Apolipoprotein M can discriminate HNF1A-MODY from Type 1 diabetes.

AIMS: Missed diagnosis of maturity-onset diabetes of the young (MODY) has led to an interest in biomarkers that enable efficient prioritization of patients for definitive molecular testing. Apolipoprotein M (apoM) was suggested as a biomarker for hepatocyte nuclear factor 1 alpha (HNF1A)-MODY because of its reduced expression in Hnf1a(-/-) mice. However, subsequent human studies examining apoM as a biomarker have yielded conflicting results. We aimed to evaluate apoM as a biomarker for HNF1A-MODY using a highly specific and sensitive ELISA. METHODS: ApoM concentration was measured in subjects with HNF1A-MODY (n = 69), Type 1 diabetes (n = 50), Type 2 diabetes (n = 120) and healthy control subjects (n = 100). The discriminative accuracy of apoM and of the apoM/HDL ratio for diabetes aetiology was evaluated. RESULTS: Mean (standard deviation) serum apoM concentration (µmol/l) was significantly lower for subjects with HNF1A-MODY [0.86 (0.29)], than for those with Type 1 diabetes [1.37 (0.26), P = 3.1 × 10(-18) ) and control subjects [1.34 (0.22), P = 7.2 × 10(-19) ). There was no significant difference in apoM concentration between subjects with HNF1A-MODY and Type 2 diabetes [0.89 (0.28), P = 0.13]. The C-statistic measure of discriminative accuracy for apoM was 0.91 for HNF1A-MODY vs. Type 1 diabetes, indicating high discriminative accuracy. The apoM/HDL ratio was significantly lower in HNF1A-MODY than other study groups. However, this ratio did not perform well in discriminating HNF1A-MODY from either Type 1 diabetes (C-statistic = 0.79) or Type 2 diabetes (C-statistic = 0.68). CONCLUSIONS: We confirm an earlier report that serum apoM levels are lower in HNF1A-MODY than in controls. Serum apoM provides good discrimination between HNF1A-MODY and Type 1 diabetes and warrants further investigation for clinical utility in diabetes diagnostics.

Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7.

BACKGROUND AND PURPOSE: The mGlu(7) receptors are strategically located at the site of vesicle fusion where they modulate the release of the main excitatory and inhibitory neurotransmitters. Consequently, they are implicated in the underlying pathophysiology of CNS diseases such as epilepsy and stress-related psychiatric disorders. Here, we characterized a selective, potent and functional anti-mGlu(7) monoclonal antibody, MAB1/28, that triggers receptor internalization. EXPERIMENTAL APPROACH: MAB1/28's activity was investigated using Western blot and direct immunofluorescence on live cells, in vitro pharmacology by functional cAMP and [(35) S]-GTPγ binding assays, the kinetics of IgG-induced internalization by image analysis, and the activation of the ERK1/2 by elisa. KEY RESULTS: mGlu(7) /mGlu(6) chimeric studies located the MAB1/28 binding site at the extracellular amino-terminus of mGlu(7) . MAB1/28 potently antagonized both orthosteric and allosteric agonist-induced inhibition of cAMP accumulation. The potency of the antagonistic actions was similar to the potency in triggering receptor internalization. The internalization mechanism occurred via a pertussis toxin-insensitive pathway and did not require Gα(i) protein activation. MAB1/28 activated ERK1/2 with potency similar to that for receptor internalization. The requirement of a bivalent receptor binding mode for receptor internalizations suggests that MAB1/28 modulates mGlu(7) dimers. CONCLUSIONS AND IMPLICATIONS: We obtained evidence for an allosteric-biased agonist activity triggered by MAB1/28, which activates a novel IgG-mediated GPCR internalization pathway that is not utilized by small molecule, orthosteric or allosteric agonists. Thus, MAB1/28 provides an invaluable biological tool for probing mGlu(7) function and selective activation of its intracellular trafficking.

Multimodal imaging of pancreatic beta cells in vivo by targeting transmembrane protein 27 (TMEM27).

