The CYP26 inhibitor R115866 potentiates the effects of all-trans retinoic acid on cultured human epidermal keratinocytes.

BACKGROUND: All-trans retinoic acid (RA) is known to regulate keratinocyte proliferation and differentiation, and retinoids are used as therapeutic agents in certain dermatological disorders, such as psoriasis and acne. Epidermal expression of the heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced by RA treatment and HB-EGF is responsible for RA-mediated epidermal hyperplasia in vivo. RA also induces HB-EGF expression in cultured keratinocytes and alters their differentiating phenotype. R115866 is a specific inhibitor of the cytochrome P450 isoform CYP26, which is involved in the metabolic inactivation pathway of RA. Thereby, R115866 is thought to be able to increase the intracellular levels of endogenous RA. OBJECTIVES: To determine whether or not R115866 potentiates the effect of low concentrations of RA on keratinocytes. METHODS: We analysed HB-EGF, involucrin and keratin 10 mRNA and protein levels in autocrine human keratinocyte cultures incubated for 18 h with RA or R115866 alone and with RA and R115866 combinations. RESULTS: RA induced HB-EGF and involucrin expression in a concentration-dependent manner, whereas it inhibited keratin 10 expression. R115866 alone had no effect on the expression of these genes. However, when R115866 was combined with low concentrations of RA, HB-EGF and involucrin expression was induced. CONCLUSION: These results strongly suggest that R115866 potentiates the effects of RA on epidermal keratinocytes when RA is present at low concentrations.

A simple reconstructed human epidermis: preparation of the culture model and utilization in in vitro studies.

The preparation of a reconstructed human epidermis is described with examples of its utilization in in vitro studies. The model was obtained by culturing normal human keratinocytes at high cell density for 14 days in serum-free and high calcium (1.5 m M) medium on an inert polycarbonate filter at the air-liquid interface. These stratified cultures showed histological features similar to those observed in vivo in the epidermis: a proliferating basal layer and differentiating spinous, granular, and cornified layers. Electron microscopy illustrated lamellar bodies, junctions and keratohyalin granules. Immunofluorescent localization of epidermal markers (keratins 14 and 10, involucrin and filaggrin) revealed typical differentiation. This in vitro reconstructed tissue was used in studies of toxic effects of chemicals. The modelled tissue showed progressive cytotoxicity of a skin irritant (benzalkonium chloride) and a sensitizer (dinitrochlorobenzene) as assessed by MTT assay. Moreover, differential release of interleukin-1alpha and interleukin-8 were measured after 20 h of incubation allowing the irritant to be distinguished from the sensitizer. Permeation studies indicated efficient barrier function of the reconstructed epidermis, as well as metabolizing properties towards hormones. This model can be custom-made and is potentially useful for studies involving keratinocytes in the epidermis, in basic science, dermatology or toxicology.

Inhibition of in-vitro simvastatin metabolism in rat liver microsomes by bergamottin, a component of grapefruit juice.

Grapefruit juice can modify the pharmacokinetic parameters of many drugs, in particular simvastatin, an orally active cholesterol-lowering agent. The exact components in grapefruit juice responsible for drug interactions are not perfectly known. However, it seems that bergamottin, a furocoumarin derivative, is one of the main active components within grapefruit juice. The objective of this paper was to quantify and to characterize in-vitro the inhibitory effect of bergamottin on simvastatin metabolism by using rat and human liver microsomes. In rat liver microsomes, the incubation conditions (+/-NADPH) of bergamottin were found to influence its inhibiting capacity. In co-incubation with simvastatin, the Ki value (the equilibrium dissociation constant for the enzyme-inhibitor complex) was higher (Ki = 174 +/- 36 microM) than in pre-incubation (Ki = 45 +/- 6 microM and 4 +/- 2 microM, without and with NADPH, respectively). It thus seems that the pre-incubation of bergamottin (in particular with NADPH) increases its inhibiting capacity on simvastatin metabolism. Bergamottin metabolism study in rat liver microsomes showed the formation of two metabolites that were CYP-450 dependent. In contrast, in human liver microsomes, the incubation conditions of bergamottin did not influence its inhibiting capacity of simvastatin metabolism (Ki = 34 +/- 5 microM, Ki = 22 +/- 5 microM, Ki = 27 +/- 11 microM in coincubation and pre-incubation without and with NADPH, respectively). In rat and man, bergamottin was found to be a mixed-type inhibitor of simvastatin hepatic metabolism. However, in rat, bergamottin was partially a mechanism-based inhibitor by involvement of either bergamottin alone or one of its metabolites. The results highlight the importance of validating in-vitro models to help verify the suitability of the in-vitro model for predicting the nature and degree of metabolic drug interactions.

