Enabling Chemistry Technologies and Parallel Synthesis-Accelerators of Drug Discovery Programmes.

There is a pressing need to improve overall productivity in the pharmaceutical industry. Judicious investments in chemistry technologies can have a significant impact on cycle times, cost of goods and probability of technical success. This perspective describes some of these technologies developed and implemented at AbbVie, and their applications to the synthesis of novel scaffolds and to parallel synthesis.

Are somatosensory evoked potentials of the tibial nerve the most sensitive test in diagnosing multiple sclerosis?

BACKGROUND: Multiple sclerosis (MS) is mostly diagnosed clinically, but the diagnosis has significantly improved through the use of brain magnetic resonance imaging (MRI), testing of cerebrospinal fluid, and multimodal evoked potentials (MEPs). Even though MRI is the superior method in diagnosing this illness, MEPs remain important because they can detect clinically silent lesions in the sensory and motor pathways of the central nervous system (CNS). AIM: The aim of the study is to test the diagnostic sensitivity of MEPs and MRI and the ratio of their sensitivity in patients with MS. MATERIALS AND METHODS: The study subjects included 293 patients with MS with disease duration of two to six years: 249 patients with relapsing-remitting (RR) MS and 44 with primary-progressive (PP) MS. All patients were subjected to an MRI brain scan, visual evoked potentials (VEPs), median somatosensory evoked potentials (SEPs), tibial somatosensory evoked potentials (SEPs), and auditory evoked potentials (AEPs). Abnormal Findings Included : changed wave morphology, interside difference in wave amplitude, absolute and interwave latency increased by 2.5 SD as compared with the control group. The control group comprised of 35 healthy subjects. Results : In this study the most abnormal findings were tibial SEPs, median SEPs, and VEPs. Our results suggest different sensitivity of MEPs in patients suffering from different forms of MS. In RR-MS the sensitivity of tibial SEPs was statically significant (Fischer's exact probability test) as compared to other evoked potential modalities. Similarly VEPs were more sensitive as compared to AEPs. In the PP-MS, median SEPs have been found to be more sensitive than VEPs, while tibial SEPs have been found to be more sensitive than AEPs. There was no significant difference in the sensitivity of MRI and MEPs both the forms of MS. CONCLUSION: Tibial SEPs produce the most abnormal results and the highest sensitivity in the RR-MS. We propose that this test as useful criterion for the diagnosis of MS.

Particle size analysis: 90Y and 99mTc-labelled colloids.

Colloidal particle size is an important characteristic to consider when choosing a radiopharmaceutical for diagnosis and therapeutic purposes in nuclear medicine. Photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) were used to determine the particle-size distribution of (90)Y- and (99m)Tc-labelled antimony trisulfide (Sb(2)S(3)) and tin colloids (Sn-colloid). (90)Y-Sb(2)S(3) and (99m)Tc-Sb(2)S(3) were found to have a diameter of 28.92 +/- 0.14 and 35.61 +/- 0.11 nm, respectively, by PCS. By TEM, (90)Y-Sb(2)S(3) particles were measured to be 14.33 +/- 0.09 nm. (90)Y-labelled Sn colloid were found to exist with a d(v(max1)) of 805 nm and a d(v(max2)) of 2590 nm, by PCS, whereas (99m)Tc-Sn colloid was shown to have more than 80% of radioactive particles of approximately 910 nm by PCS. For (90)Y-labelled Sb(2)S(3) and Sn colloid, a comparison of TEM and PCS indicates that these techniques found significantly different mean diameters. TEM has an excellent resolution necessary for radiocolloid particle-sizing analysis, and it is a desirable size-measuring technique because it is more reliable than PCS.

Epidemiology of end-stage renal disease and current status of hemodialysis in Yugoslavia.

Maintenance hemodialysis (HD) in Yugoslavia started in the sixties and followed the dialysis trends in the Western Europe. However, in the last decade the development of renal replacement therapy (RRT) slowed down. In this report the epidemiology of ESRD from 1997-1999 and the survey of the status of HD treatment in Yugoslavia in 1999 are presented. Epidemiological data are obtained by the annual center questionnaires (response rate: 92.6 -94.2%). The survey of HD status is based on a specific questionnaire and covered 2108 patients (65%). At the end of 1999 there were 56 RRT centers in Yugoslavia treating 3939 patients: 3232 (82%) patients by HD, 248 (6.3%) by peritoneal dialysis, and 459 (11.7%) living with transplanted kidney. In a three year period, incidence of ESRD ranged from 108-128 pmp, point prevalence from 435-463 pmp and mortality rate from 20.7-17.9. Numerous refugee patients were treated over the last 10 years. Main causes of ESRD were glomerulonephritis (30%); Balkan nephropathy represented 11% and diabetic nephropathy 7% of all primary renal diseases. Cardiovascular and cerebrovascular diseases were the most common causes of death of RRT patients. Most centers are overcrowded and HD machines are worn out. Mean Kt/V was 1.19+/-0.08, mean URR% 58.8+/-7.4. The shortage of drugs prevented adequate management: 83% of HD patients had hemoglobin level less than 100 g/L but only 10.3 -17.8% were treated with rHuEpo; 64.5% of patients had phosphate levels higher than 1.7 mmol/L but only 33.5% used phosphate binders; 47% of patients had hypertension despite the antihypertensive therapy. The prevalence of hepatitis B remained unchanged (about 14%) in HD population during the last three years, but the prevalence of anti-HCV positive patients decreased (31-23%). In conclusion, there is a well developed dialysis service in Yugoslavia but insufficient conditions for adequate treatment.

