[A rare cause of cryptorchidism, the persistence of müllerian ducts syndrome]. / Une cause rare de cryptorchidie le syndrome de persistance des structures müllériennes.

The Persistent Müllerian Ducts Syndrome (PMDS) is a rare congenital syndrome. It is one of abnormalities of genito-sexual development that is found on the normally virilized boy (46XY). It is characterized by the development of both Wolf structures and Müller duct. The pathophysiology can be explained by an action deficit of the anti-müllerian hormone (AMH). Its clinical presentations vary depending on the localization of the testis and the associated symptoms. Its discovery is mostly fortuitous and generally made in per-operative surgery of cryptorchidism or inguinal hernia. Treatment should be surgical. It relies on two aspects : ensuring the testicular descent and performing the excision of the müllerian duct. The follow-up is identical to the cryptorchid testes and the fertility problems will be influenced by the surgical procedure as well as the timing of the treatment.

Le syndrome de persistance des canaux mullériens (PMDS) est un syndrome congénital rare donnant des anomalies du développement génito-sexuel chez le garçon normalement virilisé (46XY). Il se caractérise par le développement à la fois des structures de Wolf et des canaux de Müller. Sa physiopathologie s'explique par un défaut d'action de l'hormone anti-müllérienne (AMH). Il existe différentes présentations cliniques qui varient en fonction de la localisation du testicule et des symptômes associés. Sa découverte est fortuite et généralement faite en per-opératoire d'une chirurgie de cryptorchidie ou d'hernie inguinale. Le traitement doit être chirurgical. Il repose sur deux aspects : assurer la descente testiculaire et réaliser l'exérèse des canaux müllériens. Le suivi est identique à celui d'un testicule cryptorchide et le risque de trouble de la fertilité varie en fonction de l'âge de prise en charge et du geste chirurgical.

[How I TREAT... A RENAL COLIC]. / COMMENT JE TRAITE ... une colique néphrétique.

Renal colic (RC) represents nearly 2% of emergency department admissions. RC is defined by the occurrence of back pain which may radiate towards the abdomen and external genitals. In adults, the obstruction is caused by a urinary stone in 80% of cases. The 20 % of non-stone related RCs are due either to an intrinsic obstruction (pyeloureteral junction stenosis, ureteral tumor, ...) or an extrinsic compression (pelvic tumor, lymphadenopathy ...). In over 90% of cases, an RC does not require hospitalization and is treated with medication. In contrast, complicated renal colic (CRC) requires hospitalization with specialized care. Obstructive pyelonephritis (OPN) is a form of CRC and the diagnosis should be considered in a clinical presentation of "renal colic" with acute pyelonephritis. This is a true emergency requiring surgical drainage of the upper urinary tract upstream of the obstacle, as well as antibiotic therapy. It must be kept in mind that some clinical presentations may be atypical, especially in the elderly, which can delay the diagnosis and, thus, the management. The gold standard for diagnosis is CT urography.

[Highflow priapism : diagnostic evaluation, contribution of ultrasound and recommendations]. / Priapisme à haut débit d'origine traumatique : mise au point diagnostique, apport de l'échographie et recommandations.

The high flow priapism (HFP) is a very rare pathology. It must be distinguished from the low flow which is a real urologic emergency. The diagnosis of HFP (most often post-trauma) remains clinical, but penile color Doppler ultrasound can confirm, identify and track the evolution of the lesion. Conservative treatment is effective and remains the first line treatment. However the different therapeutic modalities (selective embolisation, surgery) should be explained to the patient and be considered case by case.

Le priapisme à haut débit (PHD) est une pathologie très rare et il faut le différencier de celui à bas débit qui est une véritable urgence urologique. Le diagnostic de PHD, souvent d'origine post-traumatique, reste clinique, mais l'échographie Doppler couleur pénienne permet de le confirmer, d'identifier et de suivre l'évolution de la lésion. Le traitement conservateur est efficace et reste celui de première intention. Cependant les différentes modalités thérapeutiques (embolisation sélective, chirurgie) doivent être expliquées au patient et être envisagées au cas par cas.

Homocamptothecin, an E-ring modified camptothecin with enhanced lactone stability, retains topoisomerase I-targeted activity and antitumor properties.

