RESUMEN
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. Therefore, therapy tests with SUM-IAP in combination with cisplatin and N-methylformamide (NMF), which were not detoxified in the liver, were carried out. METHOD: Antitumor activity was assayed in female CD2F1 mice with advanced subcutaneously growing P388 mice leukemia cells. RESULT: The results of the therapy tests with SUM-IAP plus cisplatin were as expected: No formation of metastases and long-time survival of more than 100 days were observed; however, the toxicity was increased as measured by decrease in body weight and the number in leukocytes. The results of the tests in combination with NMF were surprising: Applying only half the dose of SUM-IAP used in the experiments with cisplatin, no metastases were found and long-time survivors did not show signs of additional toxicity. CONCLUSION: NMF strongly enhances the antitumor activity of the oxazaphosphorine cytostatic SUM-IAP in mice with subcutaneously growing P388 mice leukemia cells by an unknown mechanism of action.
Asunto(s)
Antineoplásicos/farmacología , Formamidas/farmacología , Compuestos Organofosforados/farmacología , Tiazinas/farmacología , Animales , Antineoplásicos/administración & dosificación , Cisplatino/administración & dosificación , Sinergismo Farmacológico , Femenino , Formamidas/administración & dosificación , Ratones , Compuestos Organofosforados/administración & dosificación , Tiazinas/administración & dosificaciónRESUMEN
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.
Asunto(s)
Antineoplásicos/síntesis química , Mostazas de Fosforamida/síntesis química , Profármacos/síntesis química , Tiazinas/síntesis química , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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. 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.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Experimental/tratamiento farmacológico , Mostazas de Fosforamida/farmacología , Animales , Muerte Celular , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Ifosfamida/farmacología , Leucemia Experimental/patología , Ratones , Tiazinas/farmacologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Mostazas de Fosforamida/farmacología , Tiazoles/farmacología , Animales , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Leucemia P388/tratamiento farmacológico , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Relación Estructura-ActividadRESUMEN
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).
Asunto(s)
Albúminas/química , Mesna/análogos & derivados , Mesna/química , Albúmina Sérica/química , Cromatografía de Afinidad , Humanos , Concentración de Iones de Hidrógeno , Mesna/síntesis química , Peso Molecular , Albúmina Sérica/síntesis química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
Asunto(s)
Compuestos de Mostaza Nitrogenada/toxicidad , Profármacos/toxicidad , Tiazinas/toxicidad , Tiazoles/toxicidad , Animales , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacocinética , Ciclofosfamida/toxicidad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/uso terapéutico , Inhibidores Enzimáticos/toxicidad , Femenino , Inyecciones Intraperitoneales , Leucemia P388/tratamiento farmacológico , Masculino , Sarcoma de Mastocitos/tratamiento farmacológico , Ratones , Compuestos de Mostaza Nitrogenada/química , Compuestos de Mostaza Nitrogenada/uso terapéutico , Unión Proteica , Tiazinas/química , Tiazinas/uso terapéutico , Tiazoles/química , Tiazoles/uso terapéutico , Tiazolidinas , Distribución TisularRESUMEN
Thiazolidinyl- and perhydrothiazinyl-ethyl-N-mustard-phosphamide esters were designed to act as highly specific suicide inactivators of DNA polymerase alpha holoenzymes. Acute and subacute toxicity of these drugs in mice was very small. By daily i.p. injection, on day 0-4 mice were cured of P 388 lymphatic leukaemia with no depression of blood leucocytes. The findings suggest that suicide inactivators of DNA polymerase alpha holoenzyme may be promising drugs for low toxicity cancer chemotherapy.
