Conversion of the CPT-11 metabolite APC to SN-38 by rabbit liver carboxylesterase

The anticancer drug CPT-11 (7-ethyl-[4(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is a water-soluble derivative of camptothecin. We report here the conversion of APC (7-ethyl-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin), an inactive metabolite of CPT-11, to SN-38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT-11, by a rabbit liver carboxylesterase. This reaction is not catalyzed by any known human enzyme. The formation of SN-38 from APC was characterized by an apparent Km of 37.9 +/- 7.1 microM and a Vmax of 16.9 +/- 0.9 pmol/units/min. SN-38 was confirmed as a reaction product by high-performance liquid chromatography and mass spectrometry. A 24-h incubation of 10 microM APC with 500 units/ml of rabbit carboxylesterase produced 4 microM SN-38. The product of this reaction inhibited the growth of U373 MG human glioblastoma cells in vitro. The IC50 for a 24-h exposure of U373 MG cells to APC in the presence of 50 units/ml of rabbit carboxylesterase was 0.27 +/- 0.08 microM, whereas APC alone demonstrated no inhibition of growth at concentrations up to 1 microM. The IC50 of U373 MG cells transfected with the cDNA encoding the rabbit carboxylesterase (U373pIRESrabbit) and exposed to APC for 24 h was 0.8 +/- 0.1 microM APC, whereas the growth of cells transfected with vector control (U373pIRES) was unaffected by up to 1 microM APC. Because APC is nontoxic to human cells, we are investigating the possibility of using APC/rabbit carboxylesterase in a prodrug/enzyme therapeutic approach.     

Plasma pharmacokinetics of 7-ethyl-10-hydroxycamptothecin (SN-38) after intravenous administration of SN-38 and irinotecan (CPT-11) to rats

We studied the plasma pharmacokinetics of the lactone form and the carboxylate form of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), after intravenous bolus administrations of each form of SN-38 and of CPT-11 to rats. After the SN-38 lactone injection, SN-38 lactone was predominant at first, and then the lactone to the carboxylate concentration ratio (LC ratio) was maintained from 30 to 90 min after the injection. The carboxylate was predominant throughout the period after the carboxylate dosing. Model-dependent analyses showed that the SN-38 lactone had greater plasma clearance and a greater distribution volume than the carboxylate. The CPT-11 administration resulted in a predominant plasma SN-38 lactone concentration. The contribution of the SN-38 lactone AUC to the total SN-38 AUC (57%) was independent of the dose of CPT-11. These results suggest that it is possible to estimate the SN-38 lactone concentration and AUC from the total SN-38 concentration without separate determination of the lactone and carboxylate. Our results showed that both SN-38 lactone and CPT-11 administration gave the predominant SN-38 lactone in plasma; however, only CPT-11 could sustain the lactone concentration at a high level, which is necessary for antitumor activity.     

Reversal of CPT-11 resistance of lung cancer cells by adenovirus-mediated gene transfer of the human carboxylesterase cDNA

To evaluate the concept that transfer of the human carboxylesterase (CE) gene will overcome the drug resistance of a solid tumor to CPT-11 (irinotecan), we used an adenovirus vector (AdCMV.CE) carrying human CE cDNA to infect CPT-11-resistant A549 human adenocarcinoma cells (A549/CPT) in vitro and in vivo and evaluated cell growth over time. The A549/CPT cells, selected by stepwise and continuous exposure of parental A549 cells to CPT-11 over 10 months, had a 6-fold resistance to CPT-11 and 42% CE activity in comparison with parental A549 cells. AdCMV.CE infection resulted in an increase in functional CE protein in resistant cells in vitro that was sufficient to convert CPT-11 to its active metabolite, SN-38, and effectively suppressed resistant cell growth in vitro in the presence of CPT-11. When AdCMV.CE was directly injected into established s.c. resistant A549-based tumors in nude mice receiving CPT-11, there was a 1.8-fold reduction in tumor size at day 20 compared to that of controls (P < 0.05). These observations suggest that adenovirus-mediated gene transfer of the human CE gene and concomitant administration of CPT-11 may have potential as a strategy for local control of acquired CPT-11 resistance of solid tumors.     

