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AMHRII, the anti-Müllerian hormone receptor, is selectively expressed in normal sexual organs but is also re-expressed in gynecologic cancers. Hence, we developed murlentamab, a humanized glyco-engineered anti-AMHRII monoclonal antibody currently in clinical trial. Low-fucosylated antibodies are known to increase the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) potency of effector cells, but some preliminary results suggest a more global murlentamab-dependent activation of the immune system. In this context, we demonstrate here that the murlentamab opsonization of AMHRII-expressing ovarian tumor cells, in the presence of unstimulated- or tumor-associated macrophage (TAM)-like macrophages, significantly promotes macrophage-mediated ADCC and shifts the whole microenvironment towards a pro-inflammatory and anti-tumoral status, thus triggering anti-tumor activity. We also report that murlentamab orients both unstimulated- and TAM-like macrophages to an M1-like phenotype characterized by a strong expression of co-stimulation markers, pro-inflammatory cytokines and chemokines, favoring T cell recruitment and activation. Moreover, we show that murlentamab treatment shifts CD4 + Th1/Th2 balance towards a Th1 response and activates CD8 + T cells. Altogether, these results suggest that murlentamab, through naïve macrophage orientation and TAM reprogrammation, stimulates the anti-tumor adaptive immune response. Those mechanisms might contribute to the sustained clinical benefit observed in advanced cancer patients treated with murlentamab. Finally, the enhanced murlentamab activity in combination with pembrolizumab opens new therapeutic perspectives.

The anti-Müllerian hormone (AMH) belongs to the TGF-β family and plays a key role during fetal sexual development. Various reports have described the expression of AMH type II receptor (AMHRII) in human gynecological cancers including ovarian tumors. According to qRT-PCR results confirmed by specific In-Situ Hybridization (ISH) experiments, AMHRII mRNA is expressed in an extremely restricted number of normal tissues. By performing ISH on tissue microarray of solid tumor samples AMHRII mRNA was unexpectedly detected in several non-gynecological primary cancers including lung, breast, head and neck, and colorectal cancers. AMHRII protein expression, evaluated by immunohistochemistry (IHC) was detected in approximately 70% of epithelial ovarian cancers. Using the same IHC protocol on more than 900 frozen samples covering 18 different cancer types we detected AMHRII expression in more than 50% of hepato-carcinomas, colorectal, lung, and renal cancer samples. AMHRII expression was not observed in neuroendocrine lung tumor samples nor in non-Hodgkin lymphoma samples. Complementary analyses by immunofluorescence and flow cytometry confirmed the detection of AMHRII on a panel of ovarian and colorectal cancers displaying comparable expression levels with mean values of 39,000 and 50,000 AMHRII receptors per cell, respectively. Overall, our results suggest that this embryonic receptor could be a suitable target for treating AMHRII-expressing tumors with an anti-AMHRII selective agent such as murlentamab, also named 3C23K or GM102. This potential therapeutic intervention was confirmed in vivo by showing antitumor activity of murlentamab against AMHRII-expressing colorectal cancer and hepatocarcinoma Patient-Derived tumor Xenografts (PDX) models.

Background: Angiotensin-converting enzyme 2 is the receptor that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses for entry into lung cells. Because ACE-2 may be modulated by angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs), there is concern that patients treated with ACEIs and ARBs are at higher risk of coronavirus disease 2019 (COVID-19) pneumonia., Aim: This study sought to analyze the association of COVID-19 pneumonia with previous treatment with ACEIs and ARBs., Materials and Methods: We retrospectively reviewed 684 consecutive patients hospitalized for suspected COVID-19 pneumonia and tested by polymerase chain reaction assay. Patients were split into two groups, according to whether (group 1, n = 484) or not (group 2, n = 250) COVID-19 was confirmed. Multivariable adjusted comparisons included a propensity score analysis., Results: The mean age was 63.6 ± 18.7 years, and 302 patients (44%) were female. Hypertension was present in 42.6% and 38.4% of patients in groups 1 and 2, respectively (P = 0.28). Treatment with ARBs was more frequent in group 1 than group 2 (20.7% vs. 12.0%, respectively; odds ratio [OR] 1.92, 95% confidence interval [CI] 1.23-2.98; P = 0.004). No difference was found for treatment with ACEIs (12.7% vs. 15.7%, respectively; OR 0.81, 95% CI 0.52-1.26; P = 0.35). Propensity score-matched multivariable logistic regression confirmed a significant association between COVID-19 and previous treatment with ARBs (adjusted OR 2.36, 95% CI 1.38-4.04; P = 0.002). Significant interaction between ARBs and ACEIs for the risk of COVID-19 was observed in patients aged > 60 years, women, and hypertensive patients., Conclusions: This study suggests that ACEIs and ARBs are not similarly associated with COVID-19. In this retrospective series, patients with COVID-19 pneumonia more frequently had previous treatment with ARBs compared with patients without COVID-19., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Dr. Georges has received consultant or speaker fees from AstraZeneca France, Sanofi-Aventis, Amgen, and Merck Sharpe and Dohme. The other authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Besides the interest of an early detection of ovarian cancer, there is an urgent need for new predictive and prognostic biomarkers of tumor development and cancer treatment. In healthy patients, circulating blood monocytes are typically subdivided into classical (85%), intermediate (5%) and non-classical (10%) populations. Although these circulating monocyte subsets have been suggested as biomarkers in several diseases, few studies have investigate their potential as a predictive signature for tumor immune status,tumor growth and treatment adaptation., Methods: In this study, we used a homogeneous cohort of 28 chemotherapy-naïve patients with ovarian cancer to evaluate monocyte subsets as biomarkers of the ascites immunological status. We evaluated the correlations between circulating monocyte subsets and immune cells and tumor burden in peritoneal ascites. Moreover, to validate the use of circulating monocyte subsets tofollow tumor progression and treatment response, we characterized blood monocytes from ovarian cancer patients included in a phase 1 clinical trial at baseline and following murlentamab treatment., Results: We demonstrate here a robust expansion of the intermediate blood monocytes (IBMs) in ovarian cancer patients. We establish a significant positive correlation between IBM percentage and tumor-associate macrophages with a CCR2 high /CD163 high /CD206 high /CD86 low profile. Moreover, IBM expansion is associated with a decreased effector/regulatory T-cell ratio in ascites and with the presence of soluble immunosuppressive mediators. We also establish that IBM proportion positively correlates with the peritoneum tumor burden. Finally, the study of IBMs in patients with ovarian cancer under immunotherapy during the phase clinical trial supports IBMs to follow the evolution of tumor development and the treatment adaptation., Conclusions: This study, which links IBM level with immunosuppression and tumor burden in peritoneum, identifies IBMs as apotential predictive signature of ascites immune status and as a biomarker ofovarian cancer development and treatment response., Trial Registration Number: EudraCT: 2015-004252-22 NCT02978755., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence., Methods: We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival., Results: The assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003)., Conclusions: Our TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.

