Design and Synthesis of Pyrrolo[2,3-d]pyrimidine-Derived Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors Using a Checkpoint Kinase 1 (CHK1)-Derived Crystallographic Surrogate.

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3-d]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2R)-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S cKi 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18/CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino] derivative 32. Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45, which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.

Treating Food Approval-Seeking Behavior: One Bite at a Time.

The prevalence of feeding problems in children with autism is high. The current investigation was a treatment of a unique presentation of food-related prompt dependence with a 6-year-old boy with autism who was reliant upon approval from adults for consumption of every bite of food. Instructions were used to establish independent eating, in which the number of bites specified in the instruction was systematically increased. Independent bites increased from a baseline level of 0.67% to a final phase level of almost 100%, and the instruction was faded to "eat your lunch".

Design of Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors Using a Crystallographic Surrogate Derived from Checkpoint Kinase 1 (CHK1).

Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson's disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LRRK2-pSer935 IC50 values for 22 in mouse brain and kidney being 1.3 and 5 nM, respectively.

Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells.

Genetic variation in the leucine-rich repeat kinase 2 (LRRK2) gene is associated with risk of familial and sporadic Parkinson's disease (PD). To support clinical development of LRRK2 inhibitors as disease-modifying treatment in PD biomarkers for kinase activity, target engagement and kinase inhibition are prerequisite tools. In a combined proteomics and phosphoproteomics study on human peripheral mononuclear blood cells (PBMCs) treated with the LRRK2 inhibitor Lu AF58786 a number of putative biomarkers were identified. Among the phospho-site hits were known LRRK2 sites as well as two phospho-sites on human Rab10 and Rab12. LRRK2 dependent phosphorylation of human Rab10 and human Rab12 at positions Thr73 and Ser106, respectively, was confirmed in HEK293 and, more importantly, Rab10-pThr73 inhibition was validated in immune stimulated human PBMCs using two distinct LRRK2 inhibitors. In addition, in non-stimulated human PBMCs acute inhibition of LRRK2 with two distinct LRRK2 inhibitor compounds reduced Rab10-Thr73 phosphorylation in a concentration-dependent manner with apparent IC50's equivalent to IC50's on LRRK2-pSer935. The identification of Rab10 phosphorylated at Thr73 as a LRRK2 inhibition marker in human PBMCs strongly support inclusion of assays quantifying Rab10-pThr73 levels in upcoming clinical trials evaluating LRRK2 kinase inhibition as a disease-modifying treatment principle in PD.

The design and SAR of a novel series of 2-aminopyridine based LRRK2 inhibitors.

Leucine-rich repeat kinase 2 (LRRK2) has attracted considerable interest as a therapeutic target for the treatment of Parkinson's disease. Compounds derived from a 2-aminopyridine screening hit were optimised using a LRRK2 homology model based on mixed lineage kinase 1 (MLK1), such that a 2-aminopyridine-based lead molecule 45, with in vivo activity, was identified.

Negative modulation of GABAA α5 receptors by RO4938581 attenuates discrete sub-chronic and early postnatal phencyclidine (PCP)-induced cognitive deficits in rats.