AIMS/HYPOTHESIS: Non-invasive diagnostic tools specific for pancreatic beta cells will have a profound impact on our understanding of the pathophysiology of metabolic diseases such as diabetes. The objective of this study was to use molecular imaging probes specifically targeting beta cells on human samples and animal models using state-of-the-art imaging modalities (fluorescence and PET) with preclinical and clinical perspective. METHODS: We generated a monoclonal antibody, 8/9-mAb, targeting transmembrane protein 27 (TMEM27; a surface N-glycoprotein that is highly expressed on beta cells), compared its expression in human and mouse pancreas, and demonstrated beta cell-specific binding in both. In vivo imaging was performed in mice with subcutaneous insulinomas overexpressing the human TMEM27 gene, or transgenic mice with beta cell-specific hTMEM27 expression under the control of rat insulin promoter (RIP-hTMEM27-tg), using fluorescence and radioactively labelled antibody, followed by tissue ex vivo analysis and fluorescence microscopy. RESULTS: Fluorescently labelled 8/9-mAb showed beta cell-specific staining on human and mouse pancreatic sections. Real-time PCR on islet cDNA indicated about tenfold higher expression of hTMEM27 in RIP-hTMEM27-tg mice than in humans. In vivo fluorescence and PET imaging in nude mice with insulinoma xenografts expressing hTMEM27 showed high 8/9-mAb uptake in tumours after 72 h. Antibody homing was also observed in beta cells of RIP-hTMEM27-tg mice by in vivo fluorescence imaging. Ex vivo analysis of intact pancreas and fluorescence microscopy in beta cells confirmed these findings. CONCLUSIONS/INTERPRETATION: hTMEM27 constitutes an attractive target for in vivo visualisation of pancreatic beta cells. Studies in mouse insulinoma models and mice expressing hTMEM27 demonstrate the feasibility of beta cell-targeted in vivo imaging, which is attractive for preclinical investigations and holds potential in clinical diagnostics.

Immobilized amyloid precursor protein constructs: a tool for the in vitro screening of glial cell reactivity.

Astrocytes and microglia are closely associated with amyloid plaques in Alzheimer's disease (AD). Microglia constitute the first barrier surrounding plaques, although they seem to be unable to remove them efficiently. We evaluated the reaction of microglial cells from neonatal rats and mice to plaque mimetics. The C-terminal part of the amyloid precursor protein (APP) or amyloid peptide (A beta) was immobilized to either 60-microm or 2.8-microm beads and incubated with microglial cells. Beads of 60 microm, having approximately the size of senile plaques, were not phagocytosed, in contrast to 2.8-microm beads, which were phagocytosed by microglia but not by astrocytes. Once taken up by the cells, proteins immobilized to the beads were degraded rapidly, as confirmed by mass spectrometry and immunofluorescence with an antibody against beta-amyloid. On the other hand, no protein degradation was observed with 60-microm beads. Also, probably as a reaction to its incapability to phagocytose the beads, glia organized around the beads and started to proliferate. Cell proliferation was more pronounced when the beads contained the A beta epitope compared with the beads with an inert surface. This in vitro effect could be exploited to set up a screening assay for compounds that ameliorate the adverse reaction of microglia supposed to contribute to the pathogenesis of AD.

Short report: floxacrine analog WR 243251 inhibits hematin polymerization.

Floxacrine was a promising antimalarial compound that led to the identification of WR 243251. On the basis of their structures, we suspected that these compounds might be good inhibitors of hematin polymerization. Indeed, WR 243251 was as potent and floxacrine was only 2-fold less potent than chloroquine as inhibitors of this process. However, this hematin polymerization inhibition did not completely account for the increased antimalarial potency of WR 243251 versus chloroquine. The WR 243251 ketone hydrolysis product WR 243246 was without activity against hematin polymerization. These data also confirm that hematin polymerization inhibition can be quite sensitive to small changes in inhibitor structure.

Rhoptry-associated protein 1-binding monoclonal antibody raised against a heterologous peptide sequence inhibits Plasmodium falciparum growth in vitro.

Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-1(23-711)) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y218KYSL222 as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus appears to be associated with conformational rather than sequence homology. While the anti-35.1 MAb SP8.18 exhibited parasite growth-inhibitory activity, none of the tested anti-rRAP-1(23-711) MAbs inhibited parasite growth, independently of their fine specificity for the RAP-1 sequences at positions 33 to 42, 213 to 222, 243 to 247, 280 to 287, or 405 to 446. The growth-inhibitory activity of MAb SP8.18 was, however, accelerated by noninhibitory anti-RAP-1 MAbs. Results demonstrate that in addition to fine specificity, other binding parameters are also crucial for the inhibitory potential of an antibody.

