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Aldofosfamide-perhydrothiazine derivatives are a new class of prodrugs which spontaneously, with half-life times of 2 to12 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.

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.

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.

Auf der Groshirnrinde der Katze wurden der mittlere O2- und CO2-Druck, die lokale Hirndurchblutung und der pH-Wert wahrend passiver Hyperventilation (art. CO2-Druck unter 19 mm Hg) fortlaufend gemessen und zu definierten Zeitpunkten Gewebsproben der Hirnrinde entnommen, die auf ihre Gehalte an Metaboliten des energieliefernden Stoffwechsels analysiert wurden. Ziel der Untersuchung war eine Synopsis der auf der Hirnoberflache gemessenen Daten mit geeigneten Kenngrosen des Energie-Stoffwechsels. Unmittelbar nach Beginn der Hyperventilation sank die Durchblutung, der mittlere kortikale Gewebsdruck fur O2 und CO2 bei gleichzeitiger Erhohung des pH-Wertes. Im weiteren Verlauf des Experimentes erreichte die lokale Hirndurchblutung wieder den Ausgangswert. Trotz eines Anstiegs des Gewebs-Laktatspiegels um das Funffache wahrend der Hyperventilation und des auf 5–10 mm Hg erniedrigten lokalen O2-Druckes der Hirnoberflache war der Phosphorylierungsgrad der energiereichen Phosphate nicht geringer als unter normoxyschen Bedingungen. Die Untersuchungen ergeben keinen Anhalt fur einen Energiemangel der Hirnrindenzelle bei Hyperventilation. Der Begriff der Hypoxie und ihre Kenngrosen werden fur den zellularen Bereich prazisiert. Die moglichen Ursachen des erhohten Gewebs-Laktatspiegels wahrend der Hyperventilation trotz Fehlens von Zeichen einer zellularen Hypoxie werden diskutiert.

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. 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.

“Aktivierte” N-(2-Clorathyl)-amido-oxazaphosphorine wie 4-Hydroxycyclophosphamid, 4-Hydroperoxycyclophosphamid, 4-Hydroxyifosfamid und 4-Hydroperoxyifosfamid konnen uber die Freisetzung von Acrolein und dessen Kondensation mit m-Aminophenol zu 7-Hydroxychinolin fluorometrisch bestimmt werden. Die Nachweisempfindlichkeit betragt 1x10-10 mol; der Test ist spezifisch fur “aktivierte” N-(2-Chlorathyl)-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 storen den Test. Im Mauseblut 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.

Bei krebskranken Patienten wurden wahrend 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, wahrend die Eliminationskonstante des aktivierten Cyclophosphamid (ke[M]∼6,78 h-1) weitaus groser war und die gleiche Grosenordnung wie beim Laboratoriumstier zeigte. 4-Ketocyclophosphamid, Carboxyphosphamid und N-Lost-Phosphorsaurediamid 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 uber Tage andauernder konstanter Spiegel von Protein-gebundenen Cyclophosphamid-Metaboliten erreicht, von dem die Halfte 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-Phosphorsaurediamid und Carboxyphosphamid, wohingengen der Anteil aktivierten Cyclophosphamids an den Urinmetaboliten nur 2% betrug.

Das Cytostaticum Cyclophosphamid (ENDOXAN®) wird in vivo uber 4-Hydroxycyclophosphamid zur Wirkform „gegiftet“ bzw. aktiviert. Unsere Ergebnisse zeigen, das auf biologischem Weg aktiviertes Cyclophosphamid fur in vitro Tumorsensibilitatsteste ungeeignet ist, da die Versuchsergebnisse durch die zwangslaufig mit zugesetzten Fremdeiweise bzw. Leberextrakte verfalscht werden. Diese Schwierigkeiten lassen sich mit chemisch dargestelltem 4-Hydroxycyclophosphamid umgehen. Bei 29 Tumortestungen (14 Mammacarcinome, 8 Ovarialcarcinome, 7 weitere Malignome) wurde mit Hilfe einer Kurzzeitinkubationstechnik die Einbauhemmung von 3H-Uridin und 3H-Thymidin gepruft. 12 Tumoren zeigten deutliche, 5 weitere masige Beeintrachtigungen des Einbaus der Nucleinsaureprakursoren. 11 von 12 in vitro gegen 4-Hydroxycyclophosphamid empfindliche Tumoren reagierten auch gegenuber anderen Cytostatica sensibel. Histologisch entdifferenzierte Tumoren waren empfindlicher gegenuber 4-Hydroxycyclophosphamid als hochdifferenzierte Tumoren. Erste weiterfuhrende Untersuchungen deuten darauf hin, das das nur geringe Zeit haltbare 4-Hydroxycyclophosphamid in vitro ersetzbar ist durch 4-Hydroperoxycyclophosphamid. Diese Substanz ist bei −20° C mehrere Monate haltbar.