Background: HER3/ErbB3 receptor deletion or blockade leads to tumor cell apoptosis, whereas its overexpression confers anti-cancer drug resistance through upregulation of protective mechanisms against apoptosis. We produced the anti-HER3 antibody 9F7-F11 that promotes HER3 ubiquitination and degradation via JNK1/2-dependent activation of the E3 ubiquitin ligase ITCH, and that induces apoptosis of cancer cells. Cellular FLICE-like inhibitory protein (c-FLIP) is a key regulator of apoptotic pathways. Here, we wanted to determine the mechanisms underlying the pro-apoptotic effect of 9F7-F11., Methods: Anti-HER3 antibody-induced apoptosis was assessed by western blot, and by flow cytometry measurement of Annexin V/7-AAD-labelled tumor cells (BxPC3, MDA-MB-468 and DU145 cell lines). c-FLIP/ITCH interaction and subsequent degradation/ubiquitination were investigated by co-immunoprecipitation of ITCH-silenced vs scramble control cells. The relationship between ITCH-mediated c-FLIP degradation and antibody-induced apoptosis was examined by western blot and flow cytometry of tumor cells, after ITCH RNA interference or by pre-treatment with ITCH chemical inhibitor chlorimipramine (CI)., Results: Following incubation with 9F7-F11, cancer cell apoptosis occurs through activation of caspase-8, - 9 and - 3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP). Moreover we showed that ubiquitination and proteasomal degradation of the anti-apoptotic protein c-FLIP was mediated by USP8-regulated ITCH recruitment. This effect was abrogated by ITCH- and USP8-specific RNA interference (siRNA), or by the ITCH chemical inhibitor CI. Specifically, ITCH silencing or CI blocked 9F7-F11-induced caspase-8-mediated apoptosis of tumor cells, and restored c-FLIP expression. ITCH-silencing or CI concomitantly abrogated HER3-specific antibody-induced apoptosis of Annexin V/7-AAD-labelled BxPC3 cells. 9F7-F11 favored the extrinsic apoptosis pathway by inducing TRAIL-R2/DR5 upregulation and TRAIL expression that promoted the formation of death-inducing signaling complex (DISC), leading to caspase-8-mediated apoptosis. Incubation with 9F7-F11 also induced BID cleavage, BAX upregulation and BIM expression, which initiated the caspase-9/3-mediated mitochondrial death pathway. The anti-HER3 antibody pro-apoptotic effect occurred concomitantly with downregulation of the pro-survival proteins c-IAP2 and XIAP., Conclusions: The allosteric non-neuregulin competing modulator 9F7-F11, sensitizes tumor cells to DR5/caspase-8-mediated apoptosis through ITCH-dependent downregulation of c-FLIP.

Müllerian inhibiting substance, also called anti-Müllerian hormone (AMH), inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, such as human ovarian cancers (OCs). On this basis, a humanized glyco-engineered monoclonal antibody (3C23K) has been developed. The aim of this study was therefore to experimentally confirm the therapeutic potential of 3C23K in human OCs. We first determined by immunofluorescence, immunohistochemistry and cytofluorometry analyses the expression of AMHRII in patient's tumors and found that a majority (60 to 80% depending on the detection technique) of OCs were positive for this marker. We then provided evidence that the tumor stroma of OC is enriched in tumor-associated macrophages and that these cells are responsible for 3C23K-induced killing of tumor cells through ADCP and ADCC mechanisms. In addition, we showed that 3C23K reduced macrophages induced-T cells immunosuppression. Finally, we evaluated the therapeutic efficacy of 3C23K alone and in combination with a carboplatin-paclitaxel chemotherapy in a panel of OC Patient-Derived Xenografts. In those experiments, we showed that 3C23K significantly increased the proportion and the quality of chemotherapy-based in vivo responses. Altogether, our data support the potential interest of AMHRII targeting in human ovarian cancers and the evaluation of 3C23K in further clinical trials., Competing Interests: CONFLICTS OF INTEREST This study was supported by grants from GamaMabs Pharma, in which belong two authors, Jean-Marc Barret and Jean-François Prost.