The transformation of irinotecan (CPT-11) to its active metabolite SN-38 by human liver microsomes. Differential hydrolysis for the lactone and carboxylate forms

Apolipoprotein E (apo E) is a component of all the classes of lipoproteins and can be distributed among apo B- (LpB) and non apo B-containing lipoproteins (Lp-non-B). Using a new electroimmunoassay kit, plasma apo E, apo E in Lp-non-B (apo E-Lp-non-B) and apo E in LpB (apo E-LpB) levels were measured in healthy control subjects (n=481) from 3 centers participating in the ECTIM study (Etude Cas-Témoins sur l'Infarctus du Myocarde), a population-based study on myocardial infarction. The distribution of apo E among lipoproteins was analyzed according to the apo E phenotype after adjustment for center, body mass index, tobacco use, alcohol consumption and triglycerides. Apo E was higher (average excess: + 0.32; P < 0.0001) and lower (average excess: -0.12; P < 0.0001) in subjects carrying the allele epsilon2 and the allele epsilon4 respectively, than in apo E3/3 subjects. These differences are the consequence of variations in apo E-Lp-non-B which clearly differed between the groups classified according to their apo E phenotype (P < 0.0001). The average excess of apo E Lp non-B compared to apo E3/3 subjects was + 0.43 (P < 0.0001) and -0.22 (P < 0.0001) for the epsilon2 and epsilon4 alleles respectively. Apo E-LpB was lower in subjects carrying the epsilon2 allele (P < 0.02) while the presence of the epsilon4 allele did not modify this parameter. The proportion of apo E within HDL was clearly higher and lower in subjects carrying apo E2 and apo E4 respectively than in apo E3/3 subjects. Although triglyceride levels were dependent on the apo E phenotype, the adjustment of the proportion of apo E in HDL for triglycerides hardly modified the results. For the first time, these results, using direct measurements on a large number of subjects, confirm the greater preference of apo E4 over apo E2 for LpB and vice versa for Lp-non-B. They also show a greater affinity of apo E2 for HDL compared to apo E3. This high affinity of apo E2 for HDL could be due to the formation of the apo E-A-II complex. These results indicate that apo E phenotype modulates the distribution of apo E among lipoproteins and suggest differences in lipoprotein metabolism between apo E2, apo E3 and apo E4.     

Determinants of trimetrexate lethality in human colon cancer cells

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.     

Irinotecan (CPT-11) metabolites in human bile and urine

A female patient was treated with irinotecan (CPT-11) for liver metastatic colon carcinoma. She had a percutaneous biliary catheter because of extrahepatic biliary obstruction. The patient was treated with CPT-11 for three courses at doses of 350 mg/m2 for the first course and 300 mg/m2 for the remaining courses, given as a 30-min i. v. infusion. Metabolism studies in bile and urine were performed by coupling high-performance liquid chromatography to electrospray mass spectrometry. Conventional spectra [liquid chromatography/mass spectrometry (LC/MS)] allowed on-line molecular mass determination of CPT-11 and its main metabolites, whereas structural information was obtained by tandem mass spectrometry (LC/MS/MS). At least 16 metabolites were detected in bile, while 8 of them were also detected in urine. Three compounds were identified as the parent drug, the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), and SN-38 glucuronide. The major metabolic pathway consists in oxidations of the terminal piperidine ring of the CPT-11 side chain, which eventually results in the formation of a primary amine. Other metabolites result from oxidation of the camptothecin nucleus. Finally, decarboxylation of the carboxylate form of CPT-11 was also observed. Several metabolites result from combinations of these pathways. The structures of the identified metabolites indicate for the first time a major role of monooxygenases in the elimination of a camptothecin derivative in humans. This finding will allow better understanding of interindividual variability in pharmacokinetics and intestinal toxicity of CPT-11.     

