RESUMO
A series of 2-aminoalkyl-5-nitropyrazolo [3,4,5-kl]acridines (pyrazoloacridines) were tested in vitro against a panel of multidrug-resistant cell lines comprising Adriamycin-resistant P388 leukemia, B16 melanoma, and mammary adenocarcinoma 16c. This new class of anticancer agents, particularly the 9-substituted methoxy derivatives, exhibited significant activity against all of the lines tested. The degree of cross-resistance to these compounds ranged from zero to 8-fold in the 138-fold Adriamycin-resistant P388/ADR line and was greatly diminished in the B16/ADR and 16c/ADR lines. Selected pyrazoloacridines were subsequently tested in vivo against B16 and B16/ADR cells established as solid tumors from the tissue culture line and shown to retain a significant degree of Adriamycin resistance. Whereas the B16/ADR line exhibited 2 logs less net tumor-cell kill than the B16 parent in response to Adriamycin treatment, the resistant tumor was completely sensitive to the pyrazoloacridines tested and proved in some experiments to be collaterally sensitive. The favorable activity of the pyrazoloacridines against these Adriamycin-resistant tumor lines points to the potential efficacy of these compounds against multidrug-resistant tumors encountered clinically.
Assuntos
Acridinas/farmacologia , Adenocarcinoma/patologia , Antineoplásicos , Leucemia P388/patologia , Leucemia Experimental/patologia , Neoplasias Mamárias Experimentais/patologia , Melanoma Experimental/patologia , Pirazóis/farmacologia , Acridinas/uso terapêutico , Animais , Linhagem Celular , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistência a Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Substâncias Intercalantes/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Camundongos , Transplante de Neoplasias , Pirazóis/uso terapêutico , Células Tumorais Cultivadas/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco , Vincristina/farmacologia , Vincristina/uso terapêuticoRESUMO
A series of 2-aminoalkyl-5-nitropyrazolo[3,4,5-kl]acridines (pyrazoloacridines) was evaluated in vitro for activity against a panel of human tumor cell lines of breast, colon, or lung origin. Several pyrazoloacridines were found to possess solid tumor selectivity relative to their activity against murine leukemia L1210 cells as well as human lymphoblastoid cells. The superior compounds in this regard were also found to exhibit excellent activity against primary human tumors in stem cell clonogenic assays. In addition, many of the compounds tested were found to be selectively cytotoxic to hypoxic relative to oxic HCT-8 colon adenocarcinoma cells, a property that may be a consequence of the potentially reducible 5-nitro function. A number of pyrazoloacridines were also found to exhibit potency against noncycling Chinese hamster ovary cells comparable to that observed against actively dividing cultures. Consistent with their favorable activity against nondividing cells, further testing of the pyrazoloacridines revealed that generally less drug is required to inhibit RNA synthesis than DNA synthesis in L1210 cells. Collectively these data indicate that the pyrazoloacridines represent a novel class of antitumor agents which warrant further preclinical evaluation for their potential clinical usefulness in the treatment of solid tumors.