Exploratory clinical trials using therapeutic anti-HER3 antibodies strongly suggest that neuregulin (NRG1; HER3 ligand) expression at tumor sites is a predictive biomarker of anti-HER3 antibody efficacy in cancer. We hypothesized that in NRG1-expressing tumors, where the ligand is present before antibody treatment, anti-HER3 antibodies that do not compete with NRG1 for receptor binding have a higher receptor-neutralizing action than antibodies competing with the ligand for binding to HER3. Using time-resolved-fluorescence energy transfer (TR-FRET), we demonstrated that in the presence of recombinant NRG1, binding of 9F7-F11 (a nonligand-competing anti-HER3 antibody) to HER3 is increased, whereas that of ligand-competing anti-HER3 antibodies (H4B-121, U3-1287, Ab#6, Mab205.10.2, and MOR09825) is decreased. Moreover, 9F7-F11 showed higher efficacy than antibodies that compete with the ligand for binding to HER3. Specifically, 9F7-F11 inhibition of cell proliferation and of HER3/AKT/ERK1/2 phosphorylation as well as 9F7-F11-dependent cell-mediated cytotoxicity were higher in cancer cells preincubated with recombinant NRG1 compared with cells directly exposed to the anti-HER3 antibody. This translated in vivo into enhanced growth inhibition of NRG1-expressing BxPC3 pancreatic, A549 lung, and HCC-1806 breast cell tumor xenografts in mice treated with 9F7-F11 compared with H4B-121. Conversely, both antibodies had similar antitumor effect in NRG1-negative HPAC pancreatic carcinoma cells. In conclusion, the allosteric modulator 9F7-F11 shows increased anticancer effectiveness in the presence of NRG1 and thus represents a novel treatment strategy for NRG1-addicted tumors. Mol Cancer Ther; 16(7); 1312-23. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Ovarian cancer is the leading cause of death in women with gynecological cancers and despite recent advances, new and more efficient therapies are crucially needed. Müllerian Inhibiting Substance type II Receptor (MISRII, also named AMHRII) is expressed in most ovarian cancer subtypes and is a novel potential target for ovarian cancer immunotherapy. We previously developed and tested 12G4, the first murine monoclonal antibody (MAb) against human MISRII. Here, we report the humanization, affinity maturation and glyco-engineering steps of 12G4 to generate the Fc-optimized 3C23K MAb, and the evaluation of its in vivo anti-tumor activity. The epitopes of 3C23K and 12G4 were strictly identical and 3C23K affinity for MISRII was enhanced by a factor of about 14 (KD = 5.5 × 10-11 M vs 7.9 × 10-10 M), while the use of the EMABling® platform allowed the production of a low-fucosylated 3C23K antibody with a 30-fold KD improvement of its affinity to FcγRIIIa. In COV434-MISRII tumor-bearing mice, 3C23K reduced tumor growth more efficiently than 12G4 and its combination with carboplatin was more efficient than each monotherapy with a mean tumor size of 500, 1100 and 100 mm3 at the end of treatment with 3C23K (10 mg/kg, Q3-4D12), carboplatin (60 mg/kg, Q7D4) and 3C23K+carboplatin, respectively. Conversely, 3C23K-FcKO, a 3C23K form without affinity for the FcγRIIIa receptor, did not display any anti-tumor effect in vivo. These results strongly suggested that 3C23K mechanisms of action are mainly Fc-related. In vitro, antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) were induced by 3C23K, as demonstrated with human effector cells. Using human NK cells, 50% of the maximal lysis was obtained with a 46-fold lower concentration of low-fucosylated 3C23K (2.9 ng/ml) than of 3C23K expressed in CHO cells (133.35 ng/ml). As 3C23K induced strong ADCC with human PBMC but almost none with murine PBMC, antibody-dependent cell phagocytosis (ADCP) was then investigated. 3C23K-dependent (100 ng/ml) ADCP was more active with murine than human macrophages (only 10% of living target cells vs. about 25%). These in vitro results suggest that the reduced ADCC with murine effectors could be partially balanced by ADCP activity in in vivo experiments. Taken together, these preclinical data indicate that 3C23K is a new promising therapeutic candidate for ovarian cancer immunotherapy and justify its recent introduction in a phase I clinical trial.

Background and Objectives: A human recombinant monoclonal anti-RhD IgG may be useful to prevent RhD allo-immunization. Roledumab is such an antibody with a glycosylation pattern optimized for biological activity. The objective of the study was to assess the safety and pharmacokinetics of roledumab in healthy RhD-negative volunteers., Materials and Methods: A total of 46 subjects received doses of 30-3000 μg i.v. of roledumab or placebo using a double-blind escalating single-dose design; 12 of these subjects also received 300 μg i.m. of roledumab. Subjects were followed for 6 months after administration. Serum roledumab concentrations were determined using flow cytometry., Results: Fourteen treatment-emergent adverse events related to treatment were reported in nine subjects, with no apparent difference in their frequency or nature after placebo or roledumab administration. No anti-roledumab antibodies were detected. AUC(last) increased from 4·4 ng/ml.day at 30 μg i.v. to 2257 ng/ml.day at 3000 g i.v. The t(½) ranged from 18 to 22 days, and the absolute bioavailability after i.m. administration was between 73% and 80%., Conclusion: Roledumab is safe and well tolerated in healthy RhD-negative volunteers and shows a pharmacokinetic profile similar to that of polyclonal anti-RhD immunoglobulin., (© 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.)

Although anti-CD20 monoclonal antibodies (mAbs) show promise for the treatment of chronic lymphocytic leukemia (CLL), the success of the anti-CD20 mAb rituximab in CLL treatment has been limited. Novel anti-CD20 mAbs with more potent cytotoxic activity have recently been engineered, but so far most have only been tested in vitro with natural killer (NK) cells from healthy donors. Because it is still unclear whether these optimized cytotoxic mAbs will improve NK-cell killing of tumor cells in CLL patients, we characterized the relevant phenotypic and functional features of NK cells from CLL patients in detail. Expression of inhibitory and activating NK-cell receptors and of Fc gamma receptor IIIA (FcγRIIIA) is well preserved in CD16(+)CD56(dim) cytotoxic NK cells from these patients, independently of disease progression. These cells are fully functional following cytokine stimulation. In addition, the FcγRIIIA-optimized LFB-R603 anti-CD20 mAb mediates 100 times greater antibody-dependent cell-mediated cytotoxicity by NK cells from CLL patients and healthy donors than rituximab. Enhanced degranulation against autologous B-CLL cells is observed at lower concentrations of LFB-R603 than rituximab, regardless of CLL prognostic factors. These findings strongly justify further clinical development of anti-CD20 mAbs optimized for FcγR engagement in CLL patients.

The Laboratoire français du fractionnement et des biotechnologies (LFB), the leading manufacturer of plasma-derived medicinal products in France and 6th worldwide, is strongly involved in the development of therapeutic monoclonal antibodies (mAb). For more than 15 years, LFB has been focusing its research effort on the study of structure-function relationship of antibodies. Its studies on the molecular basis of IgG interaction with the receptors for the Fc portion of IgG (FcgRs) has made it possible to develop antibodies with high antibody-dependent cellular cytotoxicity (ADCC) activity and enhanced affinity to FcgRIII (CD16), both correlated to a glycosylation pattern characterized by a low fucose content. Based on these studies, LFB has developed EMABling, a technological platform for the production of antibodies with enhanced cytotoxicity ability. Two EMABling antibodies recently entered clinical development: LFB-R593, a fully human anti-rhesus D (RhD) antibody, for the prevention of feto-maternal allo-immunization in RhD- women, as a substitute for human polyclonal anti-RhD immunoglobulins, and LFB-R603, a monoclonal antibody directed against CD20, for the treatment of B cell malignancies. LFB investment in bioproduction through the recent acquisition of MAbgène company, a fully integrated French contract biopharmaceutical manufacturing company, allows the production of antibodies to a large GMP scale. As a whole, LFB owns a portfolio of several EMABling antibodies with high therapeutic interest, in line with its public health mission.

