G. Voelcker

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

SummaryThe 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-hydroxychinolin. 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. After i.v. injection of 20 mg/kg cyclophosphamide or ifosfamide peak levels of activated cyclophosphamide (4.7 nmol/ml) and the area under the curve (c·t=16.7 nmol·ml/h) showed mean values three times higher than those found for activated ifosfamide.One per cent of the applied dosis of cyclophosphamide vs. 0.3% of ifosfamide was excreted as activated metabolites. Due to the high stability of activated cyclophosphamide and ifosfamide in urine a low liberation rate of acrolein was found, the concentration of which in urine was below 0.5 nmol/ml. Acrolein, which was directly liberated from activated cyclophosphamide or ifosfamide, does not seem to play an important role in the urotoxicity of these cytostatics.

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

Abstract 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 104–105 times higher than that of IAP. In contrast to the results of these in vivo experiments, IAP was more cytotoxic against P388 mouse leukaemia cells in vitro than was SUM-IAP. The inverse correlation of in vivo antitumour activity and in vitro cytotoxicity indicates that the factors leading to the increase of antitumour activity following chemical modification of IAP differ from those involved in cytotoxicity on the cellular level.

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. With P1, which hydrolyzes quickly in blood to yield 4‐OH‐CP, different blood level shapes of 4‐OH‐CP could be established either by single bolus injection of P1 or by repetitive injection of a loading dose followed by several maintenance doses which caused nearly constant levels of activated CP for a longer time period. With these models it was found that 4‐OH‐CP showed more therapeutic efficacy when present in blood at relatively low levels for longer times than after bolus injection of the same dose resulting in a sharp peak level of activated CP. So after single intraperitoneal (IP) injection of 300 mg/kg P1 which caused a bioavailability of 36 μmol/ml−1/minute a 67% inhibition of tumor growth was achieved, whereas a tumor growth reduction of 83% was obtained after injection of the same dose in 6 fractions resulting in constant blood levels with a bioavailability of only 17 μmol/ml−1/minute. In contrast to the significant influence on antitumor efficacy of activated CP, practically no effect of pharmacokinetics on toxicity of 4‐OH‐CP could be observed. Therefore, the bioavailability of activated CP, which killed 50% of the animals, was determined to be approximately 89 μmol/ml−1/minute after adjustment of pharmacokinetics to yield constant levels and approximately 79 μmol/ml−1/minute after single bolus injection. The experiments presented show that by adjustment of pharmacokinetics the therapeutic index of P1, defined as bioavailability causing 50% of animals to die, referred to bioavailability causing 90% tumor growth inhibition, could be more than doubled.

Über Blutspiegel und Urin-Ausscheidung von aktiviertem Cyclophosphamid und seinen Deaktivierungsprodukten beim Menschen

SummaryBlood 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 1.4 (2.6) nmol/ml. The rate constant for biotransformation (=activation) of cyclophosphamide in man (km=0.132 h-1) was only 1/50 of the value found in the mouse whereas the elimination rate constant of activated cyclophosphamide (ke[M]∼6.78 h-1) was much higher equalling that of laboratory animals.4-ketocyclophosphamide, carboxyphosphamide, and phosphoramidemustard reached their peak levels between 4 and 6 h after cyclophosphamide injection. Increasing quantities of cyclophosphamide metabolites were bound to plasma proteins reaching a constant level after 24 h lasted for several days. Fifty per cent of those metabolites were reversibly bound to plasma proteins. Within 24 h, the cumulative excretion of cyclophosphamide and its metabolites amounted to 50% of the dose applied. The main metabolites excreted were phosphoramide-mustard and carboxyphosphamide whereas only 2% consited of activated cyclophosphamide. The significance of the different pharmacokinetics of cyclophosphamide in laboratory animals and man for the therapeutic index is discussed.ZusammenfassungBei krebskranken Patienten wurden während der Chemotherapie mit Cyclophosphamid Blutspiegel und Urin-Ausscheidung von Cyclophosphamid und seinen Metaboliten bestimmt. Aktiviertes Cyclophosphamid (4-Hydroxycyclophosphamid+Aldophosphamid) wurde nach Derivatisierung zum stabilen 4-(S-Benzyl)-sulfido-Cyclophosphamid nachgewiesen. 20 min nach Injektion von 10(20) mg/kg Cyclophosphamid wurden im Mittel 1,4(2,6) nmol/ml aktiviertes Cyclophosphamid gefunden. Die Aktivierungsrate von Cyclophosphamid wies beim Menschen mit einer Konstante von km=0,132 h-1 nur 1/50 des bei der Maus gefundenen Wertes auf, während die Eliminationskonstante des aktivierten Cyclophosphamid (ke[M]∼6,78 h-1) weitaus größer war und die gleiche Größenordnung wie beim Laboratoriumstier zeigte. 4-Ketocyclophosphamid, Carboxyphosphamid und N-Lost-Phosphorsäurediamid erreichten zwischen 4 und 6 h nach Cyclophosphamid-Injektion ihre Maximalspiegel im Blut. Wachsende Antiele der metabolite werden an Plasma-Proteine gebunden und nach 24 h ein über Tage andauernder konstanter Spiegel von Protein-gebundenen Cyclophosphamid-Metaboliten erreicht, von dem die Hälfte aus reversibel gebundenen Metaboliten besteht. Die kumulative Ausscheidung von Cyclophosphamid und seinen Metaboliten betrug 50% der applizierten Dosis innerhalb 24 h. Hauptausscheidungsprodukte waren N-Lost-Phosphorsäurediamid und Carboxyphosphamid, wohingengen der Anteil aktivierten Cyclophosphamids an den Urinmetaboliten nur 2% betrug.Die Bedeutung der bei Tier und Mensch unterschiedlichen Pharmakokinetik von Cyclophosphamid für seine therapeutische Breite wird diskutiert.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 1.4(2.6) nmol/ml. The rate constant for biotransformation (=activation) of cyclophosphamide in man (km = 0.132 h-1) was only 1/50 of the value found in the mouse whereas the elimination rate constant of activated cyclophosphamide (ke[M] approximately 6.78 h-1) was much higher equalling that of laboratory animals. 4-ketocyclophosphamide, carboxyphosphamide, and phosphoramidemustard reached their peak levels between 4 and 6 h after cyclophosphamide injection. Increasing quantities of cyclophosphamide metabolites were bound to plasma proteins reaching a constant level after 24 h lasted for several days. Fifty per cent of those metabolites were reversibly bound to plasma proteins. Within 24 h, the cumulative excretion of cyclophosphamide and its metabolites amounted to 50% of the dose applied. The main metabolites excreted were phosphoramide-mustard and carboxyphosphamide whereas only 2% consisted of activated cyclophosphamide. The significance of the different pharmacokinetics of cyclophosphamide in laboratory animals and man for the therapeutic index is discussed.

Activated cyclophosphamide: An enzyme-mechanism-based suicide inactivator of DNA polymerase/3′–5′ exonuclease

SummaryDNA polymerase I fromE. 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 δ 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.

Enzymatic toxicogenation of “Activated” cyclophosphamide by 3′–5′ exonucleases

Summary3′–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 δ 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.

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

Summary“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. Blood levels of free 4-hydroxycyclophosphamide and 4-hydroxyifosfamide were determined after injection of cyclophosphamide and ifosfamide into mice.Zusammenfassung“Aktivierte” N-(2-Cloräthyl)-amido-oxazaphosphorine wie 4-Hydroxycyclophosphamid, 4-Hydroperoxycyclophosphamid, 4-Hydroxyifosfamid und 4-Hydroperoxyifosfamid können über die Freisetzung von Acrolein und dessen Kondensation mit m-Aminophenol zu 7-Hydroxychinolin fluorometrisch bestimmt werden. Die Nachweisempfindlichkeit beträgt 1x10-10 mol; der Test ist spezifisch für “aktivierte” N-(2-Chloräthyl)-amidooxazaphosphorin-Metabolite mit aldehydogener Funktion am Kohlenstoff 4 des Oxazaphosphorin-Rings, welche unter Testbedingungen Acrolein freisetzen. Weder Cyclophosphamid oder Ifosfamid, noch andere Metabolite dieser Cytostatica stören den Test. Im Mäuseblut wurden die Spiegel von freiem 4-Hydroxycyclophosphamid und 4-Hydroxyifosfamid nach Injektion von Cyclophosphamid bzw. Ifosfamid in einem Zeitraum vom 90 min nach der Injektion bestimmt.