RATIONALE: A growing body of evidence suggests that negative modulation of γ-aminobutyric acid (GABA) GABA(A) α5 receptors may be a promising strategy for the treatment of certain facets of cognitive impairment; however, selective modulators of GABA(A) α5 receptors have not yet been tested in "schizophrenia-relevant" cognitive assay/model systems in animals. OBJECTIVES: The objectives of this study were to investigate the potential of RO4938581, a negative modulator of GABA(A) α5 receptors, and to attenuate cognitive impairments induced following sub-chronic (sub-PCP) and early postnatal PCP (neo-PCP) administration in the novel object recognition (NOR) and intra-extradimensional shift (ID/ED) paradigms in rats. Complementary in vitro, ex vivo and in vivo studies were performed to confirm negative modulatory activity of RO4938581 and to investigate animal model validity, concept validity and potential side effect issues, respectively. RESULTS: In vitro studies confirmed the reported negative modulatory activity of RO4938581, whilst immunohistochemical analyses revealed significantly reduced parvalbumin-positive cells in the prefrontal cortex of sub-PCP- and neo-PCP-treated rats. RO4938581 (1 mg/kg) ameliorated both sub-PCP- and neo-PCP-induced cognitive deficits in NOR and ID/ED performance, respectively. In contrast, QH-II-066 (1 and 3 mg/kg), a GABA(A) α5 receptor positive modulator, impaired cognitive performance in the NOR task when administered to vehicle-treated animals. Additional studies revealed that both RO4938581 (1 mg/kg) and QH-II-066 (1 and 3 mg/kg) attenuated amphetamine-induced hyperactivity in rats. CONCLUSIONS: Taken together, these novel findings suggest that negative modulation of GABA(A) α5 receptors may represent an attractive treatment option for the cognitive impairments, and potentially positive symptoms, associated with schizophrenia.

Meperidine misuse in a patient with sphincter of Oddi dysfunction.

OBJECTIVE: To report a seizure occurring secondary to meperidine treatment despite normal renal and central nervous system (CNS) function, and to provide a review of meperidine's role in pain management, including its use in pancreatitis and sphincter of Oddi dysfunction. CASE SUMMARY: A 55-year-old white woman with a history of sphincter of Oddi dysfunction presented to the emergency department with severe abdominal pain. On admission to the hospital, the serum creatinine level was 0.6 mg/dL with slightly elevated aspartate aminotransferase of 56 U/L (normal range 0-31) and alanine aminotransferase of 34 U/L (0-31). The patient received repeated and escalating doses of intravenous meperidine, resulting in a generalized seizure on day 4 of hospitalization. The accumulated meperidine dose was 2125 mg. Buprenorphine was substituted in place of meperidine, and the patient had no further reported complications. She was then transferred to a tertiary-care facility for sphincter of Oddi reevaluation. An objective causality assessment revealed the adverse drug event as probable. DISCUSSION: Despite alternative opioids, meperidine continues to be used in pain management. Meperidine is different from other opioids because its active metabolite, normeperidine, is neurotoxic. Patients with renal insufficiency, liver failure, or CNS dysfunction are at increased risk for adverse drug reactions related to normeperidine accumulation. Due to normeperidine's extended half-life, however, accumulation of normeperidine can occur in any patient receiving repeated doses of meperidine. CONCLUSIONS: This case demonstrates the potential hazards that exist when using meperidine in any patient. Meperidine's inherent risks of both undertreating pain and causing adverse drug reactions should prompt clinicians and health organizations to restrict its use in pain management. This restriction should not make exceptions to meperidine's traditional use in pancreatitis or sphincter of Oddi dysfunction.

Albumin as an outcome measure in haemodialysis in patients: the effect of variation in assay method.

BACKGROUND: Serum/plasma albumin is an important predictor of future mortality/morbidity in haemodialysis (HD) patients and has been proposed as an important audit measure. Different methods of albumin assay give different results and the bias between methods may be greater in renal failure patients. METHODS: Albumin concentration in plasma was measured by three methods, two dye-binding methods (bromocresol green (BCG) and bromocresol purple (BCP)) and an immuno-turbidimetric (ITM) method, in 143 HD patients (group I) and 49 non-renal patients (group II). Comparisons were made between means, variation in differences across a range of albumin concentrations and on the percentage of patients within the normal range. RESULTS: In HD patients (group I), BCG over-estimated plasma albumin compared with the other two methods. The difference could be as much as 10 g/l and was more marked in hypoalbuminaemic patients. The BCP method gave results closer to the ITM method, particularly in HD patients. These differences were less marked in group II patients but both methods overestimated albumin compared with the ITM method. Using the BCG local laboratory normal range, 84% of HD patients had plasma albumin concentrations within the normal range but this fell to 57% if the BCP results were used. CONCLUSIONS: The method for determining albumin concentration has a marked effect on the results particularly in HD patients. BCG, the most commonly used method, gives higher results than other methods and correlates poorly with an immunological method. These differences make comparative audit between nephrology units difficult and have implications for other biochemical variables and other specialties.