Potent inhibition of NFAT activation and T cell cytokine production by novel low molecular weight pyrazole compounds.

NFAT (nuclear factor of activated T cell) proteins are expressed in most immune system cells and regulate the transcription of cytokine genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the Ca(2+)/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN). Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization. Current immunosuppressive drugs such as cyclosporin A and FK506 block CaN activity thus inhibiting nuclear translocation of NFAT and consequent cytokine gene transcription. The inhibition of CaN in cells outside of the immune system may contribute to the toxicities associated with cyclosporin A therapy. In a search for safer immunosuppressive drugs, we identified a series of 3,5-bistrifluoromethyl pyrazole (BTP) derivatives that block Th1 and Th2 cytokine gene transcription. The BTP compounds block the activation-dependent nuclear localization of NFAT as determined by electrophoretic mobility shift assays. Confocal microscopy of cells expressing fluorescent-tagged NFAT confirmed that the BTP compounds block calcium-induced movement of NFAT from the cytosol to the nucleus. Inhibition of NFAT was selective because the BTP compounds did not affect the activation of NF-kappaB and AP-1 transcription factors. Treatment of intact T cells with the BTP compounds prior to calcium ionophore-induced activation of CaN caused NFAT to remain in a highly phosphorylated state. However, the BTP compounds did not directly inhibit the dephosphorylation of NFAT by CaN in vitro, nor did the drugs block the dephosphorylation of other CaN substrates including the type II regulatory subunit of protein kinase A and the transcription factor Elk-1. The data suggest that the BTP compounds cause NFAT to be maintained in the cytosol in a phosphorylated state and block the nuclear import of NFAT and, hence, NFAT-dependent cytokine gene transcription by a mechanism other than direct inhibition of CaN phosphatase activity. The novel inhibitors described herein will be useful in better defining the cellular regulation of NFAT activation and may lead to identification of new therapeutic targets for the treatment of autoimmune disease and transplant rejection.

3,5-Bis(trifluoromethyl)pyrazoles: a novel class of NFAT transcription factor regulator.

A series of bis(trifluoromethyl)pyrazoles (BTPs) has been found to be a novel inhibitor of cytokine production. Identified initially as inhibitors of IL-2 synthesis, the BTPs have been optimized in this regard and even inhibit IL-2 production with a 10-fold enhancement over cyclosporine in an ex vivo assay. Additionally, the BTPs show inhibition of IL-4, IL-5, IL-8, and eotaxin production. Unlike the IL-2 inhibitors, cyclosporine and FK506, the BTPs do not directly inhibit the dephosphorylation of NFAT by calcineurin.

Structure-activity relationship studies on 1-[2-(4-Phenylphenoxy)ethyl]pyrrolidine (SC-22716), a potent inhibitor of leukotriene A(4) (LTA(4)) hydrolase.

Leukotriene B(4) (LTB(4)) is a pro-inflammatory mediator that has been implicated in the pathogenesis of a number of diseases including inflammatory bowel disease (IBD) and psoriasis. Since the action of LTA(4) hydrolase is the rate-limiting step for LTB(4) production, this enzyme represents an attractive pharmacological target for the suppression of LTB(4) production. From an in-house screening program, SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) was identified as a potent inhibitor of LTA(4) hydrolase. Structure-activity relationship (SAR) studies around this structural class resulted in the identification of a number of novel, potent inhibitors of LTA(4) hydrolase, several of which demonstrated good oral activity in a mouse ex vivo whole blood assay.

Benzofuran based PDE4 inhibitors.

Replacement of the 3,4-dialkoxyphenyl substructure common to a number of PDE4 inhibitors with a 2-alkyl-7-methoxybenzofuran unit is described. This substitution can result in either enhancement or substantial reductions in PDE4 inhibitory activity depending on the system to which it is applied. An in vitro SAR study of a potent series of 4-(2-heteroaryl-ethyl)-benzoiurans 26 is also presented.

Novel cytokine release inhibitors. Part IV: analogs of podocarpic acid.

Podocarpic acid derivatives as cytokine (IL-1beta) release inhibitors are discussed.

Inhibition of p56(lck) tyrosine kinase by isothiazolones.