2-substituted (2SR)-2-amino-2-((1SR,2SR)-2-carboxycycloprop-1-yl)glycines as potent and selective antagonists of group II metabotropic glutamate receptors. 2. Effects of aromatic substitution, pharmacological characterization, and bioavailability.

In this paper we describe the synthesis of a series of alpha-substituted analogues of the potent and selective group II metabotropic glutamate receptor (mGluR) agonist (1S,1'S,2'S)-carboxycyclopropylglycine (2, L-CCG 1). Incorporation of a substitutent on the amino acid carbon converted the agonist 2 into an antagonist. All of the compounds were prepared and tested as a series of four isomers, i.e., two racemic diastereomers. On the basis of the improvement in affinity realized for the alpha-phenylethyl analogue 3, in this paper we explored the effects of substitution on the aromatic ring as a strategy to increase the affinity to these compounds for group II mGluRs. Affinity for group II mGluRs was measured using [3H]glutamic acid (Glu) binding in rat forebrain membranes. Antagonist activity was confirmed for these compounds by measuring their ability to antagonize (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-induced inhibition of forskolin stimulated cyclic-AMP in RGT cells transfected with human mGluR2 and mGluR3. Meta substitution on the aromatic ring of 3 with a variety of substituents, both electron donating (e.g., methyl, hydroxy, amino, methoxy, phenyl, phenoxy) and electron withdrawing (e.g., fluorine, chlorine, bromine, carboxy, trifluoromethyl) gave from 1.5- to 4.5-fold increases in affinity. Substitution with p-fluorine, as in 97 (IC50 = 0.022 +/- 0.002), was the exception. Here, a greater increase in affinity was realized than for either the ortho- or meta-substituted analogues; 97 was the most potent compound resulting from monosubstitution of the aromatic. At best, only modest increases in affinity were realized for certain compounds bearing either two chlorines or two fluorines, and two methoxy groups gave no improvement in affinity (all examined in a variety of substitution patterns). Three amino acids, 4, 5, and 104, were resolved into their four constituent isomers, and affinity and functional activity for group II mGluRs was found to reside solely in the S,S,S-isomers of each, consistent with 1. With an IC50 = 2.9 +/- 0.6 nM, the resolved xanthylmethyl compound 168 was the most potent compound from this SAR. Amino acid 168 demonstrated high plasma levels following intraperitoneal (i.p.) administration and readily penetrated into the brain. This compound, however, had only limited (approximately 5%) oral bioavailability. Systemic administration of 168 protected mice from limbic seizures produced by the mGluR agonist 3,5-dihydroxyphenylglycine, with an ED50 = 31 mg/kg (i.p., 60 min preinjection). Thus, 168 represents a valuable tool to study the role of group II mGluRs in disease.

[Aspergillus prostatitis and prolonged corticotherapy. Apropos of a case report]. / Prostatite aspergillaire et corticothérapie prolongée. A propos d'une observation.

Aspergillosis prostatitis is rare but more frequent to immunodeficiency people. We report a case of aspergillosis prostatitis associated with pulmonary tuberculosis, after a corticosteroid treatment for retroperitoneal fibrosis to Methysergide.

Comparative metabolism of SC-42867 and SC-51089, two PGE2 antagonists, in rat and human hepatocyte cultures.