Artemether administered together with haemin damages schistosomes in vitro.

We conducted experiments in vitro to assess the effect of artemether in combination with haemin on adult Schistosoma japonicum, S. mansoni and S. haematobium. When schistosomes were maintained in a medium containing artemether at concentrations of 20 micrograms/mL or less for 72 h, no apparent effect on the schistosomes was seen. When the medium contained 50 or 100 micrograms/mL haemin as well as artemether, the schistosomes showed decreased motor activity 2-24 h after exposure, which was followed by the staining of the whole worm body a reddish-yellow colour, dilatation of the intestine, and extensive vesiculation of the tegument. Some of the schistosomes died 24 h after exposure, and almost all died within 48-72 h. When schistosomes were exposed to the same concentrations of haemin alone, they were stained a light yellow colour but there was no apparent effect on their survival. Our findings suggest that artemether interacts with haemin to exert a toxic effect on the worms, which might be of importance in the further elucidation of the mechanism of action of artemether on schistosomes.

Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores.

Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of (14)C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100, 000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program.

The synthetic, oxidized C-terminal fragment of the Plasmodium berghei circumsporozoite protein elicits a high protective response.

A polypeptide of 69 amino acids (PbCS 242-310) encompassing the C-terminal region of the circumsporozoite protein of Plasmodium berghei (PbCS) was generated using solid-phase peptide synthesis. The immunological and protective properties of peptide PbCS 242-310 were studied in BALB/c mice (H-2d). Two subcutaneous injections, in the presence of IFA at the base of the tail, generated (i) high titers of anti-peptide antibodies which also recognized the native P. berghei CS protein, (ii) cytolytic T cells specific for the Kd-restricted peptide PbCS 245-253 and (iii) partial CD8+-dependent protection against sporozoite-induced malaria. The same frequencies of peptide PbCS 245-253 specific CD8+ T cells were found by IFN-gamma ELISPOT in the draining lymph nodes of animals immunized with the short optimal CTL peptide 245-253 or with the polypeptide 242-310, indicating that the longer polypeptide can be processed and presented in vivo in the context of MHC class I as efficiently as the short CTL peptide. Interestingly, higher levels of IFN-gamma producing CD8 T cells and protection were observed when the four cysteine residues present in the C-terminal peptide were fully oxidized. These findings underline the potential importance of the chemical nature of the C-terminal fragment on the activation of the immune system and concomitant protection.

Phenyl beta-methoxyacrylates: a new antimalarial pharmacophore.

Phenyl beta-methoxyacrylates, linked to an aromatic ring via an olefinic bridge, have been identified as novel, potentially inexpensive, antimalarial agents. The compounds are believed to exert their activity by inhibition of mitochondrial electron transport at the cytochrome bc(1) complex. A series of compounds have been synthesized to define structure-activity relationships affecting antimalarial activity. It was found that the beta-methoxyacrylate was required ortho to the linker and the optimal bridge was (E,E)-butadiene. Compounds in which the second aromatic ring was ortho-substituted or ortho,para-disubstituted gave optimal potency. Several compounds were identified with potency that is superior to that of chloroquine both in culture and in a murine malaria model.

Sequence diversity of the merozoite surface protein 1 of Plasmodium falciparum in clinical isolates from the Kilombero District, Tanzania.