Homocamptothecin (hCPT) is a semisynthetic analogue of camptothecin (CPT) with a seven-membered beta-hydroxylactone resulting from the insertion of a methylene spacer between the alcohol moiety and the carboxyl function of the naturally occurring six-membered alpha-hydroxylactone of CPT. This E-ring modification provides a less reactive lactone with enhanced stability and decreased protein binding in human plasma. Biological testing against CPT revealed that, instead of being detrimental, the modified lactone of hCPT has a positive impact on topoisomerase I (Topo I) poisoning properties. In vitro tests showed hCPT to fully conserve the ability to stabilize Topo I-DNA cleavage complexes and to stimulate a higher level of DNA cleavage than CPT. A similar trend toward improvement was also observed in antiproliferative assays with human tumor cell lines (including cells overexpressing P-glycoprotein). In two distinct in vivo models, using L1210 murine leukemia or human colon carcinoma HT29, hCPT was found to be more efficacious than CPT. The slow, but irreversible, hydrolysis of hCPT, instead of the fast equilibrium of CPT, may account for its good in vivo activity. Overall, these results provide evidence that a highly reactive lactone is not a requisite for the Topo I-mediated antitumor activity of CPT analogues, and that hCPT is an interesting pharmacological tool with improved solution behavior as well as a promising new template for the preparation of more efficacious Topo I poisons.

Unusual potency of BN 80915, a novel fluorinated E-ring modified camptothecin, toward human colon carcinoma cells.

BN 80915 is the lead compound from a novel class of E-ring modified camptothecin analogues, the homocamptothecins, which show potent antitumor activities in animal models. Here, we report that BN 80915 induces up to 2-fold more cleavable complexes between plasmid DNA and purified human topoisomerase I than SN-38 and camptothecin. BN 80915 also induces DNA-topoisomerase I complexes in living HT-29 colon carcinoma cells, as shown by the in vivo link assay. BN 80915 is an extremely potent inducer of DNA-protein complexes in these cells starting at a concentration of 5 nM in the media. BN 80915 is clearly more potent than SN-38, because at least 20 times more SN-38 is needed to induce comparable levels of cleavable complexes. Kinetic experiments show that BN 80915 induces cleavable complexes within minutes that remain stable for at least 6 h in the presence of drug. Whereas the majority of the complexes are reversed within 15 min after drug removal, a substantial fraction (30%) persists for at least 4 h, in contrast with SN-38-treated cells, where all complexes have disappeared by this time. BN 80915 shows strong antiproliferative effects toward HT-29 cells with an IC50 of 0.3 nM compared with 20 nM for SN-38 and 40 nM for topotecan. BN 80915 is also potent against other colon carcinoma cells as well as toward cells growing in three dimensions as multicellular spheroids. HL-60 cells expressing functional P-glycoprotein or multidrug resistance protein show no cross-resistance toward BN 80915. Taken together, our results show that BN 80915 is unusually potent toward human colon carcinoma cells because of the formation of high levels of stable, covalent DNA-topoisomerase complexes.

Homocamptothecin, an E-ring-modified camptothecin, exerts more potent antiproliferative activity than other topoisomerase I inhibitors in human colon cancers obtained from surgery and maintained in vitro under histotypical culture conditions.

Topoisomerase I (Topo I) is overexpressed in cancer colon tissues compared with normal colon tissues. Several anti-Topo I inhibitors are already successfully used in the clinic. We illustrate here the antiproliferative activity of a new class of Topo I inhibitors, i.e., E-ring-modified camptothecins with enhanced lactone stability (L. Lesueur-Ginot et al., Cancer Res., 59: 2939-2943, 1999). Forty-three human colon cancers were obtained from surgical resection and maintained under organotypical culture conditions for 48 h. Cell proliferation was assessed in these ex vivo tumor tissue cultures by tritiated thymidine autoradiography. As a validation of the methodology, we first analyzed in our model the antiproliferative activity of two clinically active topoisomerase II (Topo II) inhibitors, Adriamycin and etoposide, which are not active for colon cancers; and three Topo I inhibitors, camptothecin (CPT) and two clinically active compounds (especially for colon cancers), i.e., topotecan and the active metabolite of irinothecan, SN-38. We then compared the antiproliferative activity of CPT, topotecan, and SN-38 against those of two investigational E-ring-modified camptothecins, i.e., BN80245 and BN80915. Three concentrations (1, 10, and 100 nM) were studied for each compound. The results indicate that the three Topo I inhibitors used as references, i.e., CPT, irinothecan, and SN-38, were much more active than the two Topo II inhibitors, i.e., Adriamycin and etoposide, with SN-38 being the most efficient. The two investigational compounds BN80245 and BN80915 exerted higher antiproliferative activity than the three anti-Topo I reference compounds, with the highest activity observed for BN80915.

Topoisomerase I-mediated antiproliferative activity of enantiomerically pure fluorinated homocamptothecins.