Assuntos
Acridinas/farmacologia , Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Pirazóis/farmacologia , Ciclo Celular , Linhagem Celular , DNA/biossíntese , Humanos , Oxigênio , RNA/biossíntese , Relação Estrutura-Atividade , Ensaio Tumoral de Célula-TroncoRESUMO
CI-940, PD 114,721, and PD 118,607 are structurally novel antibiotics, which were isolated from fermentation beers of a previously unknown actinomycete. They are highly lipophilic acids characterized by unsaturated lactone and branched, polyunsaturated aliphatic side-chain moieties. All three agents demonstrated significant cytotoxic activity in vitro against a number of human and mouse tumor lines which encompassed a wide range of tissue types. CI-940 retained full activity in vitro against lines of P388 leukemia that are resistant to Adriamycin, amsacrine, and mitoxantrone. Activity was confirmed for both CI-940 and PD 114,721 against a number of murine experimental tumor systems in vivo, which included the P388 and L1210 leukemias and also B16 melanoma, Ridgway osteogenic and M5076 sarcomas, and mammary adenocarcinoma 16/C. PD 118,607 was also highly active against B16 melanoma. All three agents demonstrated anticancer activity at very low dosages compared with current clinically useful anticancer agents. No significant activity was seen against the MX-1 human mammary xenograft or pancreas 02 tumor models. The primary target for host toxicity of CI-940 and PD 114,721 appeared to be gastrointestinal in nature. Neither CI-940 nor PD 114,721 caused delayed lethality when given either IP or IV. In schedule studies, the toxicities of both CI-940 and PD 114,721 were moderately dependent on the regimen used, with total maximum tolerated dosages for intermittent (q4dx2), daily (qdx5), and divided daily (q4hx3, qdx5) dosing schedules of 1, 0.25, and 0.12 mg/kg, respectively. CI-940 is being developed for clinical trial on the basis of its potent activity against seven different tumor models, its novel structure, and its apparently novel mechanism of action.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia L1210/tratamento farmacológico , Leucemia P388/tratamento farmacológico , Leucemia Experimental/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Animais , Antineoplásicos/toxicidade , Linhagem Celular , Diarreia/induzido quimicamente , Avaliação Pré-Clínica de Medicamentos , Ácidos Graxos Insaturados/uso terapêutico , Ácidos Graxos Insaturados/toxicidade , Injeções Intraperitoneais , Injeções Subcutâneas , Camundongos , Relação Estrutura-AtividadeRESUMO
The effect of the anti-tumor, anti-glutamine drug acivicin, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, was determined on the activity of the rate-limiting enzyme of de novo pyrimidine biosynthesis, carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) (EC 6.3.5.5), in human colon carcinoma. The synthetase II activity in human colon carcinoma was elevated 2- to 3-fold over values of the normal colon mucosa, and the substrate kinetic constants were similar for the enzyme in normal and neoplastic colon. The Km for glutamine was 17 microM (colon carcinoma) and 23 microM (normal mucosa), whereas the Km for ATP was 2.1 and 1.7 mM in tumor and mucosa respectively. The synthetase II activity in colon carcinoma was inhibited to a similar extent by UMP, UDP and UTP (36-41%). The three uracil nucleotides were also equally effective in inhibiting the enzyme from normal mucosa (39-46%). Both enzymes were activated by PRPP (63 and 57%) in mucosa and carcinoma respectively. Acivicin in vitro selectively inactivated the glutamine-dependent synthetase II from human colon carcinoma, and it did not affect the ammonia-dependent activity. The acivicin inactivation constant (Kinact) was 100 microM, and the minimum inactivation half-time (T) was 0.7 min. Acivicin most likely exerts its effect against human colon synthetase II by acting as an active site directed affinity analogue of L-glutamine.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/antagonistas & inibidores , Neoplasias do Colo/enzimologia , Isoxazóis/farmacologia , Ligases/antagonistas & inibidores , Oxazóis/farmacologia , Colo/enzimologia , Glutamina/análise , Humanos , CinéticaRESUMO
The concentration of L-glutamine was determined in freeze-clamped samples of normal liver of adult male fed rats (5.7-6.1 mumol/g) and in transplantable hepatomas of vastly different proliferative rates. The L-glutamine concentration in the slowly growing hepatomas was in the range of the normal liver and it decreased in relation to the increase of hepatoma growth rate, in the most rapidly growing tumors amounting to 12% of that of normal liver. In 24-hour regenerating liver, the glutamine content was slightly reduced (by 17%). In normal rat organs of high cell renewal, such as testis, intestinal mucosa, spleen, and thymus, the L-glutamine concentration was 18 to 46% of that of normal rat liver. The L-glutamine content was similar in rat brain and liver, but it was 1.6-fold higher in the heart, and low in the blood. Glutamine synthetase (EC 6.3. 1.3) activity in normal adult liver of ACI/N strain rats was 1,000 nmol per hr per mg protein; the activity increased in the very slowly growing hepatoma 20, but decreased markedly in all the other hepatomas. Thus, glutamine synthetase activity was essentially transformation-linked. The negative correlation of glutamine content with growth rate in transplanted hepatomas appears to be more closely linked with the activities of enzymes that utilize glutamine. The low L-glutamine concentration in the rapidly growing hepatomas provides a potential marker for anti-glutamine chemotherapy selectively targeted against the glutamine-utilizing enzymes.