Merozoite surface protein 1 of Plasmodium falciparum (PfMSP-1) is regarded as a key candidate antigen for malaria vaccine development. It exhibits significant antigenic polymorphism and has been divided into 17 building blocks based on the analysis of sequence diversity. Differences in the antigenic composition of PfMSP-1 in local P. falciparum populations may result in differences in the efficacy of vaccines, which contain sequences of particular allelic variant(s) of PfMSP-1. To contribute to the required knowledge of genetic diversity of malaria parasites in geographically diverse regions, we have used the polymerase chain reaction (PCR) to analyze the sequence diversity of blocks 1-4 of PfMSP-1 in disease isolates from the Kilombero District in Tanzania. In the semi-conserved block 1, in which dimorphic amino acid variances have been described at three positions, we found three of the five previously described combinations of these three pairs of amino acids. In addition one combination was found, which has not been reported before in parasite isolates from different locations worldwide. Of the two sequence variants, which were dominating, one (S44-Q47-V52) corresponded to the 83.1 sequence incorporated into the SPf66 malaria peptide vaccine, while the other one (G44-H47-I52) differed from the previous in all three dimorphic amino acids. The partial protection observed in a phase III SPf66 trial conducted in the Kilombero District in children aged 1-5, thus does not seem to be associated with a clear dominance of favourable variants of block 1 of PfMSP-1 in this area. All three different principle types of block 2, the major polymorphic region of PfMSP-1, were found in the Tanzanian isolates. Most of the sequences contained K1-type tripeptide repeats, but clones with MAD20-type repeats or no repetitive sequence (RO33-type block 2) were also present. K1- and MAD20-type tripeptide repeat motifs were never mixed within one parasite clone. In one sequence a hexapeptide repeat was found at the end of block 2, which has not been reported before. Dimorphism in 13 of the 17 previously described variable positions of the semi-conserved block 3 and three of four recombination types of block 4 (K/K, M/K and M/M) were found among the Tanzanian isolates. Apart from previously described dimorphic amino acid positions, polymorphism was rare in the non-repeated building blocks. Selection and spreading of parasite variants, which contain amino acid exchanges at other than the dimorphic positions thus, is not a common event. Parasite isolates frequently harboured more than one PfMSP-1 allele. Three of the four heterogeneous isolates analysed contained two different general types of sequences. One isolate contained at least four distinct clones, demonstrating the high endemicity of malaria in the Kilombero District, which is a well-established site for malaria vaccine field trials.

Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth.

Considerable data now support the hypothesis that chloroquine (CQ)-hematin binding in the parasite food vacuole leads to inhibition of hematin polymerization and parasite death by hematin poisoning. To better understand the structural specificity of CQ-hematin binding, 13 CQ analogues were chosen and their hematin binding affinity, inhibition of hematin polymerization, and inhibition of parasite growth were measured. As determined by isothermal titration calorimetry (ITC), the stoichiometry data and exothermic binding enthalpies indicated that, like CQ, these analogues bind to two or more hematin mu-oxo dimers in a cofacial pi-pi sandwich-type complex. Association constants (K(a)'s) ranged from 0.46 to 2.9 x 10(5) M(-1) compared to 4.0 x 10(5) M(-1) for CQ. Remarkably, we were not able to measure any significant interaction between hematin mu-oxo dimer and 11, the 6-chloro analogue of CQ. This result indicates that the 7-chloro substituent in CQ is a critical structural determinant in its binding affinity to hematin mu-oxo dimer. Molecular modeling experiments reinforce the view that the enthalpically favorable pi-pi interaction observed in the CQ-hematin mu-oxo dimer complex derives from a favorable alignment of the out-of-plane pi-electron density in CQ and hematin mu-oxo dimer at the points of intermolecular contact. For 4-aminoquinolines related to CQ, our data suggest that electron-withdrawing functional groups at the 7-position of the quinoline ring are required for activity against both hematin polymerization and parasite growth and that chlorine substitution at position 7 is optimal. Our results also confirm that the CQ diaminoalkyl side chain, especially the aliphatic tertiary nitrogen atom, is an important structural determinant in CQ drug resistance. For CQ analogues 1-13, the lack of correlation between K(a) and hematin polymerization IC(50) values suggests that other properties of the CQ-hematin mu-oxo dimer complex, rather than its association constant alone, play a role in the inhibition of hematin polymerization. However, there was a modest correlation between inhibition of hematin polymerization and inhibition of parasite growth when hematin polymerization IC(50) values were normalized for hematin mu-oxo dimer binding affinities, adding further evidence that antimalarial 4-aminoquinolines act by this mechanism.

Synthesis and antimalarial activity of 11 dispiro-1,2,4,5-tetraoxane analogues of WR 148999. 7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadecanes substituted at the 1 and 10 positions with unsaturated and polar functional groups.