Homocamptothecin (hCPT) is an E-ring modified camptothecin (CPT) analogue bearing a methylene spacer between the alcohol and carboxyl functions of the CPT lactone. Combining pronounced inhibitory activity of topoisomerase I (Topo I) with enhanced plasma stability, hCPT constitutes an attractive template for the elaboration of new anticancer agents. Fluorinated hCPT analogues, prepared in enantiomerically pure form, were assayed by their stimulation of Topo I-mediated DNA cleavage. Translation into cytotoxicity against tumor cells was evaluated on HT29 human colon adenocarcinoma and on the multidrug resistant lung and bladder tumor cell lines, A549 and T24r. Good correlation is observed between the ability of the drugs to stimulate Topo I-mediated DNA cleavage and the respective 50% inhibitory concentrations (IC(50) values) of the HT29, A549, and T24r cell growth. Fluorine substitution in the A-ring of hCPT was found to have a pronounced influence on biological activity, providing several compounds which are up to 100-fold more potent than CPT in terms of IC(50). Among these, 10,11-difluoro-hCPT has been selected for further development.

Homocamptothecins: synthesis and antitumor activity of novel E-ring-modified camptothecin analogues.

Homocamptothecin (hCPT), a camptothecin (CPT) analogue with a seven membered beta-hydroxylactone which combines enhanced plasma stability and potent topoisomerase I (Topo I)-mediated activity, is an attractive template for the elaboration of new anticancer agents. Like CPT, hCPT carries an asymmetric tertiary alcohol and displays stereoselective inhibition of Topo I. The preparation and biological screening of racemic hCPT analogues are described. The 10 hCPTs tested were better Topo I inhibitors than CPT. Fluorinated hCPTs 23c, d,f,g were found to have potent cytotoxic activity on A427 and PC-3 tumor cell lines. Their cytotoxicity remained high on the K562adr and MCF7mdr cell lines, which overexpress a functionally active P-glycoprotein. Fluorinated hCPTs were more efficacious in vivo than CPT on HT-29 xenografts. In this model, a tumor growth delay of 25 days was reached with hCPT 23g at a daily dose of 0.32 mg/kg, compared to 4 days with CPT at 0.625 mg/kg. Thus difluorinated hCPT 23g warrants further investigation as a novel Topo I inhibitor with high cytotoxicity toward tumor cells and promising in vivo efficacy.

Homocamptothecins: E-ring modified CPT analogues.

Homocamptothecins (hCPT) are modified camptothecins (CPT) with a seven-membered beta-hydroxylactone instead of the naturally occurring six-membered alpha-hydroxylactone. This E-ring modification fully conserves the ability to stabilize topo I-DNA single-strand breaks and stimulates high levels of DNA cleavage. A key feature is the irreversibility of E-ring opening, which should give reduced toxicity. Substituted hCPTs have been selected for their high antiproliferative activity on a panel of tumor cell lines, including those with cross resistance, and were found to be active at very low doses in a variety of human tumor xenografts when administered orally. BN 80915, a difluoro-hCPT, has entered clinical trials.

[The other camptothecins: recent advances with camptothecin analogues other than irinotecan and topotecan]. / Les autres camptothécines: avancées récentes sur les analogues de la camptothécine autres que l'irinotecan et le topotécan.

The first part of this review summarizes the clinical developments concerning camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, GG-211 and DX-8951f. The second part covers the recent developments of research on CPT analogs: CPTs with solubilizing substituents, CPTs with activating substituents, CPTs with dual activity, CPT prodrugs and CPTs with stabilized lactones.

CDC25 inhibitors as anticancer agents are moving forward.

The identification of a CDC25 inhibitor to arrest the cell cycle closely followed the discovery of CDC25 by Russell and Nurse in 1986. Recent advances at the preclinical and clinical stages reinforce the rationale to consider CDC25 as a relevant target for a cancer treatment. Here, in order to exemplify recent drug discovery efforts, we present our own experience with various chemical series of CDC25 inhibitors. We discuss how we have progressed and how we are considering the next steps to define the clinical entry points and hopefully complete this target validation to generate a new class of therapeutic agents.

Determination of the anti-platelet-activating factor BN-50727 and its metabolites in human plasma by high-performance liquid chromatography-solid-phase extraction.