Assuntos
Glutamina/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Fígado/metabolismo , Animais , Diferenciação Celular , Divisão Celular , Glutamato-Amônia Ligase/metabolismo , Hepatectomia , Fígado/citologia , Fígado/enzimologia , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/patologia , Regeneração Hepática , Masculino , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos BUF , Distribuição TecidualRESUMO
A single injection of the anti-glutamine drug, acivicin (NSC 163501), in tumor-bearing rats in 30 min decreased the activities of amidophosphoribosyltransferase, carbamoyl-phosphate synthetase II and CTP synthetase to 56, 50, and 7% of those of the controls. By 1 hr the activities were down to 32, 13 and 3% and they remained low for 12 hr, after which they slowly returned towards normal range in 72 hr. The decline of the activity of CTP synthetase (a loss of 80% in 10 min) was the most rapid, and the activity only returned to 60% of the controls by 3 days after the acivicin injection. In the hepatoma the concentrations of ATP and UTP changed little, but those of GTP and CTP rapidly decreased, reaching at the lowest point 32 and 2%, respectively, of control values 2 hr after acivicin; concentrations started to rise at 12 hr, reaching normal levels by 48 hr. The drop in enzyme activities preceded the decline in the pools of GTP and CTP. The behavior of enzyme activities and nucleotide concentrations in the host liver had a pattern similar to that in the hepatoma; however, the changes were less extensive than those in the tumor. The differential response between tumor and liver is attributed, in part at least, to the tissue L-glutamine concentration which in the hepatoma (0.5 mM) was 9 times lower than in the liver (4.5mM). The selectivity of acivicin action in inhibiting glutamine-utilizing enzymes is also demonstrated by the lack of effect on aspartate carbamoyltransferase, an enzymic activity which resides in the same complex as that of carbamoyl-phosphate synthetase II. The rapid decline in the activities of glutamine-utilizing enzymes is attributed to an inactivation of the enzymes by acivicin which functions as an active sitedirected affinity analog of L-glutamine. The rapid modulation of the enzymic phenotype and ribonucleotide concentrations by acivicin provides a useful tool for elucidating the role of enzymic and nucleotide imbalance in the commitment of cancer cells to replication and in the targeting of anticancer chemotherapy.