Die Aufnahme von tritiiertem N,N-Bis(2-chlorathyl)-phosphorsaurediamid in Ehrlich-Ascites-Tumorzellen wurde mit Hilfe der Siliconol-Filtrationsmethode untersucht. Selbst bei Konzentrationen von 10 mM konnte bei 1°C innerhalb 5 min keine Permeation in die Tumorzellen festgestellt werden. Dagegen zeigen Cyclophosphamid und 4-Hydroperoxycyclosphamid bei einer Konzentration von 1 mM unter gleichen experimentellen Bedingungen eine relativ rasche Aufnahme und erreichen nahezu den gleichen Sattigungswert. Offensichtlich ist die fehlende Aufnahme von N,N-Bis(2-chlrathyl)-phosphorsaurediamid in Tumorzellen Grund fur die auserordentlich geringe cytotoxische Aktivitat dieses Cyclophosphamid-Metaboliten.

Die Aufarbeitung von menschlichen Erythrocyten nach demWestphal schen Phenol/Wasser-Verfahren zeigt, das sich die M- bzw. N-Substanzen in der Polysaccharidfraktion befanden und weder durch die Aufarbeitung noch durch kurzes Kochen ihre Spezifitat verloren. Die M- und N-Substanzen sind folglich sicher keine Eiweise oder Glucoproteide.

The in vivo activation of cyclophosphamide (Endoxan) to cytostatic and alkylating metabolites has been studied. Endoxan is metabolized to the weak anion by microsomal dealkylation in the rat's liver; this product is the first alkylating metabolite. About 80% of the alkylating activity of the serum, occurring 30 min after injection of Endoxan is presented by this primary alkylating metabolite. Nor-N-mustard and N-2-chloroethyl-aziridine were found only in traces. Although some activation was detected in the liver, the lungs and to a minor extent the kidneys, this phenomenon has not been detected in the screened experimental rat tumors. The authors conclude that the greater specificity of Endoxan as compared to other cytostatic agents of the N-mustard group may be due to some specific permeation properties of the primary alkylating metabolite of Endoxan rather than to a possible specific mechanism of action of this compound within tumor cells.

An Ratten mit Jensen-Sarkom wurde der Einflus von Cyclophosphamid und Athyleniminobenzochinon auf den DPN-Gehalt von Tumor und Knochenmark gepruft. Bei Dosen mit vergleichbarer leukocytensenkender Wirkung sank der DPN-Gehalt im Tumor im Verlauf von 8 Tagen bei beiden Substanzen um mehr als die Halfte ab; eine Erniedrigung des DPN-Gehaltes im Knochenmark wurde dagegen nur nach Athyleniminobenzochinon, nicht nach Cyclophosphamid beobachtet. Die Befunde werden als Hinweis fur eine gesteigerte Selektivitat der cancerctoxischen Wirkung von Cyclophosphamid angesehen.

The hydroperoxy and several alkylthio derivatives of the antitumor agents cyclophosphamide (2-bis(2-chloroethyl)amino tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide), ifosfamide (3-(2-chloroethyl)-2-(2-chloroethylamino) tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) and trofosfamide (3-(2-chloroethyl)-2- (bis(2-chloroethyl)amino)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) were characterized by electron impact and field desorption mass spectrometry. The compounds, which are stabilized derivatives of the activated hydroxylated intermediates of cyclophosphamide (ifosfamide, trofosfamide), could be identified as 4-hydroperoxy and 4-alkylthio oxazaphosphorines. The existence of diastereomers of these products was demonstrated by thin-layer chromatography and f.d. mass spectra. Derivatization with benzylmercaptan was found to be an appropriate method for the quantitative isolation and mass spectral identification of the activated metabolic intermediates of cyclophosphamide from biological material. Using this reaction, 4-hydroxycyclophosphamide and its acyclic tautomer, aldophosphamide, which are too unstable for direct identification, were detected in urine and serum of patients treated with 3H-cyclophosphamide.

