Clinical phase I and pharmacokinetic study of S 16020, a new olivacine derivative: report on three infusion schedules.

S 16020, a new 9-OH olivacine derivative, is a novel topoisomerase II inhibitor with activity in cell lines presenting the classical multidrug resistance phenotype. This report summarizes, in addition to pharmacokinetic data, the whole phase I clinical experience of S 16020 using three different infusion schedules. Asthenia and skin toxicity were the main side effects. In an attempt to understand the skin toxicity mechanism, experiments in animals were performed, the results of which are reported. S 16020 showed rapid tumor necrotizing activity in some patients, with soft tissue metastases of epidermoïd tumors and pain at the tumor site. To document the side effects of S 16020 and tumor site reactions (pain, edema, inflammatory signs), inflammatory parameters and some cytokines were measured. In our patients there was no hemolysis and no detection of anti-S 16020 antibodies, confirming the absence of immunogenicity of the compound. Based on the overall data of the three infusion schedules of S 16020, the dose of 100 mg/m(2) over 3 h every 3 weeks was selected for phase II studies.

The olivacine S16020 enhances the antitumor effect of ionizing radiation without increasing radio-induced mucositis.

The combination of a novel topoisomerase II inhibitor, S16020, and ionizing radiation (IR) was investigated with the aim of assessing normal tissue tolerance using a mouse mucosal lip model and antitumor activity in a human carcinoma (HEP2) cell line. No increase of acute mucosal reactions was seen when combining S16020 with IR as compared with IR alone. Using clonogenic cell survival assay, a marked enhancement of HEP2 cell killing was found when S16020 was combined with irradiation. Additional in vivo combination of S16020-IR was able to increase markedly the antitumor efficacy as compared with S16020 or irradiation alone. Interestingly, the radiosensitization effect in vivo was observed at relatively low and nontoxic concentrations of S16020, and no dose-effect relationship was found beyond 30 mg/kg. In conclusion, the combination of IR and S16020 seems promising to enhance antitumor activity without increasing deleterious effect in normal tissues and to provide the basis for a new radio-chemotherapy combination.

Modulation of daunorubicin cellular resistance by combination of P-glycoprotein blockers acting on drug efflux and intracellular drug sequestration in Golgi vesicles.

BACKGROUND: S9788 and PSC833 were developped as P-glycoprotein (Pgp) blockers and found to act additionally on daunorubicin subcellular distribution, involving different putative targets. On this basis, combinations of S9788 and PSC833 were evaluated in Pgp-expressing MCF7(DXR) cells in which we recently demonstrated that daunorubicin was sequestered in Golgi vesicles (Bour-Dill et al.: Cytometry, 39: 16-25, 2000). METHODS: Combinations of S9788 and PSC833 consisted in complementary fractions of iso-effective concentrations (IEC) leading to 90% (IEC90) and median (IEC50) reversion of daunorubicin resistance. Resistance modulation was assessed using cytotoxicity assays, flow cytometry determination of intracellular daunorubicin, and fluorescence microscopy analysis of daunorubicin subcellular distribution. RESULTS: Individually, both S9788 and PSC833 were found to be very potent with IEC90 of 5 and 15 micromol/l, and IEC50 of 0.1 and 0.2 micromol/l, respectively, for S9788 and PSC833. When combined, synergistic cytotoxicity was observed for both IEC90 and IEC50 combinations while intracellular daunorubicin fluorescence was only synergistically increased for IEC90 combinations. For IEC50 combinations, no increase in intracellular fluorescence was observed, and fluorescence microscopy examination of the cells suggested that daunorubicin sequestration in Golgi vesicles could be modulated at concentrations that do not significantly increase daunorubicin cellular concentration. Using immunofluorescence and reverse transcription-polymerase chain reaction analyses, multidrug resistance-associated protein, major vault lung-resistance protein, and anthracycline-resistance associated protein were not found to be implicated. CONCLUSIONS: Synergistic combinations of S9788 and PSC833 might offer alternative ways to decrease the toxicity generated by high-dose Pgp-blockers without altering the efficacy of the resistance modulation.