Background: Chikungunya virus (CHIKV) is a recently reemerged arbovirus responsible for a massive outbreak of infection in the Indian Ocean region and India that has a very significant potential to spread globally because of the worldwide distribution of its mosquito vectors. CHIKV induces a usually self-limited disease in humans that is characterized by fever, arthralgia, myalgia, and rash; however, cases of severe CHIKV infection have recently been described, particularly in adults with underlying condition and neonates born to viremic mothers., Methods: Human polyvalent immunoglobulins were purified from plasma samples obtained from donors in the convalescent phase of CHIKV infection, and the preventive and curative effects of these immunoglobulins were investigated in 2 mouse models of CHIKV infection that we developed., Results: CHIKV immunoglobulins contain anti-CHIKV antibodies and exhibit a high in vitro neutralizing activity and a powerful prophylactic and therapeutic efficacy against CHIKV infection in vivo, including in the neonate., Conclusions: Administration of CHIKV immunoglobulins may constitute a safe and efficacious prevention strategy and treatment for individuals exposed to CHIKV who are at risk of severe infection, such as neonates born to viremic mothers and adults with underlying conditions. These results provide a proof-of-concept for purifying human immunoglobulins from plasma samples from patients in the convalescent phase of an emerging infectious disease for which neither prevention nor treatment is available.

A human anti-RhD immunoglobulin G1 monoclonal antibody (mAb), R297, was tested in a phase I study to assess its ability to induce the clearance of antibody-coated autologous RhD+ red blood cells (RBCs) in healthy male volunteers. The clearance potency of R297 was compared with that of a marketed human polyclonal anti-D product (Rhophylac). This mAb has been selected for its ability to strongly engage Fc-gamma receptor IIIA and to mediate a potent antibody-dependent cell cytotoxicity (ADCC) against RhD+ RBCs. Autologous RhD+ RBCs were sensitized with either Rhophylac or R297 at three different coating percentages (25, 12.5 and 6.25%), before re-infusion. This phase I study showed that the human R297 mAb promoted rapid and complete clearance of RBCs, and showed activity that was at least as potent as the human polyclonal anti-D antibody preparation. Clearance of RBCs could still be observed when the percentage of R297 used to coat the RBCs was reduced to 6.25%. Finally, none of the adverse events was severe or considered to be related to R297. Thus, R297 is a promising candidate for the prevention of allo-immunization and represents a new generation of Fc-modified monoclonal antibodies with increased FcgammaRIII binding and increased ADCC.

Patients with chronic lymphocytic leukaemia (CLL) treated with a combination of fludarabine, cyclophosphamide and rituximab show a high response rate. However, only a poor response is observed following rituximab monotherapy. The use of chemo-immunotherapy is often associated with haematological and infectious complications. Thus, an antibody with an enhanced ability to kill CLL cells could lead to better clinical responses to antibody monotherapy and the possibility of lowering drug doses during chemo-immunotherapy. We generated a chimeric anti-CD20 monoclonal antibody (mAb), EMAB-6, which has a low fucose content. Apoptosis and complement activities for EMAB-6 were similar to those seen for rituximab. By contrast, EMAB-6 mAb showed improved Fcgamma receptor IIIA (FcgammaRIIIA)/CD16 binding and FcgammaRIIIA-dependent effector functions. It induced a higher in vitro antibody-dependent cellular cytotoxicity against CLL cells and a higher FcgammaRIIIA-mediated interleukin-2 production by FcgammaRIIIA(+) Jurkat cells in the presence of CLL cells at both low and maximally saturating concentrations. Comparative studies between CLL and lymphoma cells coated with EMAB-6 or rituximab indicated that the difference of efficacy was more pronounced at low doses and when target cells expressed fewer CD20 molecules. Thus, EMAB-6 mAb represents a promising drug candidate for the treatment of CLL by inducing a strong cytotoxicity against tumour cells that express low CD20 levels.

Protein tyrosine phosphatases from several microorganisms have been shown to play a role as virulence factors by modifying the phosphorylation/dephosphorylation equilibrium in cells of their host. Two tyrosine phosphatases, MptpA and MptpB, secreted by Mycobacterium tuberculosis, have been identified. Expression of MptpA is upregulated upon infection of monocytes, but its role in host cells has not been elucidated. A eukaryotic expression vector containing the mptpA cDNA has been transfected into macrophages. We report that MptpA reduced phagocytosis of mycobacteria, opsonized zymosan or zymosan, but had no effect on phagocytosis of IgG-coated particles. We also noted that the presence of F-actin at the surface of phagosomes containing opsonized zymosan was significantly increased in cells expressing MptpA. In the presence of recombinant MptpA, the process of actin polymerization at the surface of isolated phagosomes was increased; this was not the case in the presence of the phosphatase-dead mutant MptpA(C11S). MptpA had no effect when IgG-coated particles were present inside isolated phagosomes. These results indicate that, like other tyrosine phosphatases of pathogens, MptpA plays a role in phagocytosis and actin polymerization. However, MptpA had no effect on IgG particles, suggesting that its putative substrate(s) is not linked to the signaling pathways of Fcgamma receptors.

Bacterial genomics have revealed the widespread occurrence of eukaryotic-like protein kinases in prokaryotes, but little is known about their regulation, endogenous substrates, and physiological role. The present study concerns one of these enzymes, the serine/threonine protein kinase PknF from Mycobacterium tuberculosis. It is shown that, in addition to its autokinase activity, PknF is able to phosphorylate Rv1747, a newly described ABC transporter. This reaction appears to involve two FHA domains of Rv1747. It is suggested that recruitment and phosphorylation of Rv1747 depend on the interaction between its two non-redundant FHA domains and the autophosphorylated form of PknF., (Copyright 2004 Federation of European Microbiological Societies)

In bacteria, regulatory phosphorylation of proteins at serine and/or threonine residues by Ser/Thr protein kinase (STPK) is an emerging theme in prokaryotic signaling, particularly since the prediction of the occurrence of several STPKs from genome sequencing and sequence surveys. Here we show that protein PknH possesses an autokinase activity and belongs to the large STPK family found in Mycobacterium tuberculosis. Evidence is presented that PknH can also phosphorylate EmbR, a protein suspected to modulate the level of arabinosyltransferase activity involved in arabinan biosynthesis of arabinogalactan, a key molecule of the mycobacterial cell wall. Interestingly, EmbR possesses an FHA (forkhead-associated) domain, a newly described phosphoprotein recognition domain, which plays an essential role in PknH-EmbR interaction and phosphorylation of EmbR by PknH. It is demonstrated that mutation of each of three particular residues of this FHA domain, Arg312, Ser326, and Asn348, totally abolishes the PknH-mediated phosphorylation of EmbR, thus highlighting the critical role of this domain in the direct interaction between EmbR and PknH.

The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli sigma(70) -35 elements but located much closer to the -10 element is important for optimal expression of P1, whereas the sequence at the -35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.