The existence of the cyclophosphamide (CP) metabolites, 4-hydroxycyclophosphamide and aldophosphamide, in vivo after application of CP to mice and rats has been demonstrated. The metabolite pattern in the serum and urine of rats at different times after CP application was determined. For this purpose, thin-layer chromatograpic procedure was developed which allows separation of the metabolites without decomposition. Because considerable amounts of radioactivity are bound to serum proteins after 3H-CP application, protein binding studies with CP and CP metabolites were carried out. It was found that 4-hydroxycyclophosphamide is bound to thiol groups of bovine serum albumin yielding a rather stable product which could be isolated by column chromatography on Sephadex.

Average Po2 and Pco2, local blood flow and pH values in the cerebral cortex of the cat were measured during passive hyperventilation (arterial Pco2 below 19 mm Hg). At defined intervals tissue samples were taken for metabolite analysis. The object of the study was to correlate the data obtained on the brain surface with metabolic responses. Immediately after the start of hyperventilation blood flow decreased, average cortical tissue pressures of O2 and CO2 fell, and there was a simultaneous rise in cortical pH. At a later stage in the experiment the local blood supply reverted to its resting level. Despite a fivefold rise in tissue lactate level during hyperventilation and a decrease in local O2 pressure on the brain surface to 5-10 mm Hg the degree of phosphorylation of energy rich phosphates was not less than under normal conditions of oxygenation. Our investigations showed no evidence of energy lack in cerebral cortex cells during hyperventilation. Cellular hypoxia and its characteristics are defined. The possible causes of raised tissue lactate levels during hyperventilation despite the lack of evidence of cellular hypoxia are discussed.

The relatively high oncostatic specificity of cyclophosphamide (CP) in vivo is shown to be due to the cytotoxic specificity of 4-hydroxycyclophosphamide (4-hydroxy-CP), the first product of metabolic activation of CP in the liver. This specificity can be evaluated not only in vivo by measuring the therapeutic index, but also in vitro by determining its cytotoxicity against Yoshida ascites tumor cells. Evidence is given that 4-hydroxy-CP is not an alkylating agent itself, but attains this property only by release of an alkylating N,N-(2-chloroethyl)phosphorodiamic acid moiety and acrolein. The energetic source for this rate-limiting toxication results from the resonance stabilization of the released acrolein. Reactions at the cryptoaldehyde group of 4-hydroxy-CP, which reduce or prevent the resonance stabilization of the 3-carbon unit to be released, lead to a deactivation of the primary metabolite of CP thus reducing or even preventing toxication, and hence influencing both the alkylating and cytotoxic activities of the molecule. Accordingly, it could be demonstrated by the reaction of 4-hydroxy-CP with thiols yielding 4-(S-R)-mercapto CP derivatives that the toxication of 4-hydroxy-CP can be controlled under physiologic conditions of pH and temperature. In the case of free protein sulfhydryl groups, this reaction also leads to fixation onto a macromolecule of the CP metabolite. On the basis of these peculiar reactivities of the oxazaphosphorine ring of 4-hydroxy-CP and of the partial reaction kinetics involved during toxication or deactivation, the significance of these findings to the problem of CP specificity is discussed.

a) N-2-chloroethylamido-oxazaphosphorines such as cyclophosphamide, ifosfamide and trofosfamide differ from “non-oxazaphosphorine-substituted” (alkylating) N-mustards by the selectivity of their cancerotoxic action in vivo (measured by their therapeutic index) and by the fact that for exertion of their alkylating and cytotoxic properties they must first be transformed in the body. This bio-transformation is a multi-step process which commences with enzymatic hydroxylation at carbon 4 of the oxazaphosphorine ring (activation). b) Cancerotoxic selectivity is closely bound to the oxazaphosphorine ring. Transfer of a chloroethyl group from the extracyclic amide-nitrogen (cyclophosphamide) to the cyclic amide-nitrogen (ifosfamide) or replacement of a 2-chloroethyl group by a 2-mesyloxyethyl group leaves the essential basic properties of the N-2-chloroethylamido-oxazaphosphorines largely unchanged. In spite of this, some compounds show significant pharmacotherapeutic peculiarities regarding organotropic effects and cumulative properties. c) The cancerotoxic selectivity of the N-2-chloroethylamido-oxazaphosphorines in vivo is closely related to the cytotoxic specificity of their activated primary metabolites against cancer cells in vitro. In the case of 4-hydroxycyclophosphamide, these two properties are abolished by toxification and liberation of the strongly alkylating nitrogen mustard phosphorid acid diamide. d) The toxification is the rate-determining step of the spontaneous breakdown of 4-hydroxy-cyclophosphamide. It is pH-dependent and can be indirectly controlled by competitive deactivation reactions. e) Apart from enzymatic dehydrogenation of the primary activated metabolites of cyclophosphamide into non-toxic metabolites, a reversible nonenzymatically catalysed deactivation by thiol compounds is described. This does not involve the alkylating moiety of N-2-chloroethylamido-oxazaphosphorines, but the cyclic cryptoaldehyde group of activated oxazaphosphorines, leading to the formation of 4-(SR)-mercapto-oxazaphosphorines. Since free sulfhydryl groups or proteins were found to react according to the same mechanism, activated N-2-chloroethylamido-oxazaphosphorines can thus be bound to cellular structures and enzymes. f) The significance of the various deactivation reactions for cancerotoxic selectivity and cytotoxic specificity of N-2-chloroethylamido-oxazaphosphorines is discussed with particular regard to the importance of thiol deactivation for cytotoxic specifivity and cancerotoxic selectivity against tumor cells.