Assuntos
Amidofosforribosiltransferase/antagonistas & inibidores , Antimetabólitos/farmacologia , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/antagonistas & inibidores , Carbono-Nitrogênio Ligases , Isoxazóis/farmacologia , Ligases/antagonistas & inibidores , Neoplasias Hepáticas Experimentais/enzimologia , Oxazóis/farmacologia , Pentosiltransferases/antagonistas & inibidores , Animais , Isoxazóis/sangue , Fígado/metabolismo , Masculino , Nucleotídeos/metabolismo , Fenótipo , Ratos , Ratos Endogâmicos ACIRESUMO
The antitumor drug acivicin, L-(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, in vivo irreversibly inactivated carbamoyl-phosphate synthetase II(glutamine-dependent)(EC 6.3.5.5), the first and rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, in transplantable rat hepatoma and host liver. With two injections of 0.5 mg acivicin per 100 g body weight to one group and two injections of 5 mg to another group, enzyme activity decreased to 20 and 1% in hepatoma and to 99 and 31% in liver respectively. Aspartate carbamoyltransferase (EC 2.1.3.2) activity was not affected. Acivicin in vitro selectively inactivated glutamine-dependent activity of the synthetase II from the hepatoma and liver, with an inactivation constant (Kinact) of 90 microM and a minimum inactivation half-time (T) of 0.7 min. The inactivation velocity with 10 microM acivicin was 5.0-fold stimulated by 2 mM MgATP and 18.4-fold by 2 mM MgATP plus 16.7 mM bicarbonate. MgATP at 0.5 mM caused half-maximum stimulation of the inactivation velocity. Under in vitro conditions, L-glutamine (1 mM) protected the enzyme from inactivation by 10 microM acivicin. The synthetase activity was protected in vitro by 6 mM concentrations for glycine (84%), L-glutamate (59%) and L-aspartate (51%) and by 0.5 mM UTP (35%) from inactivation by 20 microM acivicin. The results are compatible with the suggestion that acivicin is an active site-directed affinity analog of L-glutamine.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/antagonistas & inibidores , Isoxazóis/farmacologia , Ligases/antagonistas & inibidores , Neoplasias Hepáticas Experimentais/enzimologia , Fígado/enzimologia , Oxazóis/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Técnicas In Vitro , Cinética , Magnésio/farmacologia , Masculino , Ratos , Ratos Endogâmicos ACIAssuntos
Metabolismo dos Carboidratos , Neoplasias do Colo/enzimologia , Pirimidinas/biossíntese , Animais , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/tratamento farmacológico , Desoxicitidina Quinase/metabolismo , Inibidores Enzimáticos/uso terapêutico , Humanos , Mucosa Intestinal/enzimologia , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/enzimologia , Timidina Quinase/metabolismo , Uridina Quinase/metabolismo , Uridina Fosforilase/metabolismoRESUMO
On the basis of our observation of the increased specific activities of glutamine-utilizing enzymes in purine and pyrimidine metabolism in hepatoma 3924A, and because the concentration of glutamine is ten times lower in the hepatomas than in the liver, the biochemical pharmacology of the anti-glutamine agent, acivicin, was examined. (1) Acivicin competitively inhibited the activities of amidophosphoribosyl-transferase, CTP synthetase and carbamoyl-phosphate synthetase II from extracts of liver and hepatoma 3924A. (2) In addition to the competitive inhibition exerted by acivicin, evidence was obtained that this drug also irreversibly inactivated in vitro the glutamine-utilizing enzymes. It is particularly relevant for the selectivity of acivicin that the activity of aspartate carbamoyltransferase, an enzyme present in the same complex as carbamoyl-phosphate synthetase II, was not affected by the anti-glutamine agent. (3) Acivicin in vivo brought down the activities of glutamine-utilizing enzymes in a period of 10 min to 1 hr after injection. CTP synthetase activity declined to less than 10% of that observed in the uninjected rats. The decreases were not reversible by various in vitro methods, but in vivo the activities returned to normal range in 72 hr. (4) The activity of aspartate carbamoyltransferase, which exists as a multi-enzyme complex with synthetase II, was not altered by acivicin injection. Similar results were observed in transplantable sarcoma in the rat. (5) The acivicin-induced decrease in enzymic activities could not be restored by purification of the enzymes. (6) In vitro studies indicated that addition of acivicin to liver or hepatoma extracts or purified enzymes rapidly decreased enzymic activities; the activities could not be restored. These results are consistent with an interpretation that acivicin acts either as a tight-binding inhibitor or as an inactivator through alkylation of the enzymes of glutamine utilization. (7) Acivicin in combination with actinomycin provided a synergistic kill of hepatoma cells in tissue culture and also inhibited the growth of transplantable solid hepatoma 3924A in the rat. (8) The synergistic biological results of combination chemotherapy with acivicin and actinomycin can be accounted for by the action of acivicin in inhibiting GMP and CTP synthetases, resulting in a decrease in GTP and CTP content, and by the actinomycin-caused inhibition of RNA polymerase in selectively blocking the utilization of GTP and CTP.