RESUMEN
A glucuronide doxorubicin prodrug N-[4-doxorubicin-N-carbonyl (oxymethyl) phenyl] O-beta-glucuronyl carbamate (DOX-GA3) has been developed to improve the antitumor effects of doxorubicin (DOX). The prodrug was originally designed to be activated into drug by human beta-glucuronidase (GUS) released from tumor cells in necrotic areas of tumor lesions. The aim of this study was to further improve the antitumor effects of DOX-GA3 by means of antibody-directed enzyme prodrug therapy (ADEPT). We thus investigated if the administration of an enzyme-immunoconjugate prepared from the pancarcinoma Ep-CAM specific monoclonal antibody (MAb) 323/A3 and beta-glucuronidase would result in improved antitumor effects because of additional enzyme localization in tumor tissue. In vitro, the prodrug DOX-GA3 was found to be 12-times less toxic than the parent drug DOX in a human ovarian cancer cell line. Immunospecific and complete activation of the prodrug took place when the cells were pretreated with 323/A3-beta-glucuronidase conjugate. In nude mice bearing s.c. human ovarian cancer xenografts (FMa) the maximum tolerated dose (MTD) of DOX-GA3 (500 mg/kg weekly x 2) was much higher when compared with that of DOX (8 mg/kg weekly x 2). In mice bearing well-established FMa xenografts, the standard treatment of DOX at the MTD (8 mg/kg weekly x 2) resulted in a tumor growth inhibition of 67%. Treatment with DOX-GA3 at a single dose of 500 mg/kg resulted in a better tumor growth inhibition of 87%. The combination of DOX-GA3 (500 mg/kg) with 323/A3-mGUS conjugate and anti-GUS MAb 105, to clear circulating conjugate, improved the antitumor effect even further to 98%. At the lower dose of 250 mg/kg DOX-GA3 tumor growth inhibition (34%) was not better than that of DOX. The combination, however, of DOX-GA3 at 250 mg/kg and 323/A3-mGUS conjugate plus MAb 105 again greatly improved the antitumor effect (growth inhibition of 93%). DOX given at 8 mg/kg weekly x 2 did not result in tumor regressions. As a result of ADEPT, the number of regressions of tumors improved from 0 out of 12 to 9 out of 11 at a dose of 250 mg/kg DOX-GA3. At the higher prodrug dose (500 mg/kg) the number of regressions improved from 2 out of 12 to 9 out of 10 as a result from the addition of enzyme-immunoconjugate. Our studies show that the efficacy of the widely used anti-cancer agent DOX may be improved by using the prodrug DOX-GA3, in combination with the tumor-specific enzyme-immunoconjugate 323/A3-mGUS and a conjugate clearing antibody.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Doxorrubicina/análogos & derivados , Doxorrubicina/uso terapéutico , Glucuronatos/uso terapéutico , Glucuronidasa/uso terapéutico , Neoplasias/tratamiento farmacológico , Profármacos/uso terapéutico , Animales , Antibióticos Antineoplásicos/uso terapéutico , Antineoplásicos/uso terapéutico , Peso Corporal/efectos de los fármacos , División Celular , Relación Dosis-Respuesta a Droga , Femenino , Glucuronidasa/metabolismo , Humanos , Dosis Máxima Tolerada , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Desnudos , Modelos Químicos , Necrosis , Trasplante de Neoplasias , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/terapia , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The doxorubicin (DOX) prodrug N-[4-doxorubicin-N-carbonyl (oxymethyl) phenyl] O-beta-glucuronyl carbamate (DOX-GA3) was synthesised for specific activation by human beta-glucuronidase, which is released in necrotic areas of tumour lesions. This novel prodrug was completely activated to the parent drug by human beta-glucuronidase with V(max)= 25.0 micromol x min(-1) x mg(-1) and K(m) = 1100 microM. The pharmacokinetics and distribution of DOX-GA3 in nude mice bearing human ovarian cancer xenografts (OVCAR-3) were determined and compared with DOX. Administration of DOX at 8 mg x kg(-1) i.v. (maximum tolerated dose, MTD) to OVCAR-3-bearing mice resulted in a peak plasma concentration of the drug of 16.4 microM (t = 1 min). A 7.6-times lower peak plasma concentration of DOX was measured after injection of DOX-GA3 at 250 mg x kg(-1) i.v. (50% of MTD). In normal tissues the prodrug showed peak DOX concentrations that were up to 5-fold (heart) lower than those found after DOX administration. DOX-GA3 activation by beta-glucuronidase in the tumour yielded an almost 5-fold higher DOX peak concentration of 9.57 nmol x g(-1) (P< 0.05) than the peak concentration of only 2.14 nmol x g(-1) observed after DOX. As a consequence, the area under the curve of DOX calculated in tumour tissue after DOX-GA3 (13.1 micromol x min(-1) x g(-1)) was 10-fold higher than after DOX (1.31 micromol x min(-1) x g(-1)). The anti-tumour effects of DOX-GA3 and DOX were compared at equitoxic doses in OVCAR-3 xenografts at a mean tumour size of 125 mm(3). The prodrug given i.v. at 500 mg x kg(-1) weekly x 2 resulted in a maximum tumour growth inhibition of 87%, while the standard treatment with DOX at a dose of 8 mg x kg(-1) i.v. weekly x 2 resulted in a maximum tumour growth inhibition of only 56%. Treatment with DOX-GA3 was also given to mice with larger tumours containing more necrosis. For tumours with a mean size of 400 mm(3) the specific growth delay by DOX-GA3 increased from 2.7 to 3.9. Our data indicate that DOX-GA3 is more effective than DOX and suggest that the prodrug will be specifically advantageous for treatment of advanced disease.