4-Hydroperoxycyclophosphamide was obtained in approximately 20% yield by ozonization of cyclophosphamide in acetone/water at 0 degrees C. It was reduced to 4-hydroxycyclophosphamide, which was treated with several mercaptans to yield compounds of the type 4-(S-R)-mercapto-cyclophosphamide. In the solid state these compounds are stable at room temperature; in aqueous solution they are hydrolyzed to 4-hydroxycyclophosphamide or its tautomer, aldophosphamide. One of the 4-(S-R)-mercapto-cyclophosphamide compounds was tested biologically in vitro against Yoshida ascites tumor cells and showed the same cytotoxic activity as 4-hydroxycyclophosphamide.

Several important metabolites of cyclophosphamide (CP), such as 4-hydroxycyclophosphamide and phosphoramide mustard, some 4-alkyl(aryl)thio derivatives have been investigated by field-desorption and electron-impact mass spectrometry. The structural identification of synthetic compounds and of derivatives, isolated by thin-layer chromatography in vitro, was possible since complementary information can be obtained using the two ionization techniques. Whereas only the field-desorption mass spectra showed more abundant molecular ions, the electron-impact technique revealed a characteristic fragmentation pattern for most of the compounds studied. Examples are given which demonstrate the existence of stereoisomers of CP derivatives using thin-layer chromatography and field-desorption mass spectrometry. The results indicate that field-desorption mass spectrometry is also a particularly appropriate method for the characterization of unstable CP metabolites in vivo.

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-peroxy-cyclophosphamide 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.

The uptake of tritiated N,N-bis(2-chloroethyl)-diamido-phosphoric-acid into Ehrlich-Ascites-Tumor cells of mice was studied by means of the siliconoil-filtration technique. At 10 mM concentration no permeation of the metabolite into the tumor cells could be found within 5 min at 1 degrees C, while its congenors cyclophosphamide and 4-hydroperoxycyclophosphamide (1 mM) were shown to permeate into the cells very easily reaching saturation values. Thus lack of permeation into tumor cells of N,N-bis(2-chloroethyl)-diamidophosphoric-acid seems to be the reason for the poor cytotoxic activity of this metabolite of cyclophosphamide.

An assay for the characterization and quantitative determination of activated cyclophosphamide has been described. The method is based on high reactivity of the kryptaldehyde group in 4-hydroxycyclophosphamide with mercapto compounds to yield 4-(S-R)mercaptocyclophosphamide derivatives. The activated cyclophosphamide can be converted quantitatively to 4-(S-benzyl)mercaptocyclophosphamide with benzyl mercaptan, and the mercapto derivative can be separated by thin-layer chromatography on silica gel with ethyl acetate:methyl ethyl ketone as solvent. Using this assay, significant levels of activated cyclophosphamide in the blood of mice and rats, as well as of humans, were found to be present after cyclophosphamide application. The characterization of 4-(S-benzyl)mercaptocyclophosphamide formed was confirmed by comparing the thin-layer chromatographic and mass spectrometric data on this derivative obtained from an authentic sample of 4-hydroxycyclophosphamide.

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-peroxy-cyclophosphamide 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.