A sensitive and selective HPLC-solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human plasma. The procedure consisted of an automated solid-phase extraction of the drug and metabolites on disposable propylcarboxylic acid cartridges, followed by on-line chromatographic separation. The method was linear from 3.75 to 2400 ng/ml and the limit of quantitation for BN-50727 in plasma samples was 3.75 ng/ml. The within-run precision of the method, expressed as relative standard deviation, ranged from 2.1 to 8.1%. The accuracy, expressed as relative error, ranged from -3.5 to 4.0%. For the main metabolite, the O-demethylated BN-50727 product, the method was linear from 7.5 to 2400 ng/ml and the limit of quantitation in plasma was 7.5 ng/ml. The within-run precision ranged from 2.1 to 11.0% and the accuracy from -5.3 to 1.1%. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human plasma and also of its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human plasma after administration of single and multiple doses.

Determination of the anti-platelet-activating factor BN-50727 and metabolites in human urine by high-performance liquid chromatography using solid-phase extraction.

A sensitive and selective HPLC solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human urine. The procedure consisted in a double solid-phase extraction of the urine samples on cyanopropyl and silica cartridges, followed by an automated solid-phase extraction of the drug and metabolites on CBA cartridges and posterior elution on-line to the chromatographic system for its separation. The method allowed quantitation in the concentration range 10-2400 ng/ml urine for both BN-50727 and the main metabolite, the O-demethylated BN-50727 product. The limit of quantitation for both compounds was 10 ng/ml. The inter-assay precision of the method, expressed as relative standard deviation, ranged from 1.9 to 4.5% for BN-50727 and from 2.5 to 9.0% for the metabolite. The accuracy, expressed as relative error, ranged from -2.4 to 4.2% and from 0.2 to 6.2%, respectively. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human urine and also for its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human urine after single-dose administration.

Homocamptothecin, an E-ring-modified camptothecin analogue, generates new topoisomerase I-mediated DNA breaks.

Homocamptothecin (hCPT) contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone ring found in camptothecin and its tumor active analogues, including topotecan and irinotecan. The homologation of the lactone E-ring reinforces the stability of the lactone, thus reducing considerably its conversion into a carboxylate form which is inactive. We have recently shown that hCPT is much more active than the parent compound against a variety of tumor cells in vitro and in xenograft models, suggesting that a highly reactive lactone is not essential for topoisomerase I-mediated anticancer activity [Lesueur-Ginot et al. (1999) Cancer Res. 59, 2939-2943]. In the present study, we provide further evidence that hCPT has superior topoisomerase I inhibition capacities to CPT. In particular, we show that replacement of the camptothecin lactone E-ring with a homologous seven-membered lactone ring changes the sequence-specificity of the drug-induced DNA cleavage by topoisomerase I. Both CPT and hCPT stimulate the cleavage by topoisomerase I at T( downward arrow)G sites, but in addition, hCPT stabilizes cleavage at specific sites containing the sequence AAC( downward arrow)G. At low drug concentrations, the cleavage at the T( downward arrow)G sites and at the hCPT-specific C( downward arrow)G sites is more pronounced and more stable with hCPT than with CPT. The in vitro data were confirmed in cells. Higher levels of protein-DNA complexes were detected in P388 leukemia cells treated with hCPT than those treated with CPT. Immunoblotting experiments revealed that endogenous topoisomerase I was efficiently trapped onto DNA by hCPT in cells. Finally, the use of a leukemia cell line resistant to CPT provided evidence that topoisomerase I is involved in the cytotoxicity of hCPT. Altogether, the results show that the beta-hydroxylactone ring of hCPT plays an important and positive role in the poisoning of topoisomerase I. An explanation is proposed to account for such remarkable changes in the sequence specificity of topoisomerase I cleavage consequent to the modification of the lactone. The study sheds new light on the importance of the lactone ring of camptothecins for the stabilization of topoisomerase I-DNA complexes.

A novel B-ring modified homocamptothecin, 12-Cl-hCPT, showing antiproliferative and topoisomerase I inhibitory activities superior to SN-38.