Protein PknE from Mycobacterium tuberculosis has been overproduced and purified, and its biochemical properties have been analyzed. This protein is shown to be a eukaryotic-like (Hanks'-type) protein kinase with a structural organization similar to that of membrane-bound eukaryotic sensor serine/threonine kinases. It consists of a N-terminal catalytic domain located in the cytoplasm, linked via a single transmembrane-spanning region to an extracellular C-terminal domain. The full-length enzyme, as well as the cytosolic domain alone, can autophosphorylate on serine and threonine residues. Such autokinase activity requires the presence of a lysine residue at position 45 in subdomain II, which is known to be essential also for eukaryotic kinase activity. Involvement of PknE in the transduction of external signals into the cytosol of bacteria is proposed.

With the aim of identifying genes involved in cartilage differentiation, we have used a subtractive hybridization strategy with cDNAs from a chondrocytic cell line (MC615) and mRNAs from a mesenchymal precursor cell line (10T1/2). We have isolated a cDNA clone representing a novel mouse gene. The predicted 368-amino acid protein, designated ZF-12, contains four C(2)H(2)-type zinc finger motifs and one region homologous to the LeR domain, a finger-associated structural domain. ZF-12 mRNAs are expressed during embryonic development and in different organs in adult, including rib cartilage. These data suggest that ZF-12 might play an important role not only in cartilage differentiation, but also in basic cellular processes.

The regulation of transcription of the pckA gene coding for phosphoenolpyruvate carboxykinase in Escherichia coli was analysed by site-directed mutagenesis of the promoter region and measurement of in vitro transcription initiation. Mutation of the guanine residue at position -14 to either cytosine or thymine was found to result in a drastic decrease of transcription, even in the presence of the natural -35 hexamer TTTCCA that differs by only two nucleotides from the consensus sequence TTGACA. It was concluded that the promoter region of pckA contains an extended -10 module, 5'-TG-3', located one base upstream of the -10 hexamer, which is crucial for transcription.

The icd gene of Escherichia coli, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified icd P1 and a newly detected second promoter, icd P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the icd control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position -76.5 within the icd P2 promoter region and (ii) three particular mutations in the C-terminal end of the alpha subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the icd P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.

The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/phosphatase was rapidly lost after prior incubation with the ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process. About 2 mol [3H]FSBA/mol IDHK/P were bound during the time course of the inactivation. However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol. This suggested that FSBA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond. Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme insensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue. However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480.

The start site of transcription of the ppsA gene, whose expression is controlled by the regulatory protein FruR in Escherichia coli, was determined by primer extension of in vivo transcripts. The interactions of the ppsA promoter with either RNA polymerase or FruR factor were analysed by the base removal method. Our results indicate that: (i) the RNA polymerase binding site has a -10 extended module but lacks its -35 hexamer; (ii) FruR binds to a target DNA region centered around position -45.5 upstream of the ppsA gene. In addition, circular permutation analysis showed that, upon binding to its site, FruR induces a sharp bend of 120 degrees in the DNA helix, which suggests a crucial involvement of FruR-induced bending in ppsA promoter activation. Direct contacts between the upstream activating DNA and RNA polymerase were studied in an in vitro transcription assay by using reconstituted RNA polymerase mutants containing Ala substitutions in C-terminal domain of their alpha subunit. The alpha[L262A], alpha[R265A] and alpha[N268A] substitutions, which caused the most drastic reduction in the FruR-mediated activation of the ppsA promoter, had previously been shown to inhibit the upstream element-mediated activation at the rrnBP1 promoter.

Milnacipran (Ixel) is a new antidepressant with essentially equal potency for inhibiting the reuptake of both serotonin and noradrenaline, with no affinity for any neurotransmitter receptor studied. A review of the studies comparing milnacipran, placebo and active comparator antidepressants provides clear-cut evidence of its efficacy in both severe and moderate depression in hospitalized and community settings. Meta-analyses of the original data of controlled trials involving 1032 patients, comparing milnacipran with imipramine or selective serotonin reuptake inhibitors (SSRIs), show that milnacipran provides antidepressant efficacy similar to that of imipramine and significantly superior to that of the SSRIs. An analysis of a database of over 3300 patients shows that both the general and cardiovascular tolerability of milnacipran are superior to those of the tricyclic antidepressants (TCAs) with fewer cholinergic side-effects. The tolerability of milnacipran was comparable to that of the SSRIs, with a higher incidence of dysuria with milnacipran, and a higher frequency of nausea and anxiety with the SSRIs. Milnacipran is a new therapeutic option in depression, which offers a clinical efficacy in the range of the TCAs combined with a tolerability equivalent to that of the SSRIs.

The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method. Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase. A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter.

The relative benefits and risks of milnacipran, a novel antidepressant which selectively inhibits the reuptake of serotonin and noradrenaline, have been evaluated in comparative trials against tricyclic antidepressants (TCAs) or selective serotonin reuptake inhibitors (SSRIs). A total of 2462 patients with major depressive disorders have been investigated. At the optimal dose (50 mg twice a day), the efficacy of milnacipran was equivalent to that of the TCAs, with response rates of approximately 65% in both cases. Milnacipran was consistently effective against all of the principal elements of depression (anxiety, cognitive function, sleep and psychomotor retardation), and did not produce sedation or the emergence of suicidal thoughts. The Clinical Global Impression (CGI-3) score, a measure of the overall therapeutic impact of a treatment, was significantly higher with milnacipran than with TCAs (1.98 versus 1.84, p < 0.05). TCAs were associated with a higher frequency of adverse events than milnacipran, particularly with respect to anticholinergic-like effects; dysuria was the only adverse event occurring twice as frequently with milnacipran than with TCAs. Compared with TCAs, milnacipran was also associated with a lower incidence of cardiovascular adverse events. No haematological abnormalities occurred during treatment with milnacipran, and the incidence of abnormal liver function tests tended to be lower with milnacipran than with TCAs. In comparisons with SSRIs, milnacipran produced significantly higher response rates. The CGI-3 scores were significantly higher in milnacipran-treated patients (2.64 versus 2.32, p < 0.05). The adverse event profiles of the two treatments were similar, as was the incidence of abnormal liver function tests. These studies suggest that milnacipran offers clinical advantages over TCAs in terms of tolerability, and over SSRIs in terms of efficacy. In particular, the lack of cardiovascular adverse events appears to offer advantages in cases of deliberate overdose. To date, 15 such overdoses have occurred; none was fatal and each had a favourable outcome. The reproducible pharmacokinetic characteristics of milnacipran present further advantages over both groups of agents, due to lack of drug accumulation and a low risk of drug interactions.