Eleven novel dispiro-1,2,4,5-tetraoxanes 3 bearing unsaturated and polar functional groups were designed to enhance the oral antimalarial activity of the prototype tetraoxane 2 (WR 148999). With the exception of 3g and 3h, tetraoxanes 3 were available via the peroxidation of corresponding cyclohexanone derivatives in H2SO4/CH3CN. Tetraoxanes 3g and 3h were prepared by hydrolysis of ester tetraoxanes 3e and 3i, respectively. Five of the 11 tetraoxanes were inactive, but six tetraoxanes had IC50 values of 6-26 nM against the K1 and NF54 strains of Plasmodium falciparum compared to corresponding IC50 values of 28 and 39 nM for 2, and 10 and 12 nM for artemisinin (1). Ester tetraoxane 3e was the most active in vitro, some 2-fold more potent than 1. However, none of the six tetraoxanes active in vitro were as effective as either 1 or 2 in vivo; at single doses of 100 mg/kg most possessed little to no vivo activity in mice infected with Plasmodium berghei. Unsaturated tetraoxane 3a was uniquely more active when administered per os (po) than subcutan (sc). For this series of tetraoxanes, the discrepancy between vitro and vivo activities underscores the limitations of conclusions drawn solely from in vitro antimalarial data and illustrates a practical benefit of complementary single-dose in vivo antimalarial screens.

A common mechanism for blockade of heme polymerization by antimalarial quinolines.

The antimalarial quinolines are believed to work by blocking the polymerization of toxic heme released during hemoglobin proteolysis in intraerythrocytic Plasmodium falciparum. In the presence of free heme, chloroquine and quinidine associate with the heme polymer. We have proposed that this association of the quinoline-heme complex with polymer caps the growing heme polymer, preventing further sequestration of additional heme that then accumulates to levels that kill the parasite. In this work results of binding assays demonstrate that the association of quinoline-heme complex with heme polymer is specific, saturable, and high affinity and that diverse quinoline analogs can compete for binding. The relative quinoline binding affinity for heme polymer rather than free heme correlates with disruption of heme polymerization. Mefloquine, another important antimalarial quinoline, associated with polymer in a similar fashion, both in cultured parasites and in the test tube. In parasite culture, blocking heme release with protease inhibitor was antagonistic to mefloquine action, as it is to chloroquine action. These data suggest a common mechanism for quinoline antimalarial action dependent on drug interaction with both heme and heme polymer.

An assessment of drug-haematin binding as a mechanism for inhibition of haematin polymerisation by quinoline antimalarials.

Chloroquine is thought to exert its antimalarial activity by preventing the polymerisation of toxic haematin released during proteolysis of haemoglobin in the Plasmodium digestive vacuole. However, the molecular mechanisms by which this inhibition occurs and the universality of this mechanism for other quinoline antimalarials remain to be established. We demonstrate here a correlation for eight antimalarial quinolines between inhibition of haematin polymerisation in vitro and inhibition of P. falciparum growth in culture, confirming haematin polymerisation as the likely target of quinoline blood schizonticides. Furthermore, using isothermal titration microcalorimetry, a correlation was observed between the haematin binding constant of these compounds and their ability to inhibit haematin polymerisation, suggesting that these compounds mediate their activity through binding to haematin. It was also observed that the compounds bind primarily to the mu-oxo dimer form of haematin rather than the monomeric form. It is postulated that this binding inhibits haematin polymerisation by shifting the haematin dimerisation equilibrium to the mu-oxo dimer, thus reducing the availability of monomeric haematin for incorporation into haemozoin. These data reconcile the haematin polymerisation theory with the Fitch hypothesis, which states that chloroquine mediates its activity through binding to haematin.

A comparison and analysis of several ways to promote haematin (haem) polymerisation and an assessment of its initiation in vitro.