1. The metabolism of SC-42867 and SC-51089, two PGE2 antagonists, was studied in cultured rat and human hepatocytes. Both compounds possess an 8-chlorodibenzoxazepine moiety, but differ from each other by the nature of the side chain connected to the nitrogen atom. SC-42867 and SC-51089 and their in vitro metabolites were separated by reversed-phase hplc. The major metabolites of both compounds were identified by mass spectrometry (ms) analysis. 2. SC-42867 was metabolized on the tricyclic moiety only. Oxidative N-dealkylation with opening of the oxazepine ring was the major metabolic pathway obtained in rat hepatocytes. The metabolic profile obtained in cultured human hepatocytes was comparable with that of cultured rat hepatocytes. However, the compound was metabolized to a much lower extent by the human cells. 3. SC-51089 was extensively metabolized by both cultured rat and human hepatocytes. Human cells metabolized this compound quite differently than cultured rat hepatocytes. Aromatic hydroxylation with consequent glucuronidation and sulphation were the main metabolic pathways observed in cultured human hepatocytes. Oxidative N-dealkylation with opening of the oxazepine ring and consequent glucuronidation was the major metabolic pathway observed in rat hepatocytes. Further metabolism occurred, in contrast with the human hepatocytes, mainly on the side chain. 4. The present in vitro results are compared with data of previous in vivo studies performed in rat.

Irreversible binding to proteins after single and repeated daily oral administration of 4-(2,2-diphenylethyl) imidazole (SC-46264) to the Cynomolgus monkey.

SC-46264 is an antagonist of the alpha 2-adrenergic receptor. Distribution and excretion of [14C]-SC-46264 were studied after single and repeated daily oral administrations to the Cynomolgus monkey at a 1.5 mg/kg dose. After a single oral administration, more than 95% of the administered dose was recovered within 48 h in the urine (+/- 60%) and faeces (+/- 40%). Approximately 1.7% remained in the gastro-intestinal (GI) tract and 2% in the animal body. However, the radioactivity remaining in the animal body decreased very slowly from 2 to 1% between 48 and 144 h. An accumulation of very small amounts of radioactivity could be suspected in the plasma, the liver, the thyroid, the adrenals and the kidneys. In a 2 week daily oral administration of [14C]-SC-46264, the amount of total radioactivity remaining in the animal body 24, 48 and 216 h after the last administration was approximately 21, 11 and 5% of the daily administered dose, respectively. It confirmed the accumulation of [14C]-SC-46264 related compound in the plasma, the liver, the thyroid, the adrenals and the kidneys. The minimum plasma concentrations of total radioactivity observed before each administration increased during the treatment and apparently did not yet reach an equilibrium after 14 days. In these plasma samples obtained throughout the study, an increasing fraction of the total radioactivity could not be extracted and was recovered with precipitable material. These observations lead to the hypothesis of an irreversible binding of some material to the proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-9 expression in human malignant lymphomas: unique association with Hodgkin's disease and large cell anaplastic lymphoma.

To test the possibility that interleukin-9 (IL-9), the human homologue of the mouse T-cell growth factor P40, may be involved in the pathogenesis of human lymphomas, we examined IL-9 expression in a variety of tumors both by Northern blot analysis and by in situ hybridization. Of 18 B-cell non-Hodgkin's lymphomas and 11 peripheral T-cell lymphomas, none expressed IL-9 message. By contrast, IL-9 message was found in two of six cases of large cell anaplastic lymphoma (LCAL) and in 6 of 13 cases of Hodgkin's disease (HD). In HD the strongest signals were observed in Hodgkin (H) and Sternberg-Reed (SR) cells, but IL-9 mRNA was also detected in small lymphocytic cells. A search for IL-9 message in a panel of 20 cell lines derived both from hematopoietic and nonhematopoietic tumors confirmed the unique association of IL-9 expression with HD and LCAL in as much as the only two cell lines with IL-9 message were derived from cases of HD and LCAL. These results suggest that IL-9 is not involved as an autocrine growth factor in the pathogenesis of most B- and T-cell lymphomas, but that it may play a role in HD and LCAL.

Autonomous growth and tumorigenicity induced by P40/interleukin 9 cDNA transfection of a mouse P40-dependent T cell line.

To test the transforming potential of deregulated P40/Interleukin 9 expression, we transfected a mouse P40-dependent T cell line with P40 cDNA, and examined the tumorigenicity of the resulting transfectants. When the cells, which grew autonomously in vitro, were injected intraperitoneally or subcutaneously into syngeneic mice, a very high tumor incidence was observed with as few as 10(4) cells per inoculum. Animals died as a result of widespread dissemination of lymphomatous tissue to abdominal and thoracic organs. The same P40-dependent cell line transfected with a control construct did not form tumors even after injection of 10(7) cells. These results indicate that uncontrolled expression of P40 can support T cell proliferation in vivo, and may be a transforming event involved in the development of certain T cell tumors.

