Synthesis and evaluation of nicotine alpha4beta2 receptor radioligand, 5-(3'-18F-fluoropropyl)-3-(2-(S)-pyrrolidinylmethoxy)pyridine, in rodents and PET in nonhuman primate.

Nicotine alpha(4)beta(2) receptor subtypes are implicated in the study of Alzheimer's disease, schizophrenia, substance abuse, lung cancer, and other disorders. We report the development and evaluation of a putative antagonist, 5-(3'-fluoropropyl)-3-(2-(S)-pyrrolidinylmethoxy)pyridine (nifrolidine) as a PET agent for nicotine alpha(4)beta(2) receptors. METHODS: In vitro binding affinity of nifrolidine was measured in rat brain slices labeled with (125)I-iodoepibatidine or (125)I-bungaratoxin. Selectivity of binding was measured in the presence of cytisine. (18)F radiolabeling was performed by reacting the tosylate precursor with (18)F-fluoride followed by deprotection. In vitro autoradiographic studies in rat brain slices with 5-(3'-(18)F-fluoropropyl)-3-(2-(S)-pyrrolidinylmethoxy)pyridine ((18)F-nifrolidine) were read on a phosphor imager. Rats were injected with (18)F-nifrolidine (3.7 MBq each), and brain regions were counted at various times (2-120 min). Blocking studies were performed by subcutaneous injection of nicotine (10 mg/kg). A PET study of (18)F-nifrolidine (approximately 148 MBq) was performed on an anesthetized rhesus monkey using a high-resolution scanner. RESULTS: In vitro binding affinity of nifrolidine exhibited an inhibition constant of 2.89 nmol/L for the alpha(4)beta(2) sites. Radiosynthesis and high-performance liquