Design and optimization of selective azaindole amide M1 positive allosteric modulators.

Selective activation of the M1 receptor via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. A novel series of azaindole amides and their key pharmacophore elements are described. The nitrogen of the azaindole core is a key design element as it forms an intramolecular hydrogen bond with the amide N-H thus reinforcing the bioactive conformation predicted by published SAR and our homology model. Representative compound 25 is a potent and selective M1 PAM that has well aligned physicochemical properties, adequate brain penetration and pharmacokinetic (PK) properties, and is active in vivo. These favorable properties indicate that this series possesses suitable qualities for further development and studies.

Pyrolysis of solid waste residues from Lemon Myrtle essential oils extraction for bio-oil production.

Solid waste residues from the extraction of essential oils are projected to increase and need to be treated appropriately. Valorization of waste via pyrolysis can generate value-added products, such as chemicals and energy. The characterization of lemon myrtle residues (LMR) highlights their suitability for pyrolysis, with high volatile matter and low ash content. Thermogravimetric analysis/derivative thermogravimetric revealed the maximum pyrolytic degradation of LMR at 335 °C. The pyrolysis of LMR for bio-oil production was conducted in a fixed-bed reactor within a temperature range of 350-550 °C. Gas chromatography-mass spectrometry showed that the bio-oil contained abundant amounts of acetic acid, phenol, 3-methyl-1,2-cyclopentanedione, 1,2-benzenediol, guaiacol, 2-furanmethanol, and methyl dodecanoate. An increase in pyrolysis temperature led to a decrease in organic acid and ketones from 18.09% to 8.95% and 11.99% to 8.75%, respectively. In contrast, guaiacols and anhydrosugars increased from 24.23% to 30.05% and from 3.57% to 7.98%, respectively.

Global Portrait of Protein Targets of Metabolites of the Neurotoxic Compound BIA 10-2474.

Clinical investigation of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 resulted in serious adverse neurological events. Structurally unrelated FAAH inhibitors tested in humans have not presented safety concerns, suggesting that BIA 10-2474 has off-target activities. A recent activity-based protein profiling (ABPP) study revealed that BIA 10-2474 and one of its major metabolites inhibit multiple members of the serine hydrolase class to which FAAH belongs. Here, we extend these studies by performing a proteome-wide analysis of covalent targets of BIA 10-2474 metabolites. Using alkynylated probes for click chemistry-ABPP in human cells, we show that des-methylated metabolites of BIA 10-2474 covalently modify the conserved catalytic cysteine in aldehyde dehydrogenases, including ALDH2, which has been implicated in protecting the brain from oxidative stress-related damage. These findings indicate that BIA 10-2474 and its metabolites have the potential to inhibit multiple mechanistically distinct enzyme classes involved in nervous system function.

Identification and Development of an Irreversible Monoacylglycerol Lipase (MAGL) Positron Emission Tomography (PET) Radioligand with High Specificity.

Monoacylglycerol lipase (MAGL), a serine hydrolase extensively expressed throughout the brain, serves as a key gatekeeper regulating the tone of endocannabinoid signaling. Preclinically, inhibition of MAGL is known to provide therapeutic benefits for a number of neurological disorders. The availability of a MAGL-specific positron emission tomography (PET) ligand would considerably facilitate the development and clinical characterization of MAGL inhibitors via noninvasive and quantitative PET imaging. Herein, we report the identification of the potent and selective irreversible MAGL inhibitor 7 (PF-06809247) as a suitable radioligand lead, which upon radiolabeling was found to exhibit a high level of MAGL specificity; this enabled cross-species measurement of MAGL brain expression (Bmax), assessment of in vivo binding in the rat, and nonhuman primate PET imaging.

Discovery of Trifluoromethyl Glycol Carbamates as Potent and Selective Covalent Monoacylglycerol Lipase (MAGL) Inhibitors for Treatment of Neuroinflammation.

Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log  D, improving solubility, and minimizing chemical lability. Compound 15 achieves excellent CNS exposure, extended 2-AG elevation effect in vivo, and decreased brain inflammatory markers in response to an inflammatory challenge.

Discovery of the Potent and Selective M1 PAM-Agonist N-[(3R,4S)-3-Hydroxytetrahydro-2H-pyran-4-yl]-5-methyl-4-[4-(1,3-thiazol-4-yl)benzyl]pyridine-2-carboxamide (PF-06767832): Evaluation of Efficacy and Cholinergic Side Effects.