The reaction of 4-hydroxycyclophosphamide (4-hydroxy-CP) with sulfhydryl (SH) compounds was studied, and although the cytotoxic activity was lost a significant stabilization of the alkylating capacity was observed at the same time. We were able to show that the reaction of 4-hydroxy-CP with thiols lead to an equilibrium between the reaction product and the starting substrates. On the basis of this equilibrium the increased stabilization of the alkylating capacity of the 4-hydroxy-CP derivatives by raising the SH concentration can be explained. The different degrees of stabilization depending on the structure of the thiol results from different equilibria. the effect on the toxification reaction resulting from this equilibrium, in connection with the tautomeric equilibrium between 4-hydroxy-CP and aldophosphamide, is discussed.

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 permeation kinetics of cyclophosphamide and its metabolites were studied with Ehrlich ascites tumor cells, murine L1210 leukemia cells, and mouse L929 fibroblasts at 1 degrees C. In contrast to carboxyphosphamide and nor-nitrogen mustard, an equipartition of cyclophosphamide and 4-hydroperoxycyclophosphamide was observed in these cells. First experiments have been done to study the efflux of cyclophosphamide and 4-hydroperoxycyclophosphamide after incubation until saturation with Ehrlich ascites tumor cells. Cyclophosphamide could be washed out completely, whereas, 4-hydroperoxyclophosphamide shows an apparent retention in the cell at 37 degrees C.

"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.

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.

To determine whether the release of acrolein from oxazaphosphorinane-cytostatics contributes to their cytotoxic action, the effect of 4-hydroperoxycyclophosphamide, 4-hydroperoxy-semi-cyclophosphamide, 4-hydroperoxy-dechloro-cyclophosphamide, and acrolein on murine L 1210 leukemia cells in vitro was compared by measuring the median survival time (MST) after transplantation of the tumor cells in DBA2/Han mice. We found that only 4-hydroperoxycyclophosphamide, which is able to release both acrolein and the alkylating metabolite phosphoramide-mustard, decreased the transplantability of L 1210 cells, while the structurally analogous 4-hydroperoxy-dechloro-cyclophosphamide and 4-hydroperoxy-semi-cyclophosphamide, which under physiological conditions only release acrolein but no alkylating split products, showed no cytotoxicity. Acrolein itself showed only a marginal effect, when administered in concentrations equivalent to the release of acrolein from the oxazaphosphorinane-derivatives in test. In this case, however, significant lysis of the L 1210 cells was observed by estimating dye exclusion, while acrolein released intracellularly from 4-hydroperoxy-oxazaphosphorinane-compounds did not. This points to a different mechanism of the cytotoxic action of extracellular acrolein and acrolein released intracellularly from activated oxazaphosphorinane-compounds. The results suggest that the cytotoxic effect of activated cyclophosphamide is based on the alkylating moiety of the molecule. Neither the 4-hydroperoxy-group nor the activated oxazaphosphorinane-ring itself, nor acrolein released intracellularly during toxification of activated cyclophosphamide exert a direct cytotoxic effect. Thus, the release of acrolein from activated CP apparently does not contribute to the cytotoxicity of CP in vivo.

A one-step synthesis of 4-hydroperoxyifosfamide and 4-hydroperoxytrofosfamide is described. The method involves direct ozonation of ifosfamide and trofosfamide and offers improved yields in comparison with Fenton oxidation and greater convenience in comparison with ozonation of the appropriate 3-butenyl phosphorodiamidate. Evaluation of the 4-hydroperoxy derivatives of cyclophosphamide, ifosfamide, and trofosfamide against leukemia L1210 in vivo suggests a superior effect for the ifosfamide derivatie.

A new in vitro assay for screening the sensitivity of human tumour cells against Cyclophosphamide has been developed. While biologically activated Cyclophosphamide was unsuitable because of unpurities in the material, synthetic 4-Hydroxycyclophosphamide was shown to inhibit the incorporation of tritiated uridine and thymidine into the nucleic acids of human tumour cells in vitro. 29 tumours including 14 mammarial carcinomas, 8 ovarial carcinomas and 7 other malignant tumours were tested. While 12 tumours showed a significant and 5 only a slight inhibition of the 3H-uridine incorporation in vitro. 12 tumours showed no effect. Histologically none-differentiated tumours were more sensitive against 4-Hydroxycyclophosphamide as compared with the more differentiated ones. First observations point to 4-Hydroperoxycyclophosphamide instead of 4-Hydroxycyclophosphamide as a more suitable form of activated Cyclophosphamid for the in vitro assay of Cyclophosphamide sensitiveness because of the higher stability and better availability of this compound.