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Glucuronatos/farmacocinética , Neoplasias Ováricas/tratamiento farmacológico , Animales , Área Bajo la Curva , Doxorrubicina/farmacología , Femenino , Glucuronatos/farmacología , Humanos , Inyecciones Intravenosas , Ratones , Ratones Desnudos , Necrosis , Neoplasias Experimentales , Neoplasias Ováricas/patología , Distribución Tisular , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
N-[4-daunorubicin-N-carbonyl (oxymethyl)phenyl] O-beta-glucuronyl carbamate (DNR-GA3) is a glucuronide prodrug of daunorubicin (DNR) which induced a better tumor growth delay than DNR when studied at equitoxic doses in three human ovarian cancer xenografts. These results suggested that the prodrug DNR-GA3 was selectively activated by human beta-glucuronidase present in tumor tissue. We determined the pharmacokinetics and distribution of DNR-GA3 in nude mice bearing human ovarian cancer xenografts (OVCAR-3, FMa, A2780, and MRI-H-207). Administration of DNR at 10 mg/kg i.v. (maximum tolerated dose) to OVCAR-3-bearing mice resulted in a peak plasma concentration of the drug of 12.18 microM (t = 1 min). DNR-GA3 at 100 mg/kg i.v. (approximately 50% of the maximum tolerated dose [MTD]) resulted in a peak plasma concentration of DNR that was 28-fold lower than that after DNR itself; in normal tissues, prodrug injection resulted in 5- to 23-fold lower DNR concentrations. DNR showed a relatively poor uptake into OVCAR-3 tumors with a peak concentration of 2.05 nmol x g(-1) after injection. In the same xenograft, DNR-GA3 resulted in a significantly higher DNR peak concentration of 3.45 nmol x g(-1) (P < 0.05). The higher area under the curve of DNR in tumor tissue after DNR-GA3 than after DNR itself would be the result of prodrug activation by beta-glucuronidase. In this respect, a considerably higher beta-glucuronidase activity was found in tumor tissue when compared to plasma. The specific activation of DNR-GA3 by beta-glucuronidase at the tumor site relative to normal organs leads to a more tumor-selective therapy, resulting in greater efficacy without increased toxicity.
Asunto(s)
Daunorrubicina/análogos & derivados , Neoplasias Ováricas/metabolismo , Profármacos/farmacocinética , Animales , Área Bajo la Curva , Daunorrubicina/administración & dosificación , Daunorrubicina/sangre , Daunorrubicina/farmacocinética , Femenino , Glucuronidasa/análisis , Glucuronidasa/sangre , Humanos , Inyecciones Intravenosas , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Ováricas/sangre , Neoplasias Ováricas/enzimología , Profármacos/administración & dosificación , Distribución Tisular , Trasplante HeterólogoRESUMEN
The prodrug N-[4-(daunorubicin-N-carbonyl-oxymethyl)phenyl] O-beta-glucuronyl carbamate (DNR-GA3) was synthesized for specific activation by human beta-glucuronidase, released in necrotic areas of tumour lesions. In vitro, DNR-GA3 was 18 times less toxic than daunorubicin (DNR) and the prodrug was completely activated to the parent drug by human beta-glucuronidase. The maximum tolerated dose of DNR-GA3 in nude mice bearing s.c. human ovarian cancer xenografts was 6-10 times higher than that of DNR. The prodrug was cleared more rapidly from the circulation (elimination t1/2 = 20 min) than the parent drug (elimination t1/2 = 720 min). The anti-tumour effects of DNR-GA3 and DNR were investigated in four different human ovarian cancer xenografts OVCAR-3, FMa, A2780 and MRI-H-207 at a mean tumour size between 100 and 200 mm3. In three out of four of these tumour lines, the prodrug given i.v. at the maximum tolerated dose ranging from 150 to 250 mg kg(-1) resulted in a maximum tumour growth inhibition from 82% to 95%. The standard treatment with DNR at a dose of 8 mg kg(-1) given i.v. weekly x 2 resulted only in a maximum tumour growth inhibition from 40% to 47%. Tumour line FMa did not respond to DNR, nor to DNR-GA3. Treatment with DNR-GA3 was also given to mice with larger tumours that would contain more necrosis (mean size 300-950 mm3). The specific growth delay by DNR-GA3 was extended from 2.1 to 4.4 in OVCAR-3 xenografts and from 4.4 to 6.0 in MRI-H-207 xenografts. Our data indicate that DNR-GA3 is more effective than DNR and may be especially of use for treatment of tumours with areas of necrosis.