We report the synthesis and pharmacological evaluation of a novel homocamptothecin (hCPT) derivative, 12-Cl-hCPT, which contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone found in camptothecin (CPT) and bears a chloro substituent at position 12. The capacity of 12-Cl-hCPT to inhibit DNA topoisomerase I was compared with that of SN-38, the active metabolite of the clinically used antitumour prodrug CPT-11. In the DNA relaxation assay, 12-Cl-hCPT proved to be slightly more potent than SN-38 at stimulating the formation of nicked plasmid DNA molecules. A series of radiolabelled DNA restriction fragments were employed to identify and compare the position of the DNA cleavage sites induced by topoisomerase I in the presence of 12-Cl-hCPT and SN-38. These sequencing studies confirm that both 12-Cl-hCPT and SN-38 strongly promote DNA cleavage by topoisomerase I and reveal that the majority of the cleavage sites are located at the same nucleotide positions for the two drugs. However, a certain number of DNA cleavage sites were found to be specific to 12-Cl-hCPT. These sites, previously characterized with unsubstituted hCPT, generally correspond to 5'-CG sites whereas the sites common to the 12-Cl-hCPT and SN-38 essentially correspond to 5'-TG sites. We also quantified the formation of drug-induced protein-DNA complexes formed in HT29 human colon carcinoma cells. Trapping of endogenous proteins onto DNA was found to be much more efficient with 12-Cl-hCPT than with SN-38. These data provide a molecular basis to account for the enhanced antiproliferative activity of 12-Cl-hCPT compared with that of SN-38. Biological evaluation on a panel of sensitive and drug-resistant cell lines revealed 12-Cl-hCPT to be more cytotoxic to tumour cells than SN-38. 12-Cl-hCPT proved 14- and 23-fold more active than SN-38 toward the K562adr and T24anp multidrug-resistant cell lines, respectively. The marked topoisomerase I inhibitory properties of 12-Cl-hCPT coupled with its interesting antiproliferative activity, in particular against cancer cells presenting multidrug resistance phenotype with overexpression of P-glycoprotein, makes 12-Cl-hCPT a valid candidate for subsequent preclinical evaluation. Collectively, the data strengthen homocamptothecin as an extremely promising template to generate novel and potent antitumour agents.

The homocamptothecin BN 80915 is a highly potent orally active topoisomerase I poison.

BN 80915, a lead compound of the homocamptothecin (hCPT) family, has entered clinical trials. BN 80915 is a difluoro-hCPT where the six-membered alpha-hydroxylactone ring of camptothecin (CPT) is replaced by a seven-membered beta-hydroxylactone ring. Preclinical data reported here show that in spite of the modification to the crucial E-ring of CPTs, BN 80915 retains topoisomerase I poisoning activity as shown in living HT29 cells as well as in cell-free assays, where BN 80915 always performs better than SN-38 or TPT. In antiproliferative assays BN 80915 is also very potent as evidenced by IC50s values consistently lower than those of SN38 in sensitive cell lines as well as in their related multidrug-resistant lines overexpressing P-glycoprotein or multidrug resistance-associated protein. Furthermore, in human plasma, in contrast to CPT analogs, the hydrolysis of BN 80915 is slow, leading to improved plasma stability, and irreversible, thus avoiding toxicity related to the accumulation of active principle during excretion in the urinary tract. These findings may account for the good in vivo efficacy observed in PC3 xenograft experiments where BN 80915 administered orally at very low doses doubled the tumor growth delay in comparison to CPT-11 administered i.p. Altogether, these results strongly support further development of BN 80915.

Apoptosis induced by the homocamptothecin anticancer drug BN80915 in HL-60 cells.

The homocamptothecin (hCPT) derivative BN80915 containing a seven-membered lactone ring represents one of the most potent topoisomerase I inhibitors described. This anticancer agent, currently undergoing phase I clinical trials, has been shown to produce a greater number of DNA strand breaks than conventional camptothecins with a six-membered lactone ring. To shed light on the mechanism of action of hCPT at the cellular level, we compared the effects of BN80915 and the classic camptothecin SN-38, the active metabolite of irinotecan, on HL-60 human promyelocytic cancer cells. A variety of biochemical events, at both the mitochondrial and the nuclear levels, were characterized to determine how and to what extent the hCPT derivative can induce apoptotic cell death. The use of cytometry, Western blot analysis, confocal microscopy, and different colorimetric assays enabled us to demonstrate that BN80915 is a potent inducer of apoptosis in HL-60 cells. This induction of apoptosis is associated with cell cycle changes, a marked decrease of intracellular pH, activation of caspase-3 and -8, DNA fragmentation, and externalization of phosphatidylserine lipids but no significant changes of the mitochondrial membrane potential or the expression of Bcl-2. The interconnections between these different events are discussed. Collectively, the results indicate that the superior activity expressed at the topoisomerase I level leads to a more pronounced induction of apoptosis by BN80915 compared with SN-38. The study identifies and delineates signaling factors involved in BN80915-induced apoptosis in HL-60 cells.

BN 80927: a novel homocamptothecin with inhibitory activities on both topoisomerase I and topoisomerase II.

BN 80927, a novel homocamptothecin derivative, inhibits both topoisomerase I and topoisomerase II mediated DNA relaxation and shows pronounced cytotoxicity against HT29, SKOV-3, DU145 and MCF7 human tumor cell lines.