Time-schedule dependency of S 16020, a new topoisomerase II inhibitor.

S 16020 is a new olivacine derivative which has been shown to intercalate into DNA and to stabilize the cleavable complex formed by DNA and purified topoisomerase II. The aim of the present study was to estimate the impact of time exposure on the in vitro activity of S 16020. This was done on seven cancer cell lines of human origin (head and neck, kidney, and ovary). Doxorubicin was used as a reference drug. The cytotoxic activity of S 16020 remained stable during at least 3 h. A loss of activity of about 30% was apparent after 6 or 24 h preincubation. This relative loss of activity reached about 50% after 72 h preincubation. Considering all tested cell lines, the average IC50 decrease was 89+/-8% for S 16020 with incubation times between 1 and 72 h. An exposure index (El) was calculated to evaluate the effect of time on the cytotoxic efficacy. The reference time was 1 h exposure. The El values were corrected to take into account the loss of drug activity. For the majority of cell lines EI values were greater than 1 for both drugs, particularly after a 6 h exposure time. This means that, in this case as compared to the shorter exposure (1 h), increasing time has a relative detrimental effect on drug efficacy. For the two cancer cell lines of ovarian origin, El values remained close to 1 for both drugs whatever the total exposure time. This means that, in this case, time and concentration have symmetrical effects on cell survival. The pharmacological information provided by the present study may be useful in designing future clinical trials on this potentially interesting new topoisomerase II inhibitor. As a consequence of these data, 1 and 3 h drug administration schedules are currently tested during phase I trials.

S9788 modulation of P-glycoprotein- and Multidrug-related protein-mediated multidrug resistance by Servier 9788 in doxorubicin-resistant MCF7 cells.

Inherent or acquired resistance to multiple natural drugs, termed multidrug resistance (MDR), represents a major obstacle to chemotherapy. Expression of P-glycoprotein (P-gp) in MCF7mdr and MCF7R resistant cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. MCF7R, but not the MDR1 gene-transfected MCF7mdr cells, expressed multidrug-related protein (MRP) concomitantly. Efficacy of an MDR modulator, designated as Servier 9788 (S9788), was estimated by doxorubicin (Dox) sensitization, Dox incorporation, and functional rhodamine 123 assay on MCF7 cell lines. Results showed that S9788 modulates the P-gp-associated MDR of MCF7mdr cells as well as the Multidrug-related protein-associated MDR of MCF7R cells.

Inhibition of P-glycoprotein: rapid assessment of its implication in blood-brain barrier integrity and drug transport to the brain by an in vitro model of the blood-brain barrier.

PURPOSE: The objective of this work was to assess, in vitro, the passage of P-glycoprotein dependent drugs across brain capillary endothelial cells, when these drugs are associated with a reversing agent. METHODS: An in vitro model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes was used. RESULTS: We demonstrate that P-glycoprotein expression is upregulated by the presence of astrocytes. Uptake in the cells and transport across endothelial cell monolayers of vincristine, cyclosporin A and doxorubicin were studied. Using S9788 or verapamil as reversing agents, we found an increase in vincristine transport across the endothelial cell monolayers. On the other hand, the association of S9788 or verapamil with cyclosporin A failed to increase the transport of this drug. An increase in the transport of doxorubicin from luminal to abluminal compartment was also observed, due to endothelial cell monolayer breakdown. CONCLUSIONS: Using this model, it is possible to predict the passage of a P-glycoprotein dependent drug to the brain or its sequestration in brain capillary endothelial cells when this drug is associated with a reversing agent, or its toxicity on the blood-brain barrier integrity.

Activity of fotemustine in medulloblastoma and malignant glioma xenografts in relation to O6-alkylguanine-DNA alkyltransferase and alkylpurine-DNA N-glycosylase activity.