Active efflux of CPT-11 and its metabolites in human KB-derived cell lines

To investigate the possible involvement of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and/or other glutathione S-conjugate export pump (GS-X pump) family members on the active efflux of irinotecan [(7-ethyl-10-[4-(1-piperidino)-1-pipertidino)-1-piperidino]carb onylox y camptothecin (CPT-11)] and its metabolites, as well as their contribution to the acquisition of resistance, we studied the uptake of CPT-11, its active metabolite SN-38, and glucuronide conjugate (SN38-Glu) using membrane vesicles from human epidermoid KB-3-1-derived cell lines. These lines included KB-C2, C-A500, and KCP-4, which overexpress P-gp, MRP, and the unidentified GS-X pump, respectively. The carboxylate form of SN-38 exhibited significant ATP-dependent transport, with a Michaelis constant of 17 microM, into membrane vesicles from C-A500 but not from other cell lines. Among these KB-derived cells, significant ATP-dependent uptake of the carboxylate form of CPT-11 was only observed in KB-C2 vesicles. In addition, the uptake of the lactone and carboxylate forms of SN38-Glu into membrane vesicles from C-A500 and KB-C2, but not KCP-4, was ATP dependent, although the transport activity in C-A500 was much higher than that in KB-C2. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay revealed that the resistance of KB-C2 to CPT-11 and SN-38, compared with that of KB-3-1, was 6.3- and 6.8-fold, respectively; the corresponding figures for C-A500 were 12- and 27-fold, respectively, whereas those for KCP-4 were 2.3- and 20-fold, respectively. These results suggest that MRP and P-gp are involved in the active efflux of SN-38 and CPT-11, respectively, from human KB-derived cells. In addition, a difference in substrate specificity among GS-X pump members was demonstrated.     

A late phase II study of CPT-11 (irinotecan) in advanced breast cancer. CPT-11 Study Group on Breast Cancer

A late phase II study of CPT-11 for advanced breast cancer was conducted at 27 institutions. Seventy-nine patients were enrolled, 75 were eligible for the study, and 65 were evaluable for efficacy. One complete response and 14 partial responses were obtained, and the response rate was 23%. The response rate of patients with prior endocrine therapy and prior chemotherapy including adriamycin or other anthracycline drugs was 27% (11/41) and 26% (12/46), respectively. The response rate for patients with estrogen receptor-negative tumors and premenopausal patients was 32% (6/19) and 27% (4/15), respectively. Responses were observed not only for soft tissue lesions such as lymph nodes (5/17), but also for distant metastases in the lungs (8/28) and bone (1/18). The major adverse reactions were myelosuppression and gastrointestinal symptoms. The incidence of Grade 2 or higher leukopenia, anemia, nausea/vomiting, anorexia, diarrhea and alopecia was 68%, 31%, 67%, 59%, 37%, and 30%, respectively. These results suggested that CPT-11 was a promising drug for advanced breast cancer.     

Simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in rat plasma by high performance liquid chromatography

We established a high performance liquid chromatography (HPLC) method for the simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of the antitumor drug irinotecan (CPT-11), in rat plasma. Plasma samples were pretreated with chilled MeOH and zinc sulfate to precipitate protein, and were then directly injected into the HPLC system. Chromatography was carried out with a Puresil C18 column (particle size 5 microns), and the mobile phase consisted of 0.1 M ammonium acetate buffer (pH 5.5) and acetonitrile (70/30, v/v) containing 20 mM of tetra-n-pentylammonium bromide. The column effluent was monitored with a spectrofluorometer (excitation wavelength 380 nm, emission wavelength 540 nm). The method was valid for SN-38 lactone (5-2500 ng/ml) and carboxylate (5-1000 ng/ml).     

