is either detoxificated in the liver to carboxyphosphamide or bound at proteins [6]. The free concentration of OHCP/ALDO is 10 -7 -10 -8 M as determined by the equilibrium constant of protein bound OHCP [7].…”

Section: Introductionmentioning

confidence: 99%

Enzyme Catalyzed Decomposition of 4-Hydroxycyclophosphamide

Voelcker¹

2017

TOPROCJ

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According to general doctrine [1] canceroselectivity of Cyclophosphamide is based on different activities of the 4-hydroxycyclophosphamide (OHCP) detoxifying cellular enzyme aldehyde dehydrogenase in tumor and normal cells. Aldehyde dehydrogenase converts the OHCP tautomere aldophosphamide (ALDO) to the non-cytotoxic carboxyphosphamide. Due to different activities of the detoxifying enzyme more cytotoxic phosporamide mustard (PAM) is spontaneously released from OHCP/ALDO in tumor cells. PAM unfolds its cytotoxic activity by forming intrastrand and interstrand DNA crosslinks. This hypothesis is supported by in vitro experiments which show inverse correlations of aldehyde dehydrogenase activity and sensitivity of tumor cells against activated congeners of cyclophosphamide like mafosfamide which hydrolyses within a few minutes to OHCP. In protein free rat serum ultrafiltrate however free OHCP and its coexisting tautomer ALDO are stable compounds. Its half-life in protein free rat serum ultrafiltrate (pH7, 37 o C) is more than 20 h. Contrary to protein free ultrafiltrate in whole serum ALDO is enzymatically decomposed to PAM and 3-hydroxypropionaldehyde (HPA) within minutes. The decomposing enzyme was identified as 3´-5´ phosphodiesterase, the Michaelis constant was determined to be 10 -3 M in human serum.The experiments presented clearly demonstrate that ALDO is not only cleaved base catalyzed yielding acrolein and PAM [2, 3] but also cleaved enzymatically by serum phosphodiesterases yielding HPA and PAM. It is discussed that the reason of the high canceroselectivity of cyclophosphamide is not only due to enrichment of OHCP/ALDO in tumor cells due to less detoxification of ALDO in tumor cells than in normal cells. It is discussed that there is a good reason for an additional mechanism namely the amplification of apoptosis of PAM damaged cells by HPA.A two step mechanism for the mechanism of action of OHCP/ALDO is discussed. During the first step, the DNA is damaged by alkylation by PAM. During the second step the cell containing damaged DNA is eliminated by apoptosis, supported by HPA.

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Zur Bindung von Cyclophosphamid und Cyclophosphamid-Metaboliten an Serum-Albumin

Cited by 15 publications

References 6 publications

The cytotoxicity of mafosfamide on G-CSF mobilized hematopoietic progenitors is reduced by SH groups of albumin – implications for further purging strategies

The cytotoxicity of mafosfamide on G-CSF mobilized hematopoietic progenitors is reduced by SH groups of albumin – implications for further purging strategies

Thiazolidinyl- and perhydrothiazinylphosphamidesters: toxicity and preliminary antitumour evaluation

Enzyme Catalyzed Decomposition of 4-Hydroxycyclophosphamide

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