It is hypothesized that selective muscarinic M1 subtype activation could be a strategy to provide cognitive benefits to schizophrenia and Alzheimer's disease patients while minimizing the cholinergic side effects observed with nonselective muscarinic orthosteric agonists. Selective activation of M1 with a positive allosteric modulator (PAM) has emerged as a new approach to achieve selective M1 activation. This manuscript describes the development of a series of M1-selective pyridone and pyridine amides and their key pharmacophores. Compound 38 (PF-06767832) is a high quality M1 selective PAM that has well-aligned physicochemical properties, good brain penetration and pharmacokinetic properties. Extensive safety profiling suggested that despite being devoid of mAChR M2/M3 subtype activity, compound 38 still carries gastrointestinal and cardiovascular side effects. These data provide strong evidence that M1 activation contributes to the cholinergic liabilities that were previously attributed to activation of the M2 and M3 receptors.

Preferential rearrangement of developmentally regulated immunoglobulin VH1 genes in human B-lineage leukaemias.

Immunoglobulin heavy chain (IgH) variable region (VH) genes are rearranged and expressed in a programmed manner during B-cell development. In common with foetal/pre-immune B-cells, malignant B-lymphoid populations preferentially use a restricted repertoire of developmentally regulated VH genes. By nucleotide sequence analysis of polymerase chain reaction amplified IgH genes, we have compared the repertoire of VH1 family genes that are rearranged in mature, CD5+ B chronic lymphocytic leukaemia (CLL) with that in immature, CD5-B-lineage acute lymphoblastic leukaemia (ALL). The results revealed a non-random pattern pf VH1 usage in which no single VH1 family member was common to each of these disease groups. The VH1 gene, 51P1, which underlies an auto-antibody associated cross-reactive idiotype, 'G6', frequently expressed on foetal B-cells, was preferentially rearranged in CLL (three of nine rearranged alleles). Another developmentally regulated VH1 gene, 20P3, accounted for more than half of the VH1 specific IgH gene rearrangements in ALL (five of nine VH1 alleles). Such developmentally restricted VH1 genes may distinguish discrete, although not necessarily exclusive, stages or compartments in B-lymphopoiesis from which each of these disease types arise.

Immunoglobulin heavy chain gene fingerprinting reveals widespread oligoclonality in B-lineage acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) of B-cell lineage typically arises as a monoclonal expansion of committed B-lymphocyte precursors that are arrested at an immature stage of differentiation. From Southern hybridization analysis of immunoglobulin heavy chain (IgH) genes in leukaemic blasts, the occurrence of a sizeable minority of patients displaying multiple (greater than two) rearranged heavy chain alleles has been widely reported. In at least some patients these data are consistent with the presence of oligoclonal populations of precursor B-cells. We have used a more sensitive, polymerase chain reaction based immunoglobulin gene 'fingerprinting' approach in the analysis of B-cell clonality in eight patients with common ALL which were apparently monoclonal on the basis of Southern blot analysis of their IgH genes. The results revealed an oligoclonal pattern of IgH gene rearrangement in half of the patients analysed, implying that oligoclonality at the level of B-cell commitment, as defined by IgH gene rearrangement, is much more widespread in this disease than has previously been recognized.

An improved method for detection of B-lymphoid clonality by polymerase chain reaction.

Several groups have recently described methods for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements in B-cell malignancies by polymerase chain reaction (PCR) gene amplification using variable region-(VH) and joining (JH) region-specific primers. The simplest methods utilize a single VH primer specific for sequences present in most VH regions corresponding to the third framework region (FR3). An alternative approach is to use a panel of VH family-specific primers specific for the first framework regions (FR1). In the course of nucleotide sequence analysis of IgH gene rearrangements amplified using a VH FR1 primer panel, these authors previously observed 3' VH region deletion and/or base mis-matches sufficient to prevent efficient priming from the VH FR3 primer target sequence in a significant minority of cases of B-lineage malignancy. An improved PCR method has therefore been developed by using a panel of seven VH FR1 family-specific primers incorporated in a single reaction. By using this method clonal IgH gene rearrangement is detected in 15 of 16 cases of B-lineage malignancy. Significantly, this series included four cases of B-lymphoma in which previous attempts to detect PCR clonal IgH gene rearrangements using a VH FR3 primer were unsuccessful. In two of these cases, nucleotide sequence analysis of the amplified DNA showed that failure to prime with the VH FR3 primer was likely to be attributable to insufficient homology with the target sequence. The use of the approach described in this paper should significantly improve the reliability of detection of B-lymphoid clonality by PCR.