In vitro schedule-dependent interaction between paclitaxel and SN-38 (the active metabolite of irinotecan) in human carcinoma cell lines

METHODS: From 1983 to 1995, 72 patients with necrotizing pancreatitis were treated with a general approach involving planned reoperative necrosectomies and interval abdominal wound closure using a zipper. RESULTS: Hospital mortality was 25%. Multiple organ failure without sepsis caused early mortality in 3 of 4 patients and sepsis caused late mortality in 11 of the remaining 14. The mean number of reoperative necrosectomies/debridements was 2 (0 to 7). Fistulae developed in 25 patients (35%); 64% were treated conservatively. Recurrent intraabdominal abscesses developed in 9 patients (13%) but were drained percutaneously in 5. Hemorrhage required intervention in 13 patients (18%). Prognostic factors included APACHE-II score on admission < 13 (P = 0.005), absence of postoperative hemorrhage (P = 0.01), and peripancreatic tissue necrosis alone (P < 0.05). CONCLUSIONS: The zipper approach effectively maximizes the necrosectomy and decreases the incidence of recurrent intraabdominal infection requiring reoperation. APACHE-II score > or = 13, extensive parenchymal necrosis, and postoperative hemorrhage signify worse outcome.     

Effect of CPT-11 on lipid peroxide level in mouse tissues

PURPOSE: To determine the prevalence of hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV) in the serum of cornea donors who had been previously screened by social and medical history by the Eye Bank of Canada, Ontario Division. METHOD: A retrospective examination of the donor records of the Eye Bank of Canada, Ontario Division, was conducted. A total of 3,228 records were examined covering the period from May 17, 1993 to May 31, 1996. RESULTS: The prevalence of HBV was determined to be 0.25%, HCV, 0.93%; and HIV, 0.031%. CONCLUSION: The data revealed that despite the use of medical and social history to prescreen, a small percentage of prescreened donors test serologically positive. A comparison of the prevalence of HBV, HCV, and HIV in the prescreened cornea donors to a similarly screened cohort of blood donors over a similar time period reveals a higher prevalence in all three diseases in the cohort of cornea donors. An examination of the demographic characteristics of the population as a whole versus the three groups with confirmed serology failed to show a significant difference between the seropositive and seronegative groups. This study confirms the value and necessity of serologic prescreening of cornea donors as is currently the standard of practice.     

Clinical correlates of bombesin-like peptide receptor subtype expression in human lung cancer cells

The importance of the expression of the autocrine growth system for bombesin-like peptides (BLPS) to the biological behavior of human lung cancer has not been determined. Three BLP receptor subtypes have been identified in human lung and lung cancer cells: gastrin-releasing peptide (GRP) receptor, neuromedin B (NMB) receptor, and bombesin receptor subtype 3 (BRS-3). The goals of this study were: (1) to determine BLP receptor subtype expression by human lung cancer cell lines by RT/PCR; (2) to evaluate possible clinical correlates of characteristics of the patients from whom the cell lines were derived with patterns of BLP receptor expression. Degenerate PCR primers were designed to amplify all known BLP receptors and yielded products from 19/20 small cell lung carcinoma (SCLC) and 12/13 non-small cell lung carcinoma (NSCLC) cell lines. GRP receptor was the most commonly expressed BLP receptor subtype, being detected in 17/20 SCLC and 11/13 NSCLC. Eleven of 20 SCLC expressed NMB receptors, and 5/20 expressed BRS-3, compared with 4/13 and 1/13, respectively, in NSCLC cell lines. Evaluation of the clinical data of the patients from whom the cell lines were derived revealed expected age, sex, smoking history and survival based on histology and stage. Patients from whom cell lines expressed GRP receptor experienced a better survival than those whose cell lines did not (367 +/- 274 days vs. 211 +/- 114 days), but the results were not statistically significant. RT/PCR analysis is a feasible, sensitive and specific means of determining BLP receptor expression in lung cancer cells and may yield prognostic information in patient tissue.     