Asunto(s)
Daunorrubicina/análogos & derivados , Neoplasias Ováricas/tratamiento farmacológico , Profármacos/uso terapéutico , Animales , División Celular/efectos de los fármacos , Daunorrubicina/farmacocinética , Daunorrubicina/uso terapéutico , Daunorrubicina/toxicidad , Relación Dosis-Respuesta a Droga , Femenino , Semivida , Humanos , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Profármacos/farmacocinética , Profármacos/toxicidad , Trasplante HeterólogoRESUMEN
Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of a prodrug by an enzyme-immunoconjugate localized in tumor tissue. The use of an enzyme of human origin is preferable in ADEPT because it might not be immunogenic when administered to patients. In the case of human beta-glucuronidase, prodrugs should be designed that are rapidly and completely activated at a neutral pH. Four new daunorubicin glucuronides were synthesized by coupling a glucuronide group to daunorubicin via an aliphatic (GA1 and GB1) or an aromatic (GA3, GB6) carbamate spacer, to be released by electron shift (A-type) or by ring closure (B-type). These prodrugs were characterized in vitro for their usefulness in ADEPT and were compared with the previously described prodrugs epirubicin-glucuronide and doxorubicin-nitrophenyl-glucuronide. The four new prodrugs were stable in serum, hydrophilic when compared to the lipophilic daunorubicin, and at least 20-fold less toxic than the parent compound. The hydrolysis rate at clinically relevant enzyme and prodrug concentrations (1 microgram/mL human beta-glucuronidase, 100 microM prodrug) at pH 6.8 were similar for GA3 (T1/2 160 min) and higher for GB6 (T1/2 40 min) when compared to that of doxorubicin-nitrophenyl-glucuronide (T1/2 170 min). Epirubicin-glucuronide, GA1, and GB1 showed a low hydrolysis rate (T1/2 > 400 min). GA1 and GA3, but not GB1 or GB6, were activated to the parent compound. Complete activation was confirmed in OVCAR-3 cells pretreated with a specific antibody-human beta-glucuronidase conjugate, where GA3 had similar antiproliferative effects to those of daunorubicin.
Asunto(s)
Glucuronidasa/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Profármacos/farmacología , Femenino , Humanos , Hidrólisis , Factores de TiempoRESUMEN
1. The relationships between the renin-angiotensin-aldosterone system, sodium and potassium balance and systolic blood pressure were studied during development of moderate (160-180 mmHg; clip i.d. 0.25 mm) and severe (200-230 mmHg; clip i.d. 0.20 mm) renal hypertension in rats with an undisturbed contralateral kidney. 2. In severely hypertensive rats renin activity in the peripheral plasma increased from day 9, by which time the systolic blood pressure was elevated to 160-180 mmHg. The rate of total corticosteroid and aldosterone production in vitro increased from day 14 and plasma renin substrate concentration increased from day 24. In moderately hypertensive rats, none of these changes occurred. 3. During the first 10 days after the application of 0.25 and 0.20 mm clips, sodium and potassium retention/g gain in body weight were higher than in sham-operated controls. During the next 10 days, the positive balance stabilized in animals with a 0.25 mm clip whereas, in animals with a 0.20 mm clip, sodium and potassium balance returned to the level of the sham-operated controls through increased renal losses. Despite these changes the systolic pressure rose further in animals with a 0.20 mm clip. 4. The initial sodium retention could be a factor in the early rise of blood pressure and could account for the delay in the rise of peripheral plasma renin activity. The subsequent loss of the retained sodium and potassium during the development of severe hypertension could have facilitated the rise in peripheral plasma renin activity, but did not initiate this rise.