We compared several methods for producing haematin polymerisation at physiological temperatures (i.e., 37 degrees) and found that a trophozoite lysate-mediated reaction was inappropriate for measuring compound inhibition of haematin polymerisation. Using this method, we obtained significantly higher IC50 values (concentration inhibiting haematin polymerisation by 50%) for certain compounds than when other methods were used, including a food vacuole lysate-mediated reaction. This difference was probably due to the binding of these compounds to cytosolic parasite proteins, as proteinase K treatment of the trophozoite lysate reversed this effect. The initiation of haematin polymerisation was also investigated using several assays. It was found that haematin polymerisation occurred spontaneously, in the absence of preformed haemozoin, over a period of several days, but that the process was more rapid when an acetonitrile extract of malarial trophozoites was added. This extract contained no detectable protein, and its activity could be replicated using an extract from uninfected erythrocytes and by using lipids. We therefore postulate that no protein or parasite-specific material is absolutely required for the initiation of haematin polymerisation. The formation of beta-haematin de novo using the acetonitrile extract is more pH-dependent than the generation of newly synthesised beta-haematin from preformed haemozoin and cannot proceed much above pH = 6. We postulate that the initiation of haematin polymerisation is more sensitive to the equilibrium of haematin between its monomeric and mu-oxo dimer form and requires a higher concentration of monomer than for the elongation phase of polymerisation.

Elimination of P. berghei liver stages is independent of Fas (CD95/Apo-I) or perforin-mediated cytotoxicity.

Immunization of mammals with irradiated malaria sporozoites protects from a subsequent contact with the parasite. Protective immunity is directed against the pre-erythrocytic stages of the parasite, sporozoites and liver stages. Specific antibodies neutralize part of the infectious sporozoites infected by the mosquito vector, while liver stages are the target of a cellular immune response which is mediated by T cells. In this study, we evaluated the T-cell dependent protection induced by the infection of P. berghei irradiated sporozoites and the contribution of perforin and of the receptor/ligand system CD95/CD95L, two T cell-dependent mechanisms known to mediate elimination of target cells. Wild type, perforin deficient, CD95 mutant, CD95L mutant and perforin deficient/CD95L mutant mice were immunized with P. berghei irradiated sporozoites and submitted to a challenge with infectious sporozoites. All mice immunized with P. berghei irradiated sporozoites were protected against a sporozoite challenge, including perforin deficient/CD95L mutant animals. These results indicate that T cells do not kill malaria-infected hepatocytes via one of the known pathways, but rather that activated parasite-specific T cells produce cytokines which activate in cascade other mechanisms responsible for the intracellular elimination of the parasite.

Antimalarial activity of the bisquinoline trans-N1,N2-bis (7-chloroquinolin-4-yl)cyclohexane-1,2-diamine: comparison of two stereoisomers and detailed evaluation of the S,S enantiomer, Ro 47-7737.

The S,S enantiomer of the bisquinoline trans-N1,N2-bis(7-chloroquinolin-4-yl)cyclohexane-1,2-diamine, Ro 47-7737, is significantly more potent against chloroquine-resistant Plasmodium falciparum than the R,R enantiomer and the previously described racemate. Both the enantiomers and the racemate are more potent inhibitors of heme polymerization than chloroquine, and their activities are probably mediated by inhibition of this parasite-specific process. The S,S enantiomer, Ro 47-7737, was studied in more detail and proved to be a potent antimalarial in the treatment of P. vivax ex vivo and P. berghei in vivo. Its suppression of P. berghei growth in a mouse model (50% effective dose, 2.3 mg/kg of body weight) was equal to that of chloroquine and mefloquine, and Ro 47-7737 was found to be more potent than these two drugs in the Rane test, in which the curative effect of a single dose is monitored. The dose at which 50% of animals were permanently cured (34 mg/kg) was markedly superior to those of chloroquine (285 mg/kg) and mefloquine (> 250 mg/kg). When administered orally at 50 mg/kg, Ro 47-7737 also showed a faster clearance of parasites than either chloroquine or mefloquine, and unlike the other two compounds, Ro 47-7737 showed no recrudescence. In a study to compare prophylactic efficacies of oral doses of 50 mg/kg, Ro 47-7737 provided protection for 14 days compared to 3 days for mefloquine and 1 day for chloroquine. The good curative and prophylactic properties of the compound can be explained in part by its long terminal half-life. The ability to generate parasite resistance to Ro 47-7737 was also assessed. With a rodent model, resistance could be generated over eight passages. This rate of resistance generation is comparable to that of mefloquine, which has proved to be an effective antimalarial for many years. Toxicity liabilities, however, ruled out this compound as a candidate for drug development.