Multiple sclerosis, dental caries and fillings: a case-control study.

OBJECTIVES: To investigate the association between multiple sclerosis, dental caries, amalgam fillings, body mercury and lead. DESIGN: Matched case-control study. SETTING: Leicestershire in the years 1989-1990. SUBJECTS: Thirty-nine females with multiple sclerosis (of recent onset) were matched with 62 controls for age, sex and general practitioner. METHODS: Home interview of cases and controls within which there was an assessment of the DMFT index and blood and urine mercury and lead levels. RESULTS: The odds of being a MS case increased multiplicatively by 1.09 (95% CI 1.00, 1.18) for every additional unit of DMFT index of dental caries. This represents an odds ratio of 1.213 or a 21% increase in risk of MS in relation to dental caries in this population. There was no difference between cases and controls in the number of amalgam fillings or in body mercury or lead levels. There was a significant correlation between body mercury levels and the number of teeth filled with amalgam (controls: r = +0.430, P = 0.006, cases: r = +0.596, P = 0.001). CONCLUSION: There was evidence of excess dental caries among MS cases compared with the controls. This finding supports the strong geographical correlation between the two diseases. A further study of this association is recommended.

Potential impact of a change in the diagnostic criteria for diabetes mellitus on the prevalence of abnormal glucose tolerance in a local community at risk of diabetes: impact of new diagnostic criteria for diabetes mellitus.

AIMS: To compare the prevalence of diabetes and abnormal glucose metabolism using conventional and suggested new WHO and new ADA criteria in a group of people with symptoms of diabetes. METHODS: We examined retrospectively the results of 154 consecutive OGTTs in such patients performed using capillary whole blood. RESULTS: With the 1985 WHO criteria. Forty-four point eight per cent of subjects (69 subjects, with 95% confidence intervals, 37-52.6%) had diabetes, 47.8% (33 subjects, 36-59.6%) had a normal fasting glucose, 31.2% (48 subjects, 23.9-38.5%) had impaired glucose tolerance (IGT) and 76% (117 subjects, 69.3-82.7%) had abnormal glucose tolerance. Applying the ADA criteria (fasting capillary whole blood only), 33.1% (51 subjects, 25.7-40.5%) had diabetes (a 26% relative reduction) and 11% (17 subjects, 6.1-15.9%) IFG, with 44.1% (68 subjects, 36.3-51.9%) having abnormal glucose metabolism (a 42% relative reduction). If the proposed 1998 WHO criteria were used, the number with diabetes increase to 48% (74 subjects, 40.1-55.9%) a 7.2% increase on the old criteria. 27.9% (43 subjects, 20.8-35%) had IGT, so the number with some degree of abnormal glucose metabolism remains unchanged. Use of the ADA criteria, considering only the fasting glucose as suggested, will result in a significant reduction in the diagnosis of diabetes and those with abnormal glucose metabolism.

Effect of thyroid stimulating hormone on adaptive behaviour in Down's syndrome.

Patients with Down's syndrome are particularly vulnerable to the development of both hypothyroidism and Alzheimer's disease. Both hypothyroidism and Alzheimer's disease may be associated with elevated serum concentrations of thyroid stimulating hormone. In a group of institutionalized Down's syndrome patients with normal thyroid function, global scores of ability were higher than in a group of patients with elevated thyroid stimulating hormone levels in the presence of normal T3 and T4. The actual concentrations of thyroid stimulating hormone were shown to be significantly and inversely correlated with scores of global abilities. If these findings are reproducible, the authors believe that thyroid stimulating hormone estimation may provide confirmatory evidence of clinical dementia in this group of mentally handicapped individuals.