Kiln. Wschr. 53, 1075-1076 (1975) - (~) by Springer-Verlag 1975
Kurze wissenschatlliche Mitteilungen Side Chain Oxidation of Ifosfamide in Man K. Norpoth, H. Raidt, U. Witting, G. Mtiller and R. Norpoth Institut ffir Staublungenforschung und Arbeitsmedizin der WestfNischen Wilhelms-UniversitS~t Mfinster Received March 11, 1975
Seitenke'tenoxidation des IfosJhmids beim Menschen. Zusammenfassung. Ifosfamid wurde neben seinen stabilen, alkylierenden Metaboliten Carboxy-Ifosfamid und zwei dechlor~ithytierten Produklen im Urin eines Patienten mit Bronchial-Carcinom, dem 6 g des Mittels infundiert worden waren, zeitabh/ingig bestimmt. Zur quantitativen Analyse diente ein d~nnschichtspektrometrisches Verfahren, bei dem 4-Pyridinaldehyd-2-benzothiazolylhydrazon als Chromogen eingesetzt wird. Im Fall des untersuchten Patienten wurde Ifosfamid vorwiegend in zwei dechlor[ithylierte Produkte umgewandelt (48 % der gegebenen Dosis) und nur in geringem Umfang in Carboxy-Ifosfamid (2,2%). Schliisse:wSrter: Ifosfamid, Cyclophosphamid, Urinmetabolite des Ifosfamids.
sprayed with 4-pyridine-aldehyde-2-benzothiazolylhydrazone (3% in a mixture of acetophenone and demethylformam!de 7: 3), heated above hot acetophenone vapour to 200 °C tbr 20 min and after cooling down coloured with triethylamine-spray. For quantitative TL-photometric analysis the TL-chromatogram-Spectralphotometer PMQ II of C. Zeiss, Oberkochem, was used. The investigation of standard functions and details of the analytical procedure will be described in another paper (4). Results and Discussion
Fig. l shows that after being treated with 6 g of Ifosfamide the patient excreted largely the dechloroethyl-derivatives, the proporrnMol
Sumrnary. Ifosfamide as well as its stable alkylating metabolites namely carboxy-Ifosfamide and two dechloroethylated compounds were determined in the urine of a patient with progredient lung carcinoma who had been treated with 6 g of the drug. Quantitative measurements were carried out using a colorimetric procedure which consists of TL-spectrophotometric determinations on sheets which had previously been sprayed with 4-pyridinaldehyde-2-benzothiazolylhydrazone. In the case investigated the Ifosfamide was found to have been converted largely into two dechloroethylated compounds (48%) and only a small portion of carboxy-Ifosfamide (2.2%). Key words: Ifosfamide, cyclophosphamide, urine metabolites of ifosfamide.
Recently we described a method for thin-layer-chromatographic separation and quantification of Cyclophosphamide, Ifosfamide and Trofosfamide and their stable alkylating metabolites [4]. Examinations of laboratory animals and patients treated with these cytostatic drugs show that carboxy-compounds and dechloroethyl-derivatives are the main fractions of the alkylating urine metabolites. Their respective contribution to the total alkylating activity of the urine tells us whether the metabolic pathway leading to the biotogicaIly active aldo-derivatives, or the pathway of side chain oxidation is favoured. In this paper we show the results of an urine analysis of a patient suffering from bronchial-carcinoma, releasing nearly 50% of the dose as two dechloroethyl-derivatives after an infusion of 6 g Ifosfamide.
2,0
T
i,o
Material and Methods
The urine was collected at intervals of 6, 12, 24 and 48 h. Samples (0.01 ml) were applied to 10--20 TL plates for each specimen (Plates: "Alufolien-KieselgeI", Merck AG, Darmstadt, layer thickness 0.25 ram). Half of these were eluted with a chloroforme/methanol/ H~SO4 mixture (90: 52 : 7.5) and the other half with ethylacetate/acetone/tetrahydrofuran/H20 (40:20:20:20). The sheets were then
6
12
24
h
48
Fig. 1. Time functions of the renal excretion of tfosfamide (o- o), carboxy-Ifosfamide ( o - - o ) , and the dechloroethylated Metabolites a (m--m) and b="Dechloroethyl-CycIophosphamide" (~z--n) after infusion of 6,0 g Ifosfamide in the case of a male patient with lung carcinoma
1076
Kurze wissenschaftliche Mitteilungen
tions (% of the given dose) being 17% of the one and 31% of the other N dechloroethylated metabolite. On the other hand, carboxy-products and unconverted Ifosfamide accounted for only 2.2% and 4.7%. The isolated metabolites were identified using a combination of chromatographic, elemental-analytical and mass spectroscopic methods; and on this basis we postulate an oxidative mechanism of dechloroethylation. The suggestion of an oxidative process is supported by the observation that the action of KMnO 4 on Ifosfamide in vitro produces identical stable products of low toxicity. One dechloroethylated derivative of Cyclophosphamide is described already as metabolite in sheep [1] and as an oxidation-product of the turnover with