Fotemustine is a chloroethylnitrosourea with antitumor activity in disseminated melanoma and adult primary brain tumors. Because new drugs are required for the treatment of medulloblastoma in children, we evaluated the preclinical antitumor activity of fotemustine in four s.c. medulloblastoma xenografts, in comparison with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Both drugs were administered as a single i.p. injection to nude mice bearing advanced-stage tumor. Fotemustine displayed significant antitumor activity in three of four medulloblastoma xenografts; two, IGRM34 and IGRM57, were highly sensitive, with 37 and 100% tumor-free survivors, respectively, more than 120 days after treatment at the highest nontoxic dose (50 mg/kg). Fotemustine was also highly active in a malignant glioma xenograft (IGRG88; five of six tumor-free survivors on day 177). Fotemustine proved to be significantly more active than BCNU in IGRM34 and the glioma xenograft IGRG88. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) was detected in all tumor xenografts, ranging in activity from 6 to 892 fmol/mg protein. The high in vivo sensitivity to fotemustine and BCNU observed in three xenografts was clearly associated with a low ATase activity (> 20 fmol/mg), whereas the two poorly sensitive or refractory medulloblastoma xenografts showed high ATase activity (> 500 fmol/mg). Alkylpurine-DNA N-glycosylase activity was detected in all tumor xenografts but at levels ranging only from 513 to 1105 fmol/mg/h; no consistent relationship was found between alkylpurine-DNA N-glycosylase activity and the in vivo sensitivity to the two chloroethylnitrosoureas. The improved activity and tolerance of fotemustine in comparison with BCNU in pediatric medulloblastoma xenografts strongly support the clinical development of this agent in children with brain tumors, in which ATase should be examined as a potential prognostic indicator.

Influence of S9788, a new modulator of multidrug resistance, on the cellular accumulation and subcellular distribution of daunorubicin in P-glycoprotein-expressing MCF7 human breast adenocarcinoma cells.

A triazinoaminopiperidine derivative synthesized as a modulator of multidrug resistance, S9788, was investigated in the human breast adenocarcinoma MCF7DXR cell line expressing P-glycoprotein. In addition to being less sensitive to daunorubicin, the resistant cell line showed dramatic alterations in the subcellular distribution of daunorubicin, as observed via fluorescence microscopy and quantified via tritiated daunorubicin nuclear distribution analysis. Compared to verapamil and cyclosporin A at 2 and 5 mumol/liter, S9788 proved to be more potent in restoring the cellular accumulation and the subcellular distribution of daunorubicin in the resistant cells. Significant activity of S9788 was observed at 2 mumol/liter, which is clinically achievable, and S9788 restored the nuclear distribution of the drug to the level observed in the parental sensitive cell line. Consequently, the restoration of the cytotoxicity of daunorubicin by S9788 was nearly complete (> 90%) at 2 mumol/liter, wheras cyclosporin A reached this level of activity at 5 mumol/liter, and verapamil was always less active at both concentrations. These results suggest that the modulation of multidrug resistance by S9788 is not only related to the enhancement of the cellular accumulation but also especially by the restoration of the subcellular distribution of the drugs to their nuclear sites of action.

Effect of S9788, cyclosporin A and verapamil on intracellular distribution of THP-doxorubicin in multidrug-resistant K562 tumor cells, as studied by laser confocal microspectrofluorometry.

S9788 modulating resistance effect has been investigated on the activity of THP-DOX against multidrug-resistant K562R cells and compared to that of cyclosporin A and verapamil. Intracellular THP-DOX distribution and particulary its intranuclear concentration, with or without modulators, has been measured using confocal laser microspectrofluorometry. The kinetics of intranuclear accumulation of THP-DOX (1 microM in the medium), as a function of time, were rapid in K562S and K562R cells. Maximum accumulation of THP-DOX is reached in a few minutes (K562S, 400 microM; K562R, 40 microM). The addition of S9788, cyclosporin A and verapamil (5 microM) after one hour THP-DOX incubation, led to respectively 290, 250 and 114 microM. Uptake of THP-DOX was increased in K562R cells by a factor of 7 when S9788 was added. Results obtained on THP-DOX efflux from nuclei of K562S and K562R cells, after 3 hours of incubation without drug, showed a very short T1/2 (time corresponding to 50% decrease of intranuclear concentration of THP-DOX) in K562R cells (12 min) compared to that in K562S cells (150 min). The addition of S9788, cyclosporin A and verapamil (5 microM) led to a T1/2 of 90, 30 and 20 min respectively. The T1/2 of THP-DOX was increased in K562R cells by a factor of 7.5 when S9788 was added. We tried to correlate these results with those obtained in growth inhibition study. The IC50 (concentration which induces 50% growth inhibition) of THP-DOX, corresponding to one hour THP-DOX treatment and 3 days culture of K562S and K562R cells were respectively 230 and 7000 nM, and the RI (resistance index) value is 30. The addition of S9788, cyclosporin A and verapamil (5 microM), during the one hour treatment, led to an IC50 value of 350, 2000 and 5000 nM respectively. S9788 induced an IC50 value 20 times lower than without the modulator. Our study suggests that the higher activity of THP-DOX against K562R cells in the presence of S9788, compared to cyclosporin A and verapamil, is due to a higher intranuclear THP-DOX accumulation and to a strong decrease of drug efflux from K562R nuclei. This could be explained by a higher affinity of S9788 for membrane P-glycoprotein of K562R cells and/or an additional mechanism of action.

Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation.

Cryopreserved hepatocytes from various animal species and human beings were tested for their ability to survive and function in primary culture. The freeze/thaw protocol primarily designed for rat hepatocytes was used with slight modifications for the cells of all other species; it consisted of suspending parenchymal cells in the Leibovitz L15 medium containing 10% fetal calf serum and 10% to 16% dimethyl sulfoxide. After transient storage at 4 degrees C cell suspensions were transferred to -20 degrees C and then to -70 degrees C before being plunged in liquid nitrogen. Hepatocytes were stored for a few weeks to 4 yr. Prolonged storage did not augment loss of cell viability and function. Cell viability after thawing was estimated by the trypan blue exclusion test, and attachment efficiency to plastic was estimated by measuring intracellular lactate dehydrogenase content. Similar values were obtained for most species tested; after cryopreservation cell viability and attachment were decreased by 10% to 25% and by 40% to 50%, respectively. A lower attachment rate was found with dog hepatocytes. Total cytochrome P-450 and protein synthesis were compared in fresh and cryopreserved cells from four species after 4, 24, 48 or 72 hr of culture. Similar values were found in both cells after 24 or 48 hr of culture. In addition, drug-metabolizing activities were measured in human hepatocytes from five donors. In most cases phenacetin deethylation activity was decreased whereas procainamide N-acetylation and paracetamol sulfoconjugation and glucuronidation were increased in cryopreserved cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Viability and primary culture of rat hepatocytes after hypothermic preservation: the superiority of the Leibovitz medium over the University of Wisconsin solution for cold storage.

Hepatocytes isolated from adult rat livers were hypothermically preserved for 24 or 48 hr before being plated under conventional culture conditions. They were stored either in the Leibovitz medium, a cell culture medium with and without polyethylene glycol (PEG), a compound known to suppress ischemia-induced cell swelling, or in the University of Wisconsin solution, the most effective solution for cold organ preservation. After 24 or 48 hr of storage at 4.5 degrees C in Leibovitz medium, cell viability and adherence efficiency to plastic dish, were only slightly reduced, whereas University of Wisconsin hepatocytes had a decreased viability and (especially after 48-hr storage) lost their adhesion ability; they did not survive in vitro. The metabolic competence of hepatocytes maintained in Leibovitz medium was retained over the 3 days of culture, as shown by low extracellular levels of the membrane-bound and cytosolic hepatic enzymes, as well as by intracellular glutathione content, albumin secretion rate and several phase I and phase II drug metabolic reactions very close to those found with fresh hepatocytes maintained under similar culture conditions. Addition of polyethylene glycol to the Leibovitz medium resulted in slightly higher viability and function of hepatocytes after cold storage. These results clearly demonstrate that viability of a transplanted liver does not correlate with long-term in vitro viability of isolated hepatocytes after hypothermic preservation in University of Wisconsin solution. They also suggest that nutritional and energy substrates as found in the Leibovitz medium are probably required to define a suitable solution for cold preservation of isolated parenchymal cells. The findings with Leibovitz medium favor the conclusion that hypothermically preserved hepatocytes could be used for various metabolic studies and for the treatment of liver insufficiency.