A convenient multipurpose mouse restrainer.

A convenient, inexpensive, easily constructed mouse restrainer is described and illustrated. The restrainer has the advantage over other models that while the animal is effectively immobilized, time-consuming and potentially injurious binding of extremities is avoided, and selected areas such as the back, extremities and tail remain accessible for manipulations.

Effect of exercise and cavity size on right ventricular function in morbid obesity.

To assess the effect of exercise and to determine the influence of the right ventricular (RV) internal dimension on RV systolic function in morbid obesity, M-mode and 2-dimensional echocardiography and radionuclide ventriculography were performed on 22 patients whose body weight was at least twice the ideal body weight and who had no clinical or laboratory evidence of underlying organic heart disease or pulmonary disease. RV ejection fraction was measured at rest and during peak supine bicycle exercise. RV exercise response was defined as the change in RV ejection fraction during peak exercise. There was a significant negative correlation between percent over ideal body weight and RV exercise response (r = 0.86, p less than 0.00005) and between RV internal dimension and RV exercise response (r = 0.60, p less than 0.005). There were significant positive correlations between resting RV and left ventricular (LV) ejection fraction (r = 0.56, p less than 0.01) and between RV and LV exercise response (r = 0.70, p less than 0.0005). The subgroup with a high-normal or enlarged RV internal dimension (greater than or equal to 2.0 cm, n = 10) experienced no significant change in RV ejection fraction with exercise, whereas the subgroup whose RV internal dimension was less than 2.0 (n = 12) experienced a significant increase in RV ejection fraction from 44 +/- 10% at rest to 58 +/- 11% at peak exercise (p less than 0.03). The results suggest that in morbidly obese individuals without underlying cardiopulmonary disease RV dilatation may predispose to RV systolic dysfunction and assessment of RV systolic function should optimally include evaluation of RV exercise response.

Independent clonal origin of T- and B-cell clones in a composite lymphoma.

We present a detailed immunohistological and genotypic analysis of an unusual case in which a peripheral T-cell lymphoma, with features of Lennert's and angioimmunoblastic lymphoma, occurred after treatment of a low grade plasmacytoid lymphoma. By analysis of immunoglobulin and T-cell receptor genes, we show that the two diseases had an independent clonal origin at the level of lymphoid commitment. However, by employing a novel polymerase chain reaction-based technique for analysis of B-cell clonality, we show the persistence of a residual minor clonal B-cell population in the subsequent T-cell lymphoma. Only 2 previous cases of composite lymphoma involving B- and T-cell clones have been demonstrated by molecular analysis. This study underlines the immunophenotypic and genotypic heterogeneity of peripheral T-cell lymphomas and illustrates an unusual disease course in which a T-cell lymphoma has arisen in the context of, and perhaps as a consequence of, a B-cell lymphoma.

Acute skin GVHD following syngeneic BMT for CLL.

A 41-year-old woman received a syngeneic BMT for CLL and subsequently developed acute skin GVHD. Transfusion-related allogeneic GVHD was excluded on the basis of an unchanged HLA type in circulating lymphocytes. Short tandem repeat PCR was used to confirm syngeneicity between donor and recipient. The patient had a personal and family history of autoimmune disease which may have made her particularly susceptible to development of syngeneic GVHD. The distinction between allogeneic and syngeneic or autologous GVHD is important because of therapeutic implications.

The in vitro detection of anti-leukaemia-specific cytotoxicity after autologous bone marrow transplantation for acute leukaemia.