liver-microsomes [2] and with KMnO4 [3]. In the case of Cyclophosphamide, the same product arises from oxidative splitting of either of the choroethyl-side chains, whereas with Ifosfamide two different oxidation-products will arise, one of them identical with dechloroethyl-Cyclophosphamide. We assume chloroacetaldehyde to be a further splitting product. For Trofosfamide having three chloroethyl-side chains we should expect not only the monochloroethyl-derivatives, but also Ifosfamide and Cyclophosphamide as metabolites of side chain oxidation, in this patients metabolic pattern shown in the figure the fraction of the dechloroethyl-derivatives was the highest one we found within 22 urine analyses. After a second treatment we found almost the same realtions between metabolite concentrations. Investigations on a number of other patients also showed in some cases a preference for side chain oxidation [5]. These results strongly suggest therefore that the metabolism of Ifosfamide and perhaps even Cyclophosphamide via side chain oxidation is more important in man then has been believed until now. Further research should show, whether the metabolic pattern of the individual is constant and, whether the proportion of ring-
and side chain oxidation can be influenced by additional treatment too. We wish to thank Mr. Barry Laurie, M.R.C., Cambridge, for kindful revision of our manuscript. References
1. Bakke, J.E., Feil, V.J., Fjelstul, C.E., Thacker, E.J. : Metabolism of Cyclophosphamide by Sheep. J. Agr. Food Chem. 20, 384 (1972) 2. Connors, T.A, Farmer, P.B, Foster, A.B., Jarman, M.: Some Studies of the Active Intermediates Formed in the Microsomal Metabolism of CycIophosphamide and Ifosfamide. Biochem. Pharmacol. 23, 115 - 129 (1974) 3, Jarman, M. : Formation of 4-Ketocyclophosphamide by the Oxidation of Cyclophosphamide with KMnO4. Experientia 29, 812 814(1973) 4. Norpoth, K., Addicks, H.W., Witting, U., Mfiller, G., Raidt, H. : Quantitative Bestimmung des Cyclophosphamids, Ifosfamids und Trofosfamids sowie ihrer stabilen Metabolite auf der DCw Platte mit 4-Pyridinaldehyde-2-benzothiazolyl~hydrazon. Arzneim. Forsch. (in print) 5. Norpoth, K., Mfiller, U., Witting, U., Norpoth, R., Wrist, G.: In preparation Prof. Dr. K. Norpoth Institut ffir Staublungenforschung und Arbeitsmedizin D-4400 Mfinster Westring 10 Federal Republic of Germany
Kurze wissenschatlliche Mitteilungen Side Chain Oxidation of Ifosfamide in Man K. Norpoth, H. Raidt, U. Witting, G. Mtiller and R. Norpoth Institut ffir Staublungenforschung und Arbeitsmedizin der WestfNischen Wilhelms-UniversitS~t Mfinster Received March 11, 1975
Seitenke'tenoxidation des IfosJhmids beim Menschen. Zusammenfassung. Ifosfamid wurde neben seinen stabilen, alkylierenden Metaboliten Carboxy-Ifosfamid und zwei dechlor~ithytierten Produklen im Urin eines Patienten mit Bronchial-Carcinom, dem 6 g des Mittels infundiert worden waren, zeitabh/ingig bestimmt. Zur quantitativen Analyse diente ein d~nnschichtspektrometrisches Verfahren, bei dem 4-Pyridinaldehyd-2-benzothiazolylhydrazon als Chromogen eingesetzt wird. Im Fall des untersuchten Patienten wurde Ifosfamid vorwiegend in zwei dechlor[ithylierte Produkte umgewandelt (48 % der gegebenen Dosis) und nur in geringem Umfang in Carboxy-Ifosfamid (2,2%). Schliisse:wSrter: Ifosfamid, Cyclophosphamid, Urinmetabolite des Ifosfamids.
sprayed with 4-pyridine-aldehyde-2-benzothiazolylhydrazone (3% in a mixture of acetophenone and demethylformam!de 7: 3), heated above hot acetophenone vapour to 200 °C tbr 20 min and after cooling down coloured with triethylamine-spray. For quantitative TL-photometric analysis the TL-chromatogram-Spectralphotometer PMQ II of C. Zeiss, Oberkochem, was used. The investigation of standard functions and details of the analytical procedure will be described in another paper (4). Results and Discussion
Fig. l shows that after being treated with 6 g of Ifosfamide the patient excreted largely the dechloroethyl-derivatives, the proporrnMol
Sumrnary. Ifosfamide as well as its stable alkylating metabolites namely carboxy-Ifosfamide and two dechloroethylated compounds were determined in the urine of a patient with progredient lung carcinoma who had been treated with 6 g of the drug. Quantitative measurements were carried out using a colorimetric procedure which consists of TL-spectrophotometric determinations on sheets which had previously been sprayed with 4-pyridinaldehyde-2-benzothiazolylhydrazone. In the case investigated the Ifosfamide was found to have been converted largely into two dechloroethylated compounds (48%) and only a small portion of carboxy-Ifosfamide (2.2%). Key words: Ifosfamide, cyclophosphamide, urine metabolites of ifosfamide.