ND-2001 suppresses lung metastasis of human renal cancer cells in athymic mice

Explants of highly metastatic human renal cell carcinoma SN12Cpm6 cells in athymic mice were treated with sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam; ND-2001). ND-2001 (50 micrograms/ml) caused 78% inhibition of lung metastasis of SN12Cpm6 cells (two of five animals remaining metastasis free). The in vitro tumor cell invasion assay showed that ND-2001 (100 micrograms/ml) suppressed the invasive activity of SN12Cpm6 cells to Matrigel matrix at an inhibition rate of 72%. These results suggest that ND-2001 may be a new anti-metastatic drug against human cancer cells.     

Combination phase I/II study of irinotecan hydrochloride (CPT-11) and carboplatin in relapsed or refractory non-Hodgkin's lymphoma. CPT-11/Lymphoma Study Group

Irinotecan hydrochloride (CPT-11) is a new derivative of camptothecin which inhibits topoisomerase I. Phase II studies have demonstrated that CPT-11 is active against a broad spectrum of neoplasms including intractable non-Hodgkin's lymphoma. An early phase II study in lymphoma suggested that a schedule of daily infusions of 40 mg/m2/day for three or five consecutive days is more effective than a single infusion of 200 mg/m2 every three to four weeks. Carboplatin is also an active agent against lymphoma, and preclinical studies have shown that CPT-11 and its active metabolite have a synergistic effect with platinum compounds. To evaluate the maximal tolerated dose (MTD) and the therapeutic efficacy of CPT-11 in combination with carboplatin in relapsed or refractory non-Hodgkin's lymphoma, we conducted a combination phase I/II study. The starting dose of CPT-11 was 20 mg/m2/day (days 1 through 3 and 8 through 10), and dose escalations of 5 mg/m2/day increments were planned, with a fixed dose of carboplatin (300 mg/m2, day 1). Six of the eight patients receiving both agents at the starting dose level developed critical toxicities such as grade 4 hematologic (neutropenia 6/8, thrombocytopenia 1/8) and grade 3 non-hematologic toxicities (diarrhea 2/8, transaminase elevation 1/8). Further dose escalation of CPT-11 was halted, and the starting doses were judged to be the MTDs. The response rate (25%, 2/8) to the combination of the MTDs was not superior to that of CPT-11 alone in a previous phase II study (38%, 26/69), and the MTD of CPT-11 in combination with carboplatin was less than half the single-agent dose. We conclude that carboplatin is not recommendable for combination with CPT-11 in lymphoma patients. Other suitable agents for such a combination should be sought.     

Effect of nomegestrol acetate on estrone-sulfatase and 17beta-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E1S: 5 x 10(-9)M), nomegestrol acetate blocked very significantly the conversion of E1S to E2. In the MCF-7 cells, using concentrations of 5 x 10(-6)M and 5 x 10(-5) M of nomegestrol acetate, the decrease of E1S to E2 was, respectively, -43% and -77%. The values were, respectively, -60% and -71% for the T-47D cells. Using E1S at 2 x 10(-6) M and nomegestrol acetate at 10(-5) M, a direct inhibitory effect on the enzyme of -36% and -18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E1: 5 x 10(-9)M) this estrogen is converted in a great proportion to E2. Nomegestrol acetate inhibits this transformation by -35% and -85% at 5 x 10(-7)M and 5 x 10(-5)M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, -48%, at 5 x 10(-5)M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17beta-HSD activities which converts E1S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E2 can open new clinical possibilities in breast cancer therapy.     

