RESUMEN
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
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
It has been demonstrated in previous work that hexokinase is solubilized from the mitochondrial membrane in anesthesia. In the present investigation this effect was strongly correlated with the surgical stage of anesthesia by the application of thiopental to rats (80 mg/kg), mice (100 mg/kg) and guinea pigs (32 mg/kg). The solubilization of hexokinase was demonstrable, too, in different areas of rat brains under thiopental treatment (80 mg/kg). In order to elevate the cerebral concentration of glucose 6-phosphate, which is the most potent substrate for the solubilization of bound hexokinase, male Sprague-Dawley rats were pretreated with the antimetabolite 6-aminonicotinamide (6-AN). After i.p. injection of 6-AN (35 mg/kg) we measured an increased activity of soluble brain hexokinase in the same order of magnitude as it was determined in anesthesia. Thiopental application did not show a further significant increase. A solubilization of hexokinase activity by 6-AN was not measurable in vitro. Furthermore, we examined the other key enzymes in the glycolytic pathway. We found a competitive inhibition of phosphofrucktokinase activity by thiopental, but no inhibition of hexokinase and pyruvate-kinase activity.
Asunto(s)
Encéfalo/enzimología , Glucólisis/efectos de los fármacos , Tiopental/farmacología , Anestesia , Animales , Encéfalo/efectos de los fármacos , Encéfalo/ultraestructura , Cobayas , Hexoquinasa/metabolismo , Cinética , Masculino , Ratones , Mitocondrias/enzimología , Fosfofructoquinasa-1/metabolismo , Piruvato Quinasa/metabolismo , RatasAsunto(s)
Anestesia , Encéfalo/enzimología , Hexoquinasa/metabolismo , Mitocondrias/enzimología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/ultraestructura , Halotano/farmacología , Técnicas In Vitro , Masculino , Mitocondrias/efectos de los fármacos , Pentilenotetrazol/farmacología , Fenobarbital/farmacología , Ratas , SolubilidadRESUMEN
The control of hexokinase activity probably is accomplished by regulating the partitioning of the enzyme between soluble and particulate forms, the latter being more active. In the present investigation we have examined the thiopental effect on the cerebral hexokinase distribution. In anesthesia, after administration of thiopental to male Sprague Dawley rats, the increase of the soluble fraction of hexokinase was dose dependent. The change in the intracellular hexokinase distribution was reversible and lasted as long as general anesthesia existed. Also in experiments in vitro a solubilization of the mitochondrial hexokinase by thiopental (0.1--1 mM) occurred; it was depending on drug concentration. An inhibition of hexokinase was found neither in the total brain extract, nor in the soluble or the particulate fraction. The results suggest that phosphorylation of glucose in brain may be suppressed in anesthesia by shifting hexokinase activity from a more active mitochondrial form to its less active soluble form. This effect seems to be caused by a direct action of thiopental and is obviously correlated with anesthesia.
Asunto(s)
Encéfalo/enzimología , Hexoquinasa/metabolismo , Mitocondrias/enzimología , Tiopental/farmacología , Animales , Encéfalo/ultraestructura , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Masculino , Mitocondrias/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Ratas , Solubilidad , Factores de TiempoRESUMEN
The effect of 6-aminonicotinamide (6-AN) on putative amino acid neurotransmitters, namely glutamate, GABA and aspartate was studied on brains of rats treated with this antimetabolite (35 mg/kg i.p.). After 6-AN application the following substrates and metabolites were determined: phosphocreatine, ATP, ADP, AMP, glucose, glucose 6-phosphate, fructose, glutamate, GABA, aspartate, ammonia, and 6-phosphogluconate. The alterations in the cerebral energy metabolism were found as reported in the literature (increased levels of glucose, glucose 6-phosphate, decreased levels of lactate and pyruvate) and could be interpreted as the result of a reduced glycolytic flux rate. After a prolonged period of 6-AN pretreatment (16-30 h) the GABA and glutamate concentrations were significantly reduced, whereas the level of aspartate remained unchanged. From the result presented the two following conclusions may be drawn: a) The changes in the concentration of neurotransmitters as GABA and glutamate could be responsible for some neurological symptoms produced by 6-AN. b) As 6-AN seems to affect the GABA-shunt it represents a model substance for studying this pathway in the nervous system.
Asunto(s)
6-Aminonicotinamida/farmacología , Química Encefálica/efectos de los fármacos , Glutamatos/metabolismo , Niacinamida/análogos & derivados , Animales , Masculino , Ratas , Factores de Tiempo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The purpose of the present investigation was to shed some light on the suppression of the glycolytic pathway by anesthetics. The antimetabolite 6-aminonicotinamide (6-AN) was used to discriminate between the key enzymes hexokinase and phosphofructokinase which are suggested to be involved in the effect of anesthetics on glycolysis. The cerebral energy metabolism was studied in the isolated perfused rat brain after the addition of thiopental (0.15 mM) to the perfusion medium, after the administration of 6-AN (35mg/kg i.p.) to the intact animals 15 h before perfusion was started, as well as in brain preparations treated in the same manner with both 6-AN and thiopental. After a perfusion period of 30 min brain levels of the following substrates and metabolites were determined: phosphocreatine, ATP, ADP, AMP, glycogen, glucose, glucose 6-phosphate, fructose 6-phosphate, pyruvate, lactate, alpha-ketoglutarate, blutamate, ammonia, and 6-phosphogluconate. The metabolic alterations in the isolated rat brain caused by 6-AN or thiopental were such as reported in the literature. When the isolated brains of the 6-AN pretreated rats were perfused with thiopental we found as the most interesting result that the concentration of glucose 6-phosphate was reduced in comparison to that in brains only treated with 6-AN but still significantly higher than that in controls. The glucose concentration was significantly elevated and the lactate concentration decreased considerably. The effect of thiopental on cerebral glycolysis was interpreted as an inhibition of hexokinase activity.