Presence on a human melanoma of multiple antigens recognized by autologous CTL.

We derived from blood lymphocytes of a melanoma patient a large number of cytolytic T-cell clones directed against a cell line of the autologous tumor. Three distinct groups of antigens were recognized by these CTL on the autologous melanoma cells: group A consisted of stable antigens present on all sublines, whereas antigens B and C appeared unstable and were expressed by distinct sublines. In vitro immunoselections with various anti-A CTL clones were applied to the melanoma cells and variants resistant to 3 different CTL clones were obtained. These variants remained sensitive to other anti-A CTL clones, indicating that group A comprises at least 4 different antigens (D, E, F and A'). From a total of 76 CTL clones obtained from lymphocytes collected from the patient at various times, we found that 45 were anti-B, 17 were anti-C, 2 were anti-D, 9 were anti-E, 2 were anti-F and I was anti-A'. It is therefore likely that the 6 antigens identified by these CTL clones represent all or nearly all the transplantation antigens recognized by autologous CTL on this human melanoma.

Antigenic heterogeneity of a human melanoma tumor detected by autologous CTL clones.

Peripheral blood lymphocytes from a melanoma patient were stimulated with autologous melanoma cells in mixed lymphocyte tumor cultures (MLTC). After three restimulations, the lytic activity of the responder cells directed against the autologous melanoma cells was higher than that against K-562 and autologous Epstein-Barr virus-transformed B cell line (EBV-B) cells. From these MLTC-responder cells, we derived specific cytolytic T cell (CTL) clones that lysed the autologous melanoma cells and did not lyse K-562 or autologous EBV-B cells. Autologous melanoma clones were found that were resistant to some or all of these CTL clones. The autologous CTL clones recognized at least two different antigens (A, B) on the melanoma cells and three types of melanoma clones could be distinguished (A+B+, A+B-, A-B-). This antigenic heterogeneity of melanoma clones was confirmed by testing the CTL clones in cold target competition and also in antigen-dependent CTL proliferation assays performed with very small numbers of stimulator cells. The data further indicated an instability of the expression of a melanoma-associated antigen in the course of a long culture period. Among the melanoma clones that expressed antigen A, one was found to stimulate the proliferation of anti-A CTL clones much more effectively than the others. This represents a new type of heterogeneity among tumor cells which may be of significance for the elicitation of an autologous anti-tumoral immune response.

Production of stable cytolytic T-cell clones directed against autologous human melanoma.

We have attempted to optimize the production of stable human cytolytic T lymphocyte clones directed against autologous melanoma cell lines. MLTC were restimulated every week with irradiated melanoma cells in medium containing human serum and IL-2. After 21 to 35 days, in 5 out of 6 patients, these cultures expressed a preferential cytolytic activity against the autologous melanoma cells, as compared to autologous EBV-B cells or NK target K562. Limiting dilution of MLTC responder cells was performed at times varying from days 7 to 28, in medium containing IL-2 and allogeneic EBV-B cells as feeders. Approximately 1% of these responder cells gave rise to CTL clones that lysed the autologous melanoma cells, but did not lyse K562 or autologous B cells. It was possible to maintain in culture for several months a large number of CTL clones that retained this specificity with high activity, and multiplied more than 5-fold every week. Some of these CTL clones were dependent on the presence of the autologous melanoma cells for their growth. With one melanoma, the use of autologous CTL clones made it possible to identify 3 different antigens on the tumor cells.

[Sacral chordoma, a rare tumor]. / Le chordome sacré, tumeur rare.

One case of chordoma is described. A review of literature shows that this condition is rare. This malignant tumor grows slowly. The symptoms owing to the surrounding structures compression appear insidiously. Standard radiography, tomography, echography and computed tomography are an aid to diagnosis confirmed by biopsy. Surgical removal of the tumor is the primary modality of treatment. Surgical resection is carried out using a combined abdominal and trans-sacral approach. Complete tumoral resection is limited by preservation of sacral stability and urinary and fecal continence. Many patients are referred for radiation therapy after subtotal resection. Local recurrence is frequent. Metastasis is rare. 90 per cent of patients are dead in ten years.