Crystalline 4-(SR)-sulfidocyclophosphamides, sulfido derivatives of activated cyclophosphamide (4-hydroxycyclophosphamide), were synthesized by ozonation of cyclophosphamide and reaction of the intermediate 4-hydroxycyclophosphamide with various thiols (HSR). The products were characterized by elemental analysis, 1H NMR and IR spectroscopy, and mass spectrometry. 1H NMR and polarimetric analysis demonstrated that they consist of racemic cis-isomers that are stable in the crystalline state at room temperature. In aqueous solution these derivatives are hydrolyzed to 4-hydroxycyclophosphamide and the corresponding thiol, with half-lives ranging between 4 and 17 min at 37 degrees C and pH 7. The cytotoxicity of 4-(S-ethyl)- and 4-(S-ethanol)-sulfidocyclophosphamide against Yoshida sarcoma ascites cells and the toxicity in rats were found to be practically identical with those of activated cyclophosphamide. A preliminary evaluation of the curative effect after a single IV injection of 4-(S-ethane)- and 4-(S-ethanol)-sulfidocyclophosphamide in rats bearing Yoshida ascites sarcoma or of 4-(S-ethanol)-sulfidocyclophosphamide in nu/nu mice bearing human breast carcinoma xenografts suggested that these sulfido derivatives possess the same oncostatic efficacy as activated cyclophosphamide itself.

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. Thus the therapeutic index was increased (2.5 fold) by the combination of belly bath and protection by L-cysteine, contrary to 2-mercaptoethane sulfonic acid sodium salt (Mesna) as protector thiol which reduced both the acute toxicity and the curative effectiveness of P1. Pharmacokinetic parameters were determined by measuring the concentrations of P1 and 4-hydroxycyclophosphamide (4-OH-CP), carboxyphosphamide and 4-ketocyclophosphamide in blood and peritoneal fluid. As a result of these measurements the reduction of toxicity of P1 after high volume i.p. administration is due to increased enzymatic detoxification of 4-OH-CP to 4-ketocyclophosphamide and carboxyphosphamide. The effect of L-cysteine on the toxicity of P1 is mainly the consequence of transmercaptalisation of P1 to 4-(S-cysteine)-sulfido-CP. By this reaction formation of the toxic 4-OH-CP in the peritoneal cavity is diminished, and the peritoneal clearance of "activated" CP reduced.

Cyclophosphamid und Metabolite des Cyclophosphamid wurden der Gleichgewichtsdialyse mit Rinderserumalbumin und menschlichem Blutserum unterworfen. Durch Auswertung nach Scatchard wurden Bindungsfestigkeit und Bindungsstochiometrie ermittelt.

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 mumol/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 mumol/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 mumol/ml-1/minute after adjustment of pharmacokinetics to yield constant levels and approximately 79 mumol/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.

Patients with impaired liver function have a reduced biotransformation rate of the cytostatic agent cyclophosphamide. With pathologically reduced serum cholinesterase activity the half-life of the drug increases from normally 4.3 h to 6.7 h. These patients show significantly lower peak levels of activated cyclophosphamide (4-hydroxy-cyclophosphamide + aldophosphamide). Because of the low renal clearance of cyclophosphamide (16 ml/min) and equally low renal excretion of activated cyclophosphamide amounting to only 1% of the applied dose more than 80% of the drug is still metabolized and the area under the curve of activated cyclophosphamide (cXt) remains relatively constant. No change in the pharmacokinetics of cyclophosphamide and its activated metabolite is observed in an anuric patient. However, an accumulation of toxic, directly alkylating metabolites with a fourfold alkylation rate of plasma proteins is found in this case. Hemodialysis sufficiently eliminated the toxic alkylating metabolites without a measurable influence on the pharmacokinetics of activated cyclophosphamide.

With regard to the mechanism of action of cyclophosphamide some controversy exists whether 4-hydroxycyclophosphamide (“activated cyclophosphamide”) or phosphoramide mustard, a metabolite formed from it by s-elimination of acrolein, is the therapeutically effective metabolite. In the following report, pharmacokinetic measurements after intravenous injection into the mouse show that 90% of cyclophosphamide administered is activated to 4-hydroxycyclophosphamide and that 80% thereof is detoxified to 4-ketocyclophosphamide and carboxyphosphamide. From therapy tests on heterotransplanted human breast cancers only 4-hydroxycyclophosphamide (liberated in vivo from cyclophosphamide) but not phosphoramide mustard, can imitate the therapeutic effects of cyclophosphamide. These results exlude the extracellular formation of phosphoramide mustard having a significant role in the therapeutic effects of cyclophosphamide.