Anti-leukaemia activity after allogeneic bone marrow transplantation has been studied extensively but its antigen specificity and effector cell phenotype remain unknown. Here we report a study in three recipients of autologous bone marrow transplantation done as part of the treatment for acute leukaemia, in whom we were able to detect innate specific anti-leukaemia activity post-transplant. One patient maintained selective cell-mediated cytolytic activity against her autologous leukaemic cells in the absence of cytolysis of her normal bone marrow mononuclear cells (BMMC). She remains in complete remission 3 years after ABMT for acute myeloid leukaemia (M5). A second patient was transplanted for acute lymphoblastic leukaemia and had detectable anti-leukaemia activity up to 20 weeks post-ABMT. At this point anti-leukaemia activity could no longer be demonstrated and the patient suffered a relapse 2 weeks later. A third patient was transplanted for AML (M4 Eo) and lacked detectable leukaemia-specific immune reactivity at 1, 3 and 6 months post-ABMT. She relapsed 6 months after her ABMT and returned to complete remission after further chemotherapy. She commenced treatment with alpha interferon and regained NK function. Furthermore, she developed high level cytolytic activity against her autologous leukaemic cells in the absence of activity against her remission bone marrow samples. She remains in complete remission 17 months after her initial relapse. This is the first report of an apparent association between in vitro leukaemia-specific cytolytic activity in individual patients after ABMT and clinical outcome. It encourages the theory that autologous immunomodulation may be useful in the future treatment of leukaemia.

FLAG-idarubicin and allogeneic stem cell transplantation for Ph-positive ALL beyond first remission.

We describe a single centre experience of eight consecutive patients with relapsed or refractory Ph+ ALL treated with the FLAG/idarubicin regimen followed by BMT or PBSCT. Following FLAG/idarubicin, one achieved a partial response and seven CR. All patients subsequently received allogeneic transplants: one sibling BMT, three matched unrelated (MUD) BMT and four sibling PBSCT. Two patients received second transplants with PBSC from their original BM donors following FLA/Ida with no further conditioning. Three patients are alive in CR 9, 24 and 32 months after transplant. Seven of eight patients had a cytogenetic response following FLAG/Ida induction and one of seven became bcr-abl negative. All eight patients had a complete cytogenetic response following transplant. Four of five assessable patients became p190 bcr-abl negative after transplant; three of these subsequently relapsed. Both patients with the p210 bcr-abl transcript remained bcr-abl positive in CR after transplant. FLAG/Ida was well tolerated and appears to be effective in inducing remission in relapsed Ph+ ALL. The use of FDR-containing chemotherapy without further conditioning prior to PBSCT deserves further study in heavily pre-treated patients and, in patients with relapsed ALL following BMT, may be a safer option than DLI (donor lymphocyte infusion) by avoiding the associated risk of aplasia.

Observations on the haemopoietic response to critical illness.

Peripheral blood cytopenias are common in patients receiving intensive care, particularly in those with multiple organ failure. To assess the contribution of bone marrow hypoplasia in such patients 44 bone marrow samples from 24 patients under intensive care were studied by standard morphological techniques and by the granulocyte-macrophage colony forming cell (GM-CFC) assay. Frequently observed morphological abnormalities in the bone marrow included the following: (i) a reduction in overall cellularity in seven patients, with a progressive decrease in most patients studied sequentially; (ii) an increase in the number of actively phagocytic macrophages; and (iii) a disruption of normal bone marrow architecture with the accumulation of intercellular hyaluronic acid glycosaminoglycan. Mean GM-CFC growth was significantly reduced when compared with that in a group of normal controls. In four of five patients studied sequentially GM-CFC growth became subnormal in association with a reduction in bone marrow cellularity. Inhibitory serum factors were not identified. These morphological abnormalities are similar to the changes observed in gelatinous degeneration of the bone marrow. In both situations disruption of the haemopoietic microenvironment, with the accumulation of hyaluronic acid proteoglycan, may be an important factor in the inhibition of haemopoietic progenitor cell growth. The proliferation of macrophages, by the release of a variety of cytokines or reactive oxygen intermediates, may also be implicated in impaired haemopoiesis and the development of disordered erythropoiesis.

Specificity and idiotope expression of IgM produced by CD5+ and CD5- cord blood B-cell clones.