G-CSF increases secretion of urokinase-type plasminogen activator by human lung cancer cells

AIMS: To examine the feasibility of using the human iris in vivo for the assessment of the interaction between tyramine and monoamine oxidase (MAO) inhibitors. To examine the relative roles of the two forms of MAO in terminating the response to sympathomimetic amines in the iris, by comparing the effects of single oral doses of moclobemide, a selective MAO-A inhibitor, and selegiline, a selective MAO-B inhibitor, on mydriatic responses to tyramine. METHODS: Twelve healthy male volunteers participated in three monthly sessions, each associated with ingestion of one capsule (moclobemide 450 mg, selegiline 10 mg, or placebo), according to a double-blind, balanced, cross-over design. Tyramine hydrochloride eye-drops (75 mM, 2 x 10 microl) were instilled three times in the left conjuctival sac at 40 min intervals. Pupil diameter was monitored with a binocular infrared television pupillometer before and for 4.5 h after ingestion of the capsule. The pupillary response to tyramine was expressed as the area under the pupil diameter x time curve (arbitrary units). A blood sample was taken before and 2 h after ingestion of the capsule, for the assay of platelet MAO-B activity, and plasma 3,4-dihydroxyphenylglycol (DHPG) concentration, an index of MAO-A activity. Platelet MAO activity was assayed radiochemically, using [14C]-phenylethylamine as substrate, and plasma DHPG by high performance liquid chromatography (h.p.l.c.). The results were analysed using analysis of variance with repeated measures, followed by Bonferroni's corrected t-test, using a significance criterion of P < 0.05. RESULTS: Both moclobemide and selegiline, compared with placebo, caused significant miosis in the right (untreated) eye. The changes in pupil diameter (mm +/- s.e. mean) from the pretreatment measurement were: placebo -0.09 +/- 0.07, moclobemide -0.52 +/- 0.09, selegiline -0.26 +/- 0.1. The mydriatic response to tyramine was potentiated by moclobemide, compared with the response recorded in the presence of placebo. The responses to tyramine (arbitrary units +/- s.e. mean) were: placebo 77.08 +/- 11.65, moclobemide 140.25 +/- 18.9, selegiline 72.75 +/- 12.35. Both moclobemide and selegiline significantly reduced platelet MAO activity, compared with placebo. The changes in platelet MAO activity (nmol h(-1) mg(-1) protein +/- s.e. mean) from the pretreatment level were: placebo 0.5 +/- 0.62, moclobemide -6.7 +/- 0.66, selegiline -17.7 +/- 0.87. Moclobemide significantly reduced plasma DHPG concentration, compared with placebo. The changes in plasma DHPG concentration (nmol l(-1) +/- s.e. mean) from the pretreatment level were: placebo -0.01 +/- 0.24, moclobemide -4.98 +/- 0.32, selegiline -0.51 +/- 0.26. CONCLUSIONS: The potentiation of tyramine-evoked mydriasis by moclobemide is likely to reflect the inhibition of MAO-A activity in the iris, consistent with the activity of this enzyme in sympathetic nerve terminals. The lack of effect of selegiline on tyramine-evoked mydriasis argues against a role of MAO-B in terminating the effects of sympathomimetic amines in the iris. The effects of the two drugs on platelet MAO activity and plasma DHPG concentration are in agreement with previous reports and consistent with the relative selectivity of moclobemide for MAO-A and of selegiline for MAO-B. The miosis caused by the two MAO inhibitors is likely to be due to a central sympatholytic action of the drugs.     

Selective labelling of bradykinin receptor subtypes in WI38 human lung fibroblasts