Asunto(s)
Antineoplásicos , ADN Polimerasa II/antagonistas & inhibidores , Leucemia P388/tratamiento farmacológico , Leucemia Experimental/tratamiento farmacológico , Compuestos de Mostaza Nitrogenada/uso terapéutico , Tiazinas/uso terapéutico , Tiazoles/uso terapéutico , Animales , Cinética , Leucemia P388/enzimología , Ratones , TiazolidinasRESUMEN
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. From pharmacokinetics of activated CP in mice after the injection of cyclophosphamide, it was calculated that about 17% of the CP dose given was stored in a pool of protein bound activated CP lasting for several days. From therapy studies with 4-(S-ethanol)-sulfido-CP in combination with excess of cysteine, it was concluded that the protein-S-CP pool may be that form of activated CP which is mainly responsible for the specific cytotoxic effects in the tumor cells. On the other hand free unbound 4-OH-CP was shown to contribute mainly to overall toxicity. No spontaneous toxicogenation of activated CP was noted under in vivo conditions. 3'-5' Exonucleases were found to hydrolyze 4-OH-CP, yielding phosphoramide mustard and acrolein as split products. Because of the low affinity of 4-OH-CP for plain 3'-5' exonucleases, it seems however unlikely that these enzymes play a major role in the antitumor effect of CP in vivo. 3'-5' Exonucleases associated to DNA polymerase like in DNA polymerase delta from rabbit bone marrow or in DNA polymerase I from E. coli are more likely candidates for 4-OH-CP toxicogenation because of the much higher specific activity with 4-OH-CP as substrate. In experiments with DNA polymerase I from E. coli, 4-OH-CP was shown to inhibit DNA polymerase activity after toxicogenation by the 3'-5' exonuclease subsite of the enzyme. This suggests an enzyme mechanism based suicide inactivation of the DNA polymerase. Because of the close spatial cooperation of the DNA polymerase and 3'-5' exonuclease subsites with primer/template a site-specific alkylation of DNA is also postulated. Thus we raised the hypothesis that cytotoxic specificity of activated CP is based on the interaction of protein-S-CP (protein bound activated CP) with DNA polymerase/3'-5' exonuclease as the target. In a P 815 mouse mast-cell tumor we determined by means of 5' AMP agarose affinity chromatography two/third of total DNA polymerase to be associated with 3'-5' exonuclease.
Asunto(s)
Ciclofosfamida/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Cromatografía de Afinidad , Ciclofosfamida/análogos & derivados , Ciclofosfamida/metabolismo , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/antagonistas & inhibidores , Exonucleasas/metabolismo , Sustancias Macromoleculares , Sarcoma de Mastocitos/enzimología , Ratones , Inhibidores de la Síntesis del Ácido Nucleico , Proteínas/metabolismo , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
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. During the histiotrophic phase of nutrition on the 9th day of gestation, the yolk sac wall seems to be a barrier for activated CP and dechlor-CP. This phenomenon has to be traced back on the oxazaphosphorinring activated in position C4 and not on the alkylating activity. Therefore, a direct effect of activated CP can be excluded as the main reason for the embryotoxicity of CP. Consequently, the effects of iv-injected CP on the entoderm of the visceral layer of the yolk sac placenta were investigated. Three, 6, 12, and 24 hours after CP injection, the maternal animals were laparotomized and the entoderm of the visceral layer of the yolk sac placenta in the mesometral parts of the blastoderms were prepared for electron microscopy. Like in the control group, 3 hours after CP injection no differences are found. Six hours after CP injection, a relatively flat cell-type can be observed, which is probably based on the reduced absorptive capability of the entoderm. Twelve hours after CP injection the entoderm cells are nearly uniformly of the columnar type; this is interpreted as a restored absorptive activity. Twenty-four hours after CP injection the columnar form of the entoderm cells and the reduced pinocytotic activity are interpreted as a state of secretion. During the histiotrophic phase of nutrition (9th day of gestation in rabbits), CP-induced inhibition of the absorptive activity of the entoderm cells might lead to a quantitatively and/or qualitatively changed nutrition of the developing embryo. This changed nutrition may be the source of the embryotoxic effects of CP.
Asunto(s)
Ciclofosfamida/toxicidad , Embrión de Mamíferos/efectos de los fármacos , Animales , Biotransformación , Ciclofosfamida/metabolismo , Embrión de Mamíferos/ultraestructura , Femenino , Edad Gestacional , Microscopía Electrónica , Embarazo , Conejos , Factores de TiempoRESUMEN
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.
Asunto(s)
Ciclofosfamida/análogos & derivados , Animales , Ciclofosfamida/metabolismo , Ciclofosfamida/uso terapéutico , Ciclofosfamida/toxicidad , Cisteína/administración & dosificación , Esquema de Medicación , Femenino , Cinética , Masculino , Ratones , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
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. Due to its rapid hydrolysis to 4-OH-CP, P1 exhibits severe local toxicity which is decreased by the protector thiol L-cystein. Simultaneous application of double molar amounts of L-cystein reduces toxicity in nude mice to approximately one-third. Therapeutic activity is not affected by this ratio of L-cystein so that the protector thiol increases the therapeutic efficacy of P1. Higher amounts of L-cystein reduce both the acute toxicity in nude mice and the therapeutic efficacy against the human xenograft. The perfusion experiments demonstrate that a P1 concentration necessary to cure rats with tumor bearing limb is only tolerated in combination with L-cystein.