Milnacipran (Ixel) is a new antidepressant which has been developed for its selective inhibition of both serotonin and noradrenaline reuptake and its lack of affinity for neurotransmitter receptors. It inhibits virtually equipotently the reuptake of serotonin and noradrenaline both in vitro and in vivo, as demonstrated by the antagonism of centrally acting monoamine displacers. It has no effect on dopamine reuptake. In addition, milnacipran has been shown by intracerebral microdialysis to increase the extracellular levels of both serotonin and noradrenaline after acute administration. Milnacipran is devoid of interactions at any known neurotransmitter receptor or ion channel. In particular, and unlike tricyclic antidepressants, it does not act at noradrenergic, muscarinic or histaminergic receptors. Contrary to tricyclic antidepressants, chronic administration of milnacipran does not modify beta-adrenoceptor binding or second messenger function. Milnacipran is active on various animal models of depression such as the forced swimming test in the mouse, learned helplessness in the rat and the olfactory bulbectomized rat model. This pharmacological profile, associated with an excellent bioavailability in man, was predicted to be that required for a powerful and well-tolerated antidepressant. Subsequent clinical development has shown this prediction to be well founded.

In drug development the move from tricyclic antidepressants (TCAs) to selective serotonin reuptake inhibitors (SSRIs) involved not only the loss of the direct receptor interactions responsible for the adverse side effects of TCAs, but also the ability to inhibit the reuptake of noradrenaline. Selectivity for the single neurotransmitter, serotonin, may explain why SSRIs tend to be less efficacious than the TCAs, especially in more serious forms of depression. The advent of selective serotonin and noradrenaline reuptake inhibitors (SNRIs) has tended to confirm the idea that an action on both monoamine systems is important for maximal antidepressant efficacy. This paper reviews clinical trials comparing the new SNRI milnacipran with the SSRIs fluoxetine and fluvoxamine. A meta-analysis of the principal trials shows greater response rates (the proportion of patients with a decrease in symptom scores of 50% or more) with milnacipran (50 mg twice a day) than with fluoxetine (20 mg once a day), or fluvoxamine (100 mg twice a day) (milnacipran: 64%; SSRIs: 50%). Remission rates (the proportion of patients with Hamilton Depression Rating Scores of 7 or below) were also higher with milnacipran than with SSRIs (39 versus 28%). In one study, in which 100 mg milnacipran was given once a day in the evening, the higher response rate obtained with fluoxetine appears to be largely attributable to an inappropriate milnacipran dosage regimen. Data from a pharmacovigilance database including all patients participating in clinical trials with milnacipran (n = 5732) showed that, compared with the SSRIs, milnacipran produced fewer gastrointestinal side effects, such as nausea, and less anxiety. Milnacipran was, however, associated with a higher incidence of headache, dry mouth and dysuria. The results of these studies suggest that milnacipran is superior in efficacy to SSRIs and is equally well tolerated. Milnacipran, therefore, appears to offer a therapeutic advantage over the SSRIs.

Since tachykinins released from lung sensory nerve endings are thought to play a role in inflammatory diseases of airways via NK1 and NK2 receptors, dual tachykinin NK1 and NK2 receptor antagonists may have a great therapeutic potential. In vitro, the cyclopeptide S 16474 (cyclo-[Abo-Asp(D-Trp(Suc0Na)-Phe-N-(Me)Bzl)]) bound to both human tachykinin NK1 and NK2 receptors expressed in two lines of transfected Chinese hamster ovary cells (IC50 values 85 nM and 129 nM, respectively), while showing a poor affinity for the rat tachykinin NK1 receptor. S 16474 inhibited the contractions induced by substance P in isolated rabbit vena cava (pA2 7.0) and by neurokinin A in rabbit pulmonary artery (pA2 5.6). In vivo in anaesthetized guinea-pigs, S 16474 was found to dose dependently inhibit the bronchoconstrictions induced by intravenously administered substance P, neurokinin A and capsaicin. Plasma extravasation evoked in bronchi by endogenously released tachykinins under vagus nerve stimulation was abolished by S 16474 (10 mu mol/kg i.v.). These results demonstrate clearly that S 16474 is a tachykinin receptor antagonist exhibiting, in vitro and in vivo, a dual inhibitory effect on NK1 and NK2 receptors.

The effects of S 16118 (p-guanidobenzoyl-[Hyp3,Thi5,D-Tic7, Oic8]bradykinin (BK)], a new, potent and long-acting BK B2 antagonist, were tested in some in vivo models of inflammation. In rats, S 16118 (0.1 and 1 mg/kg) given i.v. or s.c. delayed the edema formation induced by intraplantar carrageenan injections up to 4 hr after administration, confirming the involvement of kinins in this inflammatory reaction. In guinea pigs treated with atropine, vagal stimulation induced bronchial microvascular leakage. Aerosolization of S 16118 (5 x 10(-3) M for 20 sec), 4 min before vagus nerve stimulation, induced a 60% decrease in the Evans blue extravasation, demonstrating the modulatory role of BK in neurogenic inflammation. In rats, caerulein infusion (4 nmol/kg/hr) induced hypotension, massive pancreatic edema, hypovolemia due to plasma leakage and an increase in serum lipase and amylase activity. S 16118 (100 nmol/kg s.c.) prevented the hypotension, the pancreatic edema and the hypovolemia and induced a marked increase in the serum lipase and amylase activity. This confirms that BK, acting on BK B2 receptors, is involved in this model of pancreatitis. In rabbits, the injection of lipopolysaccharides (LPS; 600 micrograms/kg i.v.) induced hypotension, metabolic acidosis and leukopenia. S 16118 (1.73 mumol/kg i.v.) did not influence the effects of LPS injection. In mice, i.p. LPS (25 mg/kg) administration induced over 90% mortality in 96 hr. S 16118 (1 mg/kg x 4), given 30 min before LPS injection and 4, 8 and 24 hr after LPS injection, did not influence the mortality rate.(ABSTRACT TRUNCATED AT 250 WORDS)

The selective dopamine D3 receptor agonist, 7-OH-DPAT ((+)-7-hydroxy-2-(di-n-propylamino)tetralin) and the novel naphthofurane, S 14297 ((+)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro- naphtho(2,3b)dihydro,2,3-furane]), bound with high affinity and selectivity to recombinant, human dopamine D3 versus D2 receptors stably transfected into Chinese hamster ovary cells: Ki values = 2 versus 103 nM for 7-OH-DPAT and 13 versus 297 nM for S 14297. In contrast, the putative dopamine D3 receptor antagonist, AJ 76 (cis-(+)-5-methoxy-1-methyl-2-(n- propylamino)tetralin), displayed low affinity and selectivity for dopamine D3 versus D2 sites (70 versus 154 nM). 7-OH-DPAT (0.01-0.16 mg/kg s.c.) provoked hypothermia in rats, an action abolished by S 14297 (0.04-0.63 mg/kg s.c.) and, less potently, by AJ 76 (0.16-2.5 mg/kg s.c.). S 14297 (20.0 mg/kg s.c.) did not modify prolactin secretion. These data suggest that dopamine D3 receptors mediate hypothermia in the rat and that S 14297 acts as a selective antagonist at these sites.