Recently we described a method for thin-layer-chromatographic separation and quantification of Cyclophosphamide, Ifosfamide and Trofosfamide and their stable alkylating metabolites [4]. Examinations of laboratory animals and patients treated with these cytostatic drugs show that carboxy-compounds and dechloroethyl-derivatives are the main fractions of the alkylating urine metabolites. Their respective contribution to the total alkylating activity of the urine tells us whether the metabolic pathway leading to the biotogicaIly active aldo-derivatives, or the pathway of side chain oxidation is favoured. In this paper we show the results of an urine analysis of a patient suffering from bronchial-carcinoma, releasing nearly 50% of the dose as two dechloroethyl-derivatives after an infusion of 6 g Ifosfamide.
2,0
T
i,o
Material and Methods
The urine was collected at intervals of 6, 12, 24 and 48 h. Samples (0.01 ml) were applied to 10--20 TL plates for each specimen (Plates: "Alufolien-KieselgeI", Merck AG, Darmstadt, layer thickness 0.25 ram). Half of these were eluted with a chloroforme/methanol/ H~SO4 mixture (90: 52 : 7.5) and the other half with ethylacetate/acetone/tetrahydrofuran/H20 (40:20:20:20). The sheets were then
6
12
24
h
48
Fig. 1. Time functions of the renal excretion of tfosfamide (o- o), carboxy-Ifosfamide ( o - - o ) , and the dechloroethylated Metabolites a (m--m) and b="Dechloroethyl-CycIophosphamide" (~z--n) after infusion of 6,0 g Ifosfamide in the case of a male patient with lung carcinoma
1076
Kurze wissenschaftliche Mitteilungen
tions (% of the given dose) being 17% of the one and 31% of the other N dechloroethylated metabolite. On the other hand, carboxy-products and unconverted Ifosfamide accounted for only 2.2% and 4.7%. The isolated metabolites were identified using a combination of chromatographic, elemental-analytical and mass spectroscopic methods; and on this basis we postulate an oxidative mechanism of dechloroethylation. The suggestion of an oxidative process is supported by the observation that the action of KMnO 4 on Ifosfamide in vitro produces identical stable products of low toxicity. One dechloroethylated derivative of Cyclophosphamide is described already as metabolite in sheep [1] and as an oxidation-product of the turnover with liver-microsomes [2] and with KMnO4 [3]. In the case of Cyclophosphamide, the same product arises from oxidative splitting of either of the choroethyl-side chains, whereas with Ifosfamide two different oxidation-products will arise, one of them identical with dechloroethyl-Cyclophosphamide. We assume chloroacetaldehyde to be a further splitting product. For Trofosfamide having three chloroethyl-side chains we should expect not only the monochloroethyl-derivatives, but also Ifosfamide and Cyclophosphamide as metabolites of side chain oxidation, in this patients metabolic pattern shown in the figure the fraction of the dechloroethyl-derivatives was the highest one we found within 22 urine analyses. After a second treatment we found almost the same realtions between metabolite concentrations. Investigations on a number of other patients also showed in some cases a preference for side chain oxidation [5]. These results strongly suggest therefore that the metabolism of Ifosfamide and perhaps even Cyclophosphamide via side chain oxidation is more important in man then has been believed until now. Further research should show, whether the metabolic pattern of the individual is constant and, whether the proportion of ring-
and side chain oxidation can be influenced by additional treatment too. We wish to thank Mr. Barry Laurie, M.R.C., Cambridge, for kindful revision of our manuscript. References
1. Bakke, J.E., Feil, V.J., Fjelstul, C.E., Thacker, E.J. : Metabolism of Cyclophosphamide by Sheep. J. Agr. Food Chem. 20, 384 (1972) 2. Connors, T.A, Farmer, P.B, Foster, A.B., Jarman, M.: Some Studies of the Active Intermediates Formed in the Microsomal Metabolism of CycIophosphamide and Ifosfamide. Biochem. Pharmacol. 23, 115 - 129 (1974) 3, Jarman, M. : Formation of 4-Ketocyclophosphamide by the Oxidation of Cyclophosphamide with KMnO4. Experientia 29, 812 814(1973) 4. Norpoth, K., Addicks, H.W., Witting, U., Mfiller, G., Raidt, H. : Quantitative Bestimmung des Cyclophosphamids, Ifosfamids und Trofosfamids sowie ihrer stabilen Metabolite auf der DCw Platte mit 4-Pyridinaldehyde-2-benzothiazolyl~hydrazon. Arzneim. Forsch. (in print) 5. Norpoth, K., Mfiller, U., Witting, U., Norpoth, R., Wrist, G.: In preparation Prof. Dr. K. Norpoth Institut ffir Staublungenforschung und Arbeitsmedizin D-4400 Mfinster Westring 10 Federal Republic of Germany
![[Ifosfamide and the metabolites of side chain oxidation--excretion in urine in various pediatric therapeutic protocols].](/assets/img/hold.png)