Lck encodes a 56-kDa protein-tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56(lck), an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56(lck) kinase activity with IC50 = 1-7 microM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-Jun N-terminal kinase 2alpha. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50 = 35 microM) and blocked T cell proliferation in response to alloantigen (IC50 = 14 microM) and CD3/CD28-induced IL-2 secretion (IC50 = 2.2 microM) in primary T cell cultures. Inhibition of p56(lck )by A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56(lck) kinase activity and TCR-dependent signal transduction. Incubation with thiols such as beta-ME or DTT also blocked the ability of the isothiazolone to inhibit p56(lck) kinase activity. LC/MS analysis established the covalent modification of p56(lck) at cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56(lck) catalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56(lck) enzyme. Loss of p56(lck) activity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56(lck) inhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.

Ex vivo assessment of immunosuppression in undiluted whole blood from pigs dosed with tacrolimus (FK506).

To assess the duration of immunosuppression in FK506-dosed pigs, an undiluted whole blood assay was established to measure reactivities of T cells in their physiological milieu. PMA and ionomycin were shown to induce IL-2 production in swine blood. The IC50 of FK506 in inhibiting IL-2 production in whole blood and isolated PBMC stimulated with PMA and ionomycin measured 1.2 and 0.04 nM, respectively. These data underscore the influence of red blood cells and plasma proteins on drug potency. IL-2 levels were determined in blood drawn immediately before and 1, 24, 48, and 72 h after iv dosing. For pigs dosed with 0.05 mg/kg, 50% recovery of IL-2 production was observed at 16 h and 100% at 35 h after dosing. For pigs dosed with 0.15 mg/kg, 50% recovery was observed at 38 h and 100% at 72 h. Blood concentrations of FK506 at 50 and 100% recovery of IL-2 production measured 10.8 and 2.2 nM for pigs dosed with 0.05 mg/kg and 6.1 and 1.1 nM for pigs dosed with 0.15 mg/kg, respectively. These concentrations are severalfold higher than predicted from the IC50 of FK506 for inhibiting IL-2 production in the whole blood assay. These data suggest that the true potency of FK506 in blood after dosing is influenced by additional factors, which could include plasma protein binding, the presence of active or interfering metabolites, serum interfering factors, and sequestration of drug in blood cells. Our results demonstrate the utility of an undiluted whole blood assay for assessing the duration of immunosuppression in drug-dosed animals and emphasize the importance of assessing drug potency in the whole blood environment ex vivo.

Novel cyclic compounds as potent phosphodiesterase 4 inhibitors.

The synthesis and biological activity of a novel series of 2, 2-disubstituted indan-1,3-dione-based PDE4 inhibitors are described. This structurally unique class of PDE4 inhibitors is markedly different from the known PDE4 inhibitors such as RP 73401 (2) and CDP 840 (3). Structure-activity relationship (SAR) studies led to the identification of inhibitors with nanomolar potency and oral activity in a murine endotoxemia model for TNF-alpha inhibition. Unlike other classical PDE4 inhibitors, several analogues were found to be nonemetic in a canine emesis model at intravenous doses of up to 3 mg/kg.

The synthesis and biological evaluation of a novel series of indole PDE4 inhibitors I.

This communication describes the synthesis and in vitro evaluation of a novel potent series of phosphodiesterase type (IV) (PDE4) inhibitors. The compounds described contain an indole moiety which replaces the 'rolipram-like' 3-methoxy-4-cyclopentoxy motif. Several of the compounds presented possess low nanomolar IC50's for PDEIV inhibition. In vivo activities determined from measurement of serum TNF-alpha levels in LPS challenged mice (mouse endotoxemia model) are also reported.

Orally active indole N-oxide PDE4 inhibitors.

This communication describes the synthesis and in vitro and in vivo evaluation of a novel potent series of phosphodiesterase type (IV) (PDE4) inhibitors. Several of the compounds presented possess low nanomolar IC50's for PDE4 inhibition and excellent in vivo activity for inhibition of TNF-alpha levels in LPS challenged mice (mouse endotoxemia model). Emesis studies (dog) and efficacy in a SCW arthritis model for the most potent PDE4 inhibitors are presented.

Quaternary substituted PDE4 inhibitors I: the synthesis and in vitro evaluation of a novel series of oxindoles.

The following letter presents the synthesis and in vitro evaluation of a novel quaternary substituted series of phosphodiesterase type (IV) (PDE4) inhibitors. The compounds represent conformationally constrained analogues of the Celltech PDE IV inhibitor, CDP 840. Examples with sub-micromolar IC50's for PDE4 inhibition are reported.

Quaternary substituted PDE IV inhibitors II: the synthesis and in vitro evaluation of a novel series of gamma-lactams.

This communication describes the synthesis and in vitro evaluation of a novel potent series of phosphodiesterase type (IV) (PDE IV) inhibitors. Several of the quaternary substituted lactams presented possess low nanomolar IC50's for PDE IV inhibition.

2-Substituted-4-methoxybenzimidazole-based PDE4 inhibitors.

A new family of PDE4 inhibitors based on a benzimidazole framework is described. Several of these compounds are orally bioavailable and show efficacy in in vivo models of inflammatory disease.