Starting from 2-(2-aminoethyl)-6-methoxy-1-methylcarbazole, ethyl 9-methoxy-5-methyl-6H-pyrido[4,3-b]carbazole-1-carboxylate was obtained through a three-step sequence. This compound and its 6-methyl derivative react with (dialkylamino)alkylamines to provide various 9-methoxy-5-methyl-6H-pyrido[4,3-b]carbazole-1-(N-substituted carboxamides) whose boron tribromide demethylation afforded corresponding 9-hydroxy-1-(N-substituted carbamoyl)-olivacines. The same pathway but starting from 2-(2-aminoethyl)-6-methoxy-1,4-dimethylcarbazole led to ethyl 9-methoxy-5,11-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxylate which did not normally react with amines. It provided either the recovered starting material at 120 degrees C or 9-methoxyellipticine resulting from an unexpected decarboethylation in a steel vessel at 180 degrees C. Biological testing of the newly obtained 1-carbamoylolivacine derivatives showed that 9-hydroxylated compounds displayed high cytotoxicity for cultured L1210 and colon 38 cells (IC50 range 5-10 nM) and good antitumor activity in vivo in the P388 leukemia and colon 38 models when administered by the iv route. The most active compound in these series is 9-hydroxy-5,6-dimethyl-1-[N-[2-(dimethylamino)ethyl]carbamoyl]-6H- pyrido[4,3-b]carbazole which was selected for further evaluation on murine solid tumors and for toxicological studies.

1. S 16257 is a new bradycardic agent. Its electropharmacological profile has been compared to that of the known bradycardic compound UL-FS 49 (Zatebradine). Intracellular recordings of action potentials (APs) were performed with conventional glass microelectrodes. 2. In the rabbit isolated sino-atrial node (SAN) tissue, S 16257 and UL-FS 49 (1 microM, 3 microM and 10 microM) were equipotent in slowing spontaneous APs firing predominantly by decreasing the rate of diastolic depolarization (at 3 microM, -23.8 +/- 3.9% and -27.9 +/- 2.6%, respectively). For the two compounds a maximal effect was obtained at 3 microM. In these preparations, action potential duration at 50% of total repolarization (APD50) was more affected by UL-FS 49 than S 16257 at any concentration tested (at 3 microM, +8.9 +/- 2.9% and +29.1 +/- 3.7% for S 16257 and UL-FS 49, respectively; P < or = 0.01). 3. To estimate the direct effects on AP duration, driven cardiac preparations were exposed to these agents. In guinea-pig papillary muscles, paced at a frequency of 1 Hz, increasing concentrations of S 16257 or UL-FS 49 (0.1 to 10 microM, 30 min exposure for each concentration) slightly prolonged AP repolarization. This prolongation was more marked for UL-FS 49 (at 1 microM, +6.1 +/- 0.6% and +11.2 +/- 1.3% elevation of APD50, for S 16257 and UL-FS 49, respectively). 4. Application of UL-FS 49 (3 microM) to rabbit Purkinje fibres, triggered at a frequency of 0.25 Hz, induced a marked prolongation of APD50 and APD90 (+149.4 +/- 51.2% and +86.0 +/- 15.4%, respectively). S 16257 (3 MicroM) induced only a weak prolongation of AP (+ 14.1 +/- 5.0% and + 14.8 +/- 3.3% for APD50 and APD90, respectively) significantly smaller than in the case of UL-FS 49.5. These results show that S 16257 slows the rate of spontaneous AP firing in isolated SAN mainly by a reduction of the diastolic depolarization of the cells, which suggests an inhibition of the pace-maker current (If). S 16257 and UL-FS 49 are equipotent in their bradycardic effect but S 16257 is more specific as it induces less increase in myocardial repolarization time.

We report on the synthesis and pharmacological properties of a new series of thrombin inhibitors derived from hirudin carboxyl-terminal fragments. Two (arylphosphono)phenylalanines, p-PO3H2-L-Phe1 and m-PO3H2-L-Tyr, and one (carboxymethyl)phenylalanine, p-CH2COOH-L-Phe, were prepared and incorporated into position 63 of the modified hirudin's C-terminal dodecapeptide using the Fmoc solid-phase synthesis strategy. Substitution by any one of the residues led to very active analogs which doubled the thrombin time at low micromolar concentration (Ctt2) in vitro (1 microM < Ctt2 < 3 microM) and potently increased the activated partial thromboplastin time (APTT) ex vivo. These compounds displayed a higher potency in vitro and a longer duration of action in vivo than both the corresponding sulfated or phosphorylated tyrosine counterparts.

S9788, a new triazinoaminopiperidine derivative, was 250-fold more active than verapamil in sensitizing DC-3F/AD cells to actinomycin D. This multidrug-resistance modulating activity of S9788 was confirmed on DC-3F/AD, P388/ADR-10, P388/VCR-20, KB/A1, K562/R, S1/tMDR, and COLO320DM cells, with respect to actinomycin D, doxorubicin, vincristine, vinblastine and etoposide. S9788 is 2-255-fold more active than verapamil, depending on the cell line and the cytotoxic agent used, potentiating preferentially the cytotoxicity of the vinca-alkaloid derivatives. S9788 only had a slight effect on vincristine accumulation and retention by parental sensitive cells, as well as on their sensitivity to cytotoxic drugs. S9788 fully restored vincristine accumulation and retention by S1/tMDR cells (S1 cells transfected by the mdr 1 gene) to a level similar to that measured in S1 cells, leading to a blockade in the G2 + M phase of the cell cycle. Therefore, S9788 enhances vincristine activity by restoring its cellular accumulation in resistant cells. After a short incubation period of cells with vincristine (4 h), a condition close to the clinical administration of this cytotoxic agent, a post-incubation for 20 hours more with 5 microM S9788, fully restored the sensitivity of S1/tMDR cells to vincristine. After an exposure of 4 hours at equal concentrations, the accumulation of S9788 was 13-fold that of verapamil in S1/tMDR cells, and 20 hours after the removal of the modulators, S9788 was retained at a concentration 20-fold that of verapamil. S9788 is therefore a powerful new modifier of the P-gp-mediated multidrug-resistance, which by its pharmacological properties (cellular kinetics and activity), might be used in combination with existing chemotherapy against tumors displaying the MDR phenotype.