Effects of dietary whey proteins, their peptides or amino-acids on the ileal mucosa of normally fed and starved rats.

The effects of three liquid diets, differing only in the molecular form of the nitrogen source (whole whey proteins, WP; trypsic whey protein hydrolysate, WPH, and amino-acid mixture, AAM) were studied on the mucosa morphology and brush border hydrolase (BBH) activities (disaccharidases, peptidases) of the ileum of normally fed male Wistar rats (controls) and during refeeding of rats starved for 72h. All three diets produced repair of the fasting induced mucosal atrophy; the AAM diet gave the most rapid response and highest villus height (p < 0.01). This was correlated with an increase in crypt mitoses (p < 0.01). Similar results were obtained in controls with AAM. The sucrase (S) and acid amino peptidase (AAP) specific activities of controls were higher (p < 0.01) on the WPH diet; neutral amino peptidase (NAP) was unaffected. Dipeptidyl peptidase IV (DDP) was lowest on AAM while glucoamylase (G) highest on WP. Fasting increased S and DDP activity, and produced no change in the other BBH. Large variations in BBH occurred during refeeding except for NAP which remained stable. Control values were restored at 96h, except for AAP. The results show that BBH and mucosa morphology of the ileum in the rat can be modified by the molecular form of the nitrogen source and that the nutritional status interferes with this adaptation. These data could have implications for the therapy of small bowel disease.

Detection of growth-stimulating activity in the proximal small intestine during weaning in the suckling rat.

Adaptive changes in enzyme expression and cell proliferation occur in the small intestine of the suckling rat at the beginning of the 3rd postnatal week. This physiological adaptation can be modulated by factors including diet or glucocorticoids. We have previously described an intestinal growth-stimulating fraction derived from the remnant small bowel after resection and found that enteral nutrition is critical for its detection. In view of the similarity between the changes in cell proliferation that occur between 15 and 22 days postnatally and those immediately after resection, we sought to determine whether the small intestinal mucosa of the neonatal rat also contains a similar growth-stimulating fraction. Our results show that extracts of the proximal intestine prepared from 15-day-old rats do contain the growth-stimulating fraction. The activity was not detectable in maternal milk nor in the intestinal extract from 8-day-old rats. When the suckling rats were deprived of solid food, the activity was not detectable in the 15-day-old group. Gel filtration of the acidic extract on a G-25 Sephadex column revealed that the active component is made up of two molecular weight species (approximately 4,500 and 1,500 daltons) similar to that described in the proximal intestine of the postresectional model. These findings suggest that dietary factors may play a role in modulating the proliferative changes that occur at the time of weaning by way of the growth-stimulating fraction.

Serum lipids and apolipoproteins in the rat refed after starving: influence of the molecular form of nitrogen (protein, peptides, or free amino acids).

Efficient treatment of deep denutrition should promote the restoration of normal intestinal villous structure and the return to a positive nitrogen balance. To determine whether the plasma measurement of lipoproteins could serve as sensitive indexes of villous architecture and/or nitrogen balance, these parameters were compared in rats starved for three days and refed three types of diets containing either whey proteins (WP), whey protein hydrolysate (WPH), or amino acids, known to differ in their capacity to promote restoration of normal villous architecture. Starvation lowered the concentration of triglycerides and phospholipids but not cholesterol. Apolipoprotein AI and AIV concentrations were also significantly lowered (30% and 40%, respectively), but ApoE was significantly increased by 40%. Upon refeeding with all three diets, plasma lipids progressively returned to control values except for triglycerides, which were significantly elevated by the protein and peptide diets. Apoprotein AI continued to decrease for 24 hours on the peptide and amino acid diets. Control levels were restored in all groups after 48 hours. ApoAIV increased progressively in parallel with the restoration of the intestinal mucosa; after 48 hours of refeeding, plasma concentrations of apo AIV were significantly correlated with jejunal villous height and protein content (P less than .01). ApoE was depressed below control levels in the WP and WPH groups at 24 and 48 hours and restored only after 96 hours. Because ApoE was affected, both in the fed state and during refeeding by the form of dietary nitrogen, it may prove to reflect nitrogen balance and/or insulin: glucagon balance.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of whey proteins, their oligopeptide hydrolysates and free amino acid mixtures on growth and nitrogen retention in fed and starved rats.