Epstein-Barr virus (EBV)-immortalized monoclonal B-cell lines were established from CD5+ and CD5- cord-blood B cells. IgM from many of both CD5+ and CD5- clones reacted with IgG-Fc, ssDNA, and a variety of other autoantigens. More CD5+ B cells that used light chains of the kappa isotype reacted with IgG-Fc and ssDNA than kappa-bearing CD5- B cells. Because many of the clones reacted with IgG-Fc, they were analyzed for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor and cold agglutinin paraproteins using murine antibodies (mAb) recognizing V kappa and VH subgroup-associated determinants. Expression of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17.109 mAb was expressed at significantly higher frequency (32%; p less than 0.05) and IgM antibodies derived from the CD5+ compared with the CD5- clones (5%). Both CD5+ and CD5- clones expressed the RF paraprotein-associated idiotope recognized by G8 mAb to the same extent. Similar results were obtained using binding to SpA as a marker of VH III family usage. Furthermore, no differences in frequency of expression of RF paraprotein-associated idiotopes recognized by B6 and/or D12, and characteristic of some antibodies using VH III family genes, were found between the CD5+ and CD5- populations. Although a higher than expected frequency of VH IV-gene expression was demonstrated (around 30%) in both CD5+ and CD5- cells, there were differences in expression of CRI recognized by mAb Lc1 and R2.1A2 with specificities for two VH IV subfamilies. While some CD5+ and CD5- clones were identified in which their IgM reacted with mAb Lc1, only CD5+ clones were recognized by another mAb R2.1A2. Analysis of the relationships between antigen specificities and V kappa- and VH-family gene usage indicated that auto- or polyreactivity was not associated with V kappa III nor any particular VH family. The higher frequency of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17-109 in the CD5+ clones and the association of CD5+ B cells with the VH IV subfamily recognized by mAb R2.1A2 and 9G4 may suggest that CD5+ B cells in cord blood are expanded as a result of recruitment within the fetal environment.

Distinctive features of immunoglobulin heavy chain variable region gene rearrangement in multiple myeloma.

We have analysed the rearranged Ig heavy chain (IgH) genes in a series of 28 cases of multiple myeloma (MM), in order to extend the study of Ig heavy chain variable (VH) gene usage in B lymphoid malignancies and to explore the ontogenic compartment from which transformed precursor cells arise in this disease. We were able to amplify 28 rearranged alleles by polymerase chain reaction from 23 of these cases, using a common joining region (JH) amplimer together with a panel of VH family-specific amplimers. The pattern of VH family usage was similar to that reported in normal peripheral blood B cells with infrequent usage of VH5 and VH6 genes. However, nucleotide sequence analysis of 17 IgH alleles revealed rearrangement of other VH family members, closely related to known developmentally regulated VH genes, some of which are known to be associated with autoimmune specificities. In contrast to previous findings on more immature B lineage malignancies, the rearranged genes diverged extensively from consensus germline sequences, consistent with somatic mutation. These findings support the hypothesis that the major proliferating precursor in MM arises at, or following a stage of T cell-dependent germinal centre proliferation in lymphoid follicles.

Trypanosoma cruzi in the opossum Didelphis marsupialis: an indirect fluorescent antibody test for the diagnosis and follow-up of natural and experimental infections.

The use of an indirect fluorescent antibody test (IFAT) performed in a "sandwich" technique has demonstrated: (i) the usefulness of the test for the diagnosis of Trypanosoma cruzi infection in the opossum Didelphis marsupialis; (ii) the existence of differences in the serological response of the opossum, that were related to the parasite strain and were clearly evident during the follow-up of experimental infections in laboratory born specimens; (iii) that, despite a good correlation between serological and parasitological examinations, IFAT was the most sensitive diagnostic test used, followed by xenodiagnosis; and, (iv) that in general, the opossum D. marsupialis seems to be a good responder to T. cruzi antigens.

Frequency of blood group antigens in Nigerian children with falciparum malaria.

The frequencies of the following blood group antigens: A, B, O, M, N, S, s, U, Fya, FyB, Lea, Jsa and K have been determined in Nigerian children with severe falciparum malaria. The frequency distribution of M, N, S, s, U, Fya and Fyb were not significantly different in children with life-threatening falciparum malaria and controls. The frequencies of A, B, O, Lea, Jsa and K found in the children with severe malaria were similar to those previously reported for healthy adults in this population. The Duffy blood group antigens Fya and Fyb were virtually absent from both infected and control children. This finding is in variance with a Fya frequency of 23% reported by Worlledge et al. (1974) for healthy adults in this population.