1. Binding of the B1 bradykinin receptor radioligand, [3H]-des-Arg10-kallidin (-KD) and the B2 receptor radioligand [3H]-bradykinin (-BK) was investigated in membranes prepared from WI38 human foetal lung fibroblasts. 2. One-site analysis of the saturation data for [3H]-des-Arg10-KD gave an equilibrium dissociation constant (KD) value of 0.51 +/- 0.12 nM and a maximum receptor density (Bmax) of 260 +/- 49 fmol mg-1 of protein. [3H]-des-Arg10-KD binding was displaced by ligands in the order: des-Arg10-KD > KD > > des-Arg9[Leu8]-BK > des-Arg9-BK > Hoe 140 > > BK, implying that it was binding selectively to B1 receptors. 3. One-site analysis of the binding of [3H]-BK to W138 membranes indicated that it had a KD value of 0.25 +/- 0.06 nM and a Bmax of 753 +/- 98 fmol mg-1 of protein. The potencies for displacement of [3H]-BK binding were: Hoe 140 > > BK = KD > > > des-Arg10-KD = des-Arg9[Leu8]-BK = des-Arg9-BK, which was consistent with binding to B2 receptors. 4. This is the first characterization of [3H]-des-Arg10-KD binding to include both kinetic and equilibrium data, and demonstrates that [3H]-des-Arg10-KD has a high affinity for human B1 bradykinin receptors and is sufficiently selective to be used as a radioligand for B1 receptors in human cells or tissues expressing an excess of B2 BK receptors.     

Synergistic cytotoxicity of cisplatin and topotecan or SN-38 in a panel of eight solid-tumor cell lines in vitro

A number of ultraviolet light-induced tumors that do not grow progressively in 3- or 9-month-old animals grow progressively in animals starting at approximately 15 months of age. We investigated the immune response to one such tumor, 6130, in young and old mice to determine in nature of this breakdown in immune protection with age. The 6130 tumor cells that grow progressively in 22-month-old animals still retain sensitivity of cytotoxic T lymphocytes (CTLs) generated against the highly immunogenic "6130-A" antigen and consequently are still immunologically rejected when transplanted into 3- and 9-month-old animals, but not in mice older than 15 months. Investigation of the CD8+ CTL response found that in animals 3-12 months old, primary and secondary tumor-specific CTL responses to 6130 can be generated in vivo and in vitro. On the other hand, the CD8+ CTL responses to 6130 seen in animals 15 months of age or older were significantly depressed or absent. These results indicate that a decline in antigen-specific CD8+ CTL is the major cause of increased susceptibility to 6130 tumor growth in older animals.     

Cellular interactions of 5-fluorouracil and the camptothecin analogue CPT-11 (irinotecan) in a human colorectal carcinoma cell line

CPT-11 (irinotecan) is a DNA topoisomerase I inhibitor active against metastatic colorectal carcinoma. We investigated, in a human colon carcinoma cell line, HT-29, the effects of CPT-11 and 5-fluorouracil (5FU) combinations. A strong synergism between CPT-11 and 5FU was observed after sequential exposure and only additivity or antagonism after simultaneous exposure. When cells were first exposed to 5FU, the product of cellular CPT-11 concentrations versus time (CxT) was 6895 +/- 1020 pmol x hr/10(6) cells, while it was 3875 +/- 121 pmol x hr/10(6) cells with CPT-11 alone (p < 0.01). The same phenomenon was observed with SN-38: 148.2 +/- 49.5 versus 83.4 +/- 23.6 pmol x hr/10(6) cells (p < 0.05). Consequently, the formation of protein-DNA complexes was 1.4 times greater with 5FU pretreatment than with CPT-11 alone (p = 0.03). Moreover, the incorporation of 5FU derivatives into DNA was multiplied by a factor of 1.5 24 hr after CPT-11 exposure. When cells were first incubated with CPT-11, the decrease in thymidylate synthase (TS) activity was identical to that obtained after 5FU exposure (1.09 to 0.023 pmol/min/mg protein), but this decrease persisted for 24 hr (0.014 pmol/min/mg protein) (p = 0.035). At the same time, a 1.8-fold increase in the incorporation of 5FU derivatives into DNA and a 2-fold increase in DNA-protein complex formation were evidenced. With the two sequential associations, we observed a persistent S-phase arrest, as compared with CPT-11 alone. These results suggest that CPT-11 and 5FU combinations are of clinical interest, and mechanisms of interaction between the two drugs seem to be multifactorial.