The effects of alimentary whey proteins given, as whole proteins (WP), controlled trypsin and chymotrypsin hydrolysate oligopeptides (WPH), or a free amino acid mixture (AAM), on growth, nitrogen retention, and steatorrhea were assessed in 24 Wistar rats (250 to 300 g) after 72 hr of starvation and 24 to 96 hr of realimentation and in 24 controls. The three diets had the same caloric, nitrogen, vitamin, and mineral contents. Rats had free access to the liquid diets. Only rats which ate the whole diet (90 cal) were included in the study. No differences in steatorrhea and fecal nitrogen were observed. The absorption rate was over 95% on the three diets. In contrast, weight gain was statistically better on WPH (+9% after 96 hr of realimentation) than on WP (+5%) or AAM (+2%). This was associated with a statistically higher nitrogen retention at all time periods studied, which was a result of a significant lower nitrogen urinary excretion. Similar results were obtained in controls. This better growth was a result of a better protein synthesis and lower ureagenesis.

Dietary whey proteins and their peptides or amino acids: effects on the jejunal mucosa of starved rats.

The effects of starvation (72 h) and refeeding with three liquid diets, differing only in the molecular form of the nitrogen source (whole whey proteins, WP; tryptic whey protein hydrolysate, WPH; and amino acid mixture, AAM), on the jejunal mucosal morphology and brush border enzyme activities (sucrase, S; maltase, M; and neutral aminopeptidase, NA) of male Wistar rats were studied. All three diets produced repair of the fasting-induced mucosal atrophy; the WP diet gave the most rapid growth with maximum villus height (VH) and protein content after 48 h (p less than 0.01). AAM gave the fastest and greatest stimulation of sucrase and maltase activities (p less than 0.01). There were no significant differences in NA activity. In control rats the WPH and AAM diets produced significantly greater villus height and disaccharidase activities than did the WP diet. Jejunal morphology and disaccharidase activities can be modified by the molecular form of alimentary protein and nutritional status interferes with these modifications.

[Effect of fasting and refeeding on the adaptation of the small intestine in rats. A model for physiopathologic studies]. / Effet du jeûne et de la réalimentation sur l'adaptation de l'intestin grêle chez le rat. Un modèle d'études physio-pathologiques.

A chronological study was carried out on 50 male Wistar rats (350 g) to determine the effects of 3 days of fasting and 16 h to 9 days of refeeding on the morphology of jejunal and ileal mucosa (villus, crypt and enterocyte heights; number of mitosis), on some aspects of their functional adaptation (sucrase, maltase, protein) and on nitrogen and lipid absorptions. Three days of fasting resulted in weight loss (12 p. 100), in a jejunal mucosa atrophy (villus height: 376 +/- 18 vs. 492 +/- 4 microns in controls; enterocyte height: 31 +/- 2 vs. 41 +/- 0.3 micron in controls) and a decrease in disaccharidases activities (sucrase: 927 +/- 90 vs. 3,363 +/- 21 mU/10 cm length in controls). No change in ileal mucosa morphometry was noticed. Ad libitum refeeding caused a rapid and progressive weight gain, a jejunal morphometric regrowth identical to control values at 16 h (villus height: 521 +/- 20, enterocyte height 42 +/- 0.9 microns), and maximum at 40 h of refeeding (villus height: 601 +/- 5 microns). Disaccharidases adaptation was delayed, with a maximum at 64 h of refeeding (sucrase: 3,524 +/- 56 mU/10 cm length). Despite a 30 p. 100 increase of food consumption over the whole study (45 p. 100 during the first 16 h of refeeding), nitrogen and lipid absorption coefficients remained identical to those found in controls with an increased nitrogen balance of 70 p. 100 at 16 h and 54 p. 100 at 40 h refeeding, as compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)