Structure/activity studies with thiazolidinyl- and perhydrothiazinyl-phosphamide ester

Abstract 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.

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

Cyclophosphamid und Metabolite des Cyclophosphamid wurden der Gleichgewichtsdialyse mit Rinderserumalbumin und menschlichem Blutserum unterworfen. Durch Auswertung nach Scatchard wurden Bindungsfestigkeit und Bindungsstochiometrie ermittelt.Cyclophosphamide and some of its major metabolites were dialyzed against bovine-serum-albumin and human-blood-serum until equilibrium. From Scatchard plots the strength and stoechiometry of protein-binding were determined. Cyclophosphamide, carboxyphosphamide and 4-ketocyclophosphamide were found to be bound only weakly and without specific binding sites. N-mustard-diamido-phosphoric-acid is bound to serum-albumin with 1,5 binding sites per protein molecule. The binding is strongly pH-dependend. The binding constant obtained from the Scatchard plot is 3.2×104 l/mol at 4° C and pH 7. No alkylation of the protein occured under the conditions of equilibrium dialysis but a strong reduction of the alkylating activity of N-mustard-diamido-phosphoric-acid was found to result from protein-binding. 4-hydroxycyclophosphamide was bound to albumin, both, by specific reaction with free thiol-groups of the protein and by unspecific weak absorption. The specific binding is based on substitution of the activated oxazaphosphorin ring by free thiol-groups of the protein forming a thio-glycosid-like protein derivate. A binding constant of 2×104 l/mol at 4° C, pH 7 was determined. The reversibility of the reaction was proven by exchange of albumin-bound unlabelled 4-hydroxycyclophosphamide by tritiated 4-hydroxy-cyclophosphamide. A strong stabilisation of 4-hydroxycyclophosphamide and decrease in alkylating activity was found to result from protein-binding. In human-blood-serum only 4-hydroxycyclophosphamide and N-mustard-diamido-phosphoric-acid showed a specific binding to protein. Free and protein-bound cyclophosphamide and metabolites could be separated by charcoal absorption. It could be demonstrated that 48 h after cyclophosphamide-injection blood-serum contained significant levels of protein-bound cyclophosphamide metabolites. Cyclophosphamid und Metabolite des Cyclophosphamid wurden der Gleichgewichtsdialyse mit Rinderserumalbumin und menschlichem Blutserum unterworfen. Durch Auswertung nach Scatchard wurden Bindungsfestigkeit und Bindungsstöchiometrie ermittelt. Cyclophosphamid selbst, sowie seine Entgiftungsprodukte Carboxyphosphamid und 4-Ketocyclophosphamid wurden nur schwach und ohne spezifische Bindungsstellen durch Absorption an Protein gebunden. N-Lost-Phosphorsäurediamid bindet sich im Verhältnis 1,5∶1 an Rinderserumalbumin. Die Bindung ist reversibel und stark pH-abhängig. Die Assoziationskonstante beträgt 3,2×104 l/Mol bei 4° C und pH 7. Eine Alkylierung des Proteins wurde unter den angewandten Versuchsbedingungen ausgeschlossen, vielmehr tritt als Folge der Protein-Bindung eine Verminderung der alkylierenden Aktivität von N-Lost-Phosphorsäurediamid ein. 4-Hydroxycyclophosphamid, der primäre Metabolit des Cyclophosphamid wird neben einer unspezifischen schwachen Absorption reversibel an freie SH-Gruppen des Albumins gebunden. Die Assoziationskonstante beträgt 2×104 l/Mol bei 4° C, pH 7. Auch hier ist eine Alkylierung des Proteins ausgeschlossen, vielmehr handelt es sich um eine Thioglycosid-artige Substitution des aktivierten Oxazaphosphorinrings durch die freien Thiol-Gruppen des Proteins. Die Gleichgewichtsnatur der Reaktion wurde durch Austauschversuche nachgewiesen. Die Protein-Bindung bewirkt eine Inaktivierung des 4-Hydroxycyclophosphamid mit verlangsamter Giftung (Alkylans-Freisetzung). Im menschlichen Blutserum ist die Bindung von Cyclophosphamid und Metaboliten an Proteine qualitativ vergleichbar mit der Bindung an Rinderserumalbumin; eine spezifische und stöchiometrische Bindung erfolgt nur mit 4-Hydroxycyclophosphamid und N-Lost-Phosphorsäurediamid. Durch Absorption an Aktivkohle kann freies 4-Hydroxycyclophosphamid von Protein-gebundenem abgetrennt werden. Nach Injektion von Cyclophosphamid lassen sich im Blutserum der Ratte noch nach 48 Std Protein-gebundene Cyclophosphamid-Metabolite nachweisen.