Asunto(s)
Ciclofosfamida/análogos & derivados , Sarcoma de Yoshida/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Animales , Quimioterapia del Cáncer por Perfusión Regional , Ciclofosfamida/administración & dosificación , Ciclofosfamida/uso terapéutico , Ciclofosfamida/toxicidad , Cisteína , Femenino , Humanos , Dosificación Letal Mediana , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Ratas , Ratas Endogámicas , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Ciclofosfamida/uso terapéutico , Sarcoma 180/tratamiento farmacológico , Compuestos de Sulfhidrilo/uso terapéutico , Animales , Ciclofosfamida/toxicidad , Cisteína/uso terapéutico , Quimioterapia Combinada , Femenino , Cinética , Mesna/uso terapéutico , Ratones , Compuestos de Sulfhidrilo/administración & dosificaciónRESUMEN
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.
Asunto(s)
Ciclofosfamida/metabolismo , ADN Polimerasa I/antagonistas & inhibidores , Exodesoxirribonucleasas/antagonistas & inhibidores , Acroleína/farmacología , Alquilación , Animales , Biotransformación , Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacología , ADN Polimerasa I/metabolismo , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/metabolismo , HumanosRESUMEN
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.
Asunto(s)
Ciclofosfamida/metabolismo , Exodesoxirribonucleasas/metabolismo , Animales , Biotransformación , Ciclofosfamida/análogos & derivados , Ciclofosfamida/toxicidad , ADN Polimerasa Dirigida por ADN/metabolismo , Endonucleasas/metabolismo , Exodesoxirribonucleasa V , Humanos , Linfocitos/metabolismo , Tejido Linfoide/metabolismo , Ratas , Ribonucleasas/metabolismoRESUMEN
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. 2. The therapeutic effect is not proportional to the product of concentration and time (cXt) in blood as tumor growth was more inhibited with lower cXt when high concentrations were present over a short time than with higher cXt and adjustment of low concentrations over a long time. Pharmacokinetic measurements in mice demonstrated that this behaviour is due to the elimination of "activated" cyclophosphamide by Michaelis-Menten kinetics (Km = 146 nmol X ml-1, Vmax = 18 nmol X ml-1 X min-1) and to the fact that increasing blood concentration of "activated" cyclophosphamide is accompanied by its increased distribution towards the peripheral compartment as may be seen from the volumes of distribution of the peripheral and central compartments. From blood concentration curves of cyclophosphamide and cyclophosphamide metabolites we calculated that 91% of the cyclophosphamide administered is activated. 81% of the "activated" cyclophosphamide is detoxified to 4-keto-CP and carboxyphosphamide (Carboxyph). Since the detoxifying enzymatic system is more active against aldophosphamide than against 4-hydroxycyclophosphamide the non-detoxified rest of "activated" cyclophosphamide is composed of 80% of 4-hydroxycyclophosphamide and 20% of aldophosphamide.
Asunto(s)
Ciclofosfamida/análogos & derivados , Ciclofosfamida/metabolismo , Animales , Biotransformación , Ciclofosfamida/uso terapéutico , Femenino , Cinética , Neoplasias Mamarias Experimentales/tratamiento farmacológico , RatonesRESUMEN
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.
Asunto(s)
Ciclofosfamida/análogos & derivados , Ciclofosfamida/metabolismo , Ifosfamida/metabolismo , Acroleína/metabolismo , Biotransformación , Femenino , Humanos , Cinética , Masculino , Persona de Mediana Edad , Espectrometría de FluorescenciaRESUMEN
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.
Asunto(s)
Ciclofosfamida/metabolismo , Neoplasias/metabolismo , Anciano , Animales , Biotransformación , Proteínas Sanguíneas/metabolismo , Ciclofosfamida/análogos & derivados , Femenino , Humanos , Cinética , Masculino , Ratones , Persona de Mediana Edad , Mostazas de Fosforamida/metabolismo , Unión Proteica , Ratas , Especificidad de la EspecieRESUMEN
"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.