The role of angiotensin receptor subtypes 1 and 2 was assessed on neointima formation after injury in rat carotid artery. The effects of angiotensin converting enzyme inhibition by perindopril (3 mg.kg-1 x day-1 p.o.) and selective blockade of angiotensin subtype 1 receptors by DuP 753 (5 and 30 mg.kg-1 x day-1 p.o.) were compared on proliferative response to balloon injury. In rats treated 6 days before and for 14 days after injury, perindopril significantly reduced (-76%, p < 0.01) myointimal hyperplasia. In contrast, DuP 753 at 5 mg.kg-1 x day-1 did not modify the hyperplastic response to balloon catheterization. Only at 30 mg.kg-1 x day-1 was DuP 753 able to reduce neointima formation (-47%, p < 0.05). This dose was equipotent to perindopril on the renin-angiotensin system as assessed by the pressor response to angiotensin II and angiotensin I. Therefore, blockade of subtype 1 receptors was a less effective means of suppression of myointimal growth than angiotensin converting enzyme inhibition, suggesting that another angiotensin receptor subtype or converting enzyme substrates are involved in this process. For the determination of whether angiotensin subtype 2 receptors were implicated, the specific subtype 2 receptor antagonist CGP 42112A (1 mg.kg-1 x day-1) was continuously infused perivascularly for 14 days in the vicinity of the injured carotid artery. CGP 42112A was as effective in preventing neointima formation as perindopril (-73%, p < 0.01, versus -76%, p < 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

A series of 70 triazine derivatives have been synthesized and tested for their capacity to modulate multidrug resistance (MDR) in DC-3F/AD and KB-A1 tumor cells in vitro, in comparison with verapamil (VRP), a calcium channel antagonist currently used in therapy as an antihypertensive drug, which also shows MDR modulating activity. Among the 12 selected compounds, 16 (S9788) showed high MDR reversing properties in vitro (300- and 6-fold VRP at 5 microM in DC-3F/AD and KB-A1 cells, respectively) and induced a strong accumulation of adriamycin. The relationship between the increase of ADR accumulation and the fold reversal induced by these compounds and their lack of effects on the sensitive DC-3F cells suggest that they act mainly by inhibiting the P-glycoprotein (Pgp) catalyzed efflux of cytotoxic agents, as already described for a majority of MDR modulators. In vivo, in association with the antitumor drug vincristine (0.25 mg/kg), 16 (100 mg/kg) increased the T/C by 39% in mice bearing the resistant tumor cell line P388/VCR. According to these interesting properties, 16 was selected for a clinical development because it was more bioavailable than 34, even though it was less active.

Tertatolol is a noncardioselective beta-adrenergic blocking agent without partial agonist activity. Its central and renal hemodynamic effects were compared to those of an equipotent dosage of propranolol in two groups of 10 patients each who developed arterial hypertension and a hyperdynamic circulatory state after head injury. After tertatolol, 5 mg orally, mean arterial blood pressure was unaffected, heart rate decreased by 22% (p less than 0.01) and cardiac index by 24% (p less than 0.01) while renal blood flow remained unchanged (-5%; not significant) so that the renal fraction of cardiac index was increased by 22% (p less than 0.05). After propranolol, 160 mg orally, mean arterial blood pressure was not modified, heart rate decreased by 12% (p less than 0.01), cardiac index by 16% (p less than 0.01) and renal blood flow by 17% (p less than 0.01) so that the renal fraction of cardiac index remained unchanged (-3%; not significant). Tertatolol is a potent beta-blocking agent comparable to propranolol apart from the fact that it preserves renal perfusion; this peculiar effect is related to a redistribution of the reduced cardiac output to the benefit of the kidney.

The acute systemic and renal hemodynamic effects of tertatolol, a new noncardioselective beta-blocker without partial agonist activity, were compared to those of an equipotent dose of nadolol in eight patients with essential hypertension. Tertatolol (5 mg) or nadolol (80 mg) were administered orally at an interval of 1 week in a random order as a double-blind, cross-over study. Cardiac output was measured by Doppler echography, and renal blood flow and glomerular filtration rate were measured by constant infusion techniques using 123I-iodohippurate and 51CR-EDTA, respectively. Measurements were performed before and then successively 2 and 4 hours after ingestion of the drugs. Both nadolol and tertatolol decreased blood pressure and cardiac output to a comparable extent. Renal blood flow remained unchanged, so that the renal fraction of cardiac output increased from 14.4 +/- 1.5% to 21.3 +/- 2% after nadolol and from 14.8 +/- 2.4% to 20.5 +/- 1.8% after tertatolol (mean +/- SE, P less than 0.01 before vs. after; nadolol vs. tertatolol was not significant). The glomerular filtration rate remained unchanged, from 68 +/- 9 to 64 +/- 6 mL/min.m2 after nadolol and from 71 +/- 8 to 67 +/- 7 mL/min.m2 after tertatolol (before vs. after and nadolol vs. tertatolol levels were not significant). These results show that both tertatolol and nadolol redistribute cardiac output to the kidneys in patients with essential hypertension.

We compared the central and renal haemodynamic effects of tertatolol, a new non-cardioselective beta-adrenergic blocking drug without partial agonist activity, with those of an equipotent dosage of propranolol in two groups of 10 patients each with acute cerebral injury who had developed systemic hypertension. After tertatolol, 5 mg orally, mean arterial pressure was unchanged, heart rate decreased by 22% (P less than 0.01) and cardiac index by 24% (P less than 0.01), while renal blood flow remained unchanged (-5%, NS). After 160 mg propranolol orally, mean arterial pressure was unchanged, heart rate decreased by 12% (P less than 0.01), cardiac index by 16% (P less than 0.01) and renal blood flow by 17% (P less than 0.01). There was a moderate rise in norepinephrine levels after tertatolol only. Thus in this particular model of acute hypertension, tertatolol acted as a potent beta-blocking agent but differed from propranolol by preserving renal perfusion.