Zum Problem der in vitro-Sensibilitätstestung von Tumoren gegen Cyclophosphamid. 3H-Uridineinbau in Ribonucleinsäure menschlicher Tumorzellen nach Inkubation mit 4-Hydro-peroxy-Cyclophosphamid

The utility of 4-hydro-peroxy-cyclophosphamide for testing the selectivity of human tumours cells against cyclophosphamide in vitro was studied. 4-hydro-peroxy-cyclophosphamide in aqueous solution spontaneously decomposes to 4-hydroxy-cyclophosphamide, the primary product of metabolic activation of cyclophosphamide thus representing a new form of “activated” cyclophosphamide. From 31 human tumours including 21 mammarian-, 4 ovarial-, 2 uterine-carcinomas, 2 seminomas, 1 hypernephroma and 1 rectum-carcinoma cell suspensions were made and the effect on the 3H-Uridine- and the 3H-Thymidine-incorporation into the nucleic acids after short time incubation with the effect of 4-hydro-peroxycyclophosphamide and 4-hydroxy-cyclophosphamide. 7 malignomas showed high sensitivity both against 4-hydro-peroxy-cyclophosphamide and against 4-hydroxy-cyclophosphamide. No additional inhibitory effect of the peroxyde function besides of that of the alkylating moiety of the molecule was found. Accordingly 4-hydro-peroxy-cyclophosphamide because of its better availibility and stability may be used as “activated” cyclophosphamide in screening tests for cyclophosphamide-sensivity of human tumours in vitro. Es werden Versuche beschrieben, anstelle von 4-Hydroxy-Cyclophosphamid (dem biologischen Aktivierungsprodukt von Cyclophosphamid in vivo), das synthetisch besser zugängliche und stabilere 4-Hydroperoxy-Cyclophosphamid für die Sensibilitätstestung menschlicher Tumoren gegen Cyclophosphamid einzusetzen. 4-Hydro-peroxy-Cyclophosphamid geht in wässriger Lösung unter physiologischen Temperatur- und pH-Bedingungen spontan in 4-Hydroxy-Cyclophosphamid und dessen Folgeprodukte über und stellt demnach eine weitere Form von „aktiviertem“ Cyclophosphamid dar. Unsere Untersuchungen an 31 Tumoren (21 Mamma-, 4 Ovarial-, 2 Korpuscarcinome, 2 Seminome, 1 Hypernephrom und 1 Rektumcarcinom) zeigen, daß die Hemmung des 3H-Uridin- und des 3H-Thymidin-Einbaus in die Nukleinsäuren der Tumorzellen nach Kurzzeitinkubation mit 4-Hydro-peroxy-Cyclophosphamid und 4-Hydroxy-Cyclophosphamid praktisch gleich ist. 7 Malignome erwiesen sich in diesem Test als hochempfindlich gegenüber beiden Formen von aktiviertem Cyclophosphamid. Ein zusätzlicher Effekt der Peroxy-Funktion über den der alkylierenden N-Lostgruppe des 4-Hydro-peroxy-Cyclophosphamid hinaus war nicht meßbar. Wegen seiner besseren Verfügbarkeit und Haltbarkeit erscheint deshalb 4-Hydro-peroxy-Cyclophosphamid als aktivierte Form von Cyclophosphamid für die Sensibilitätstestung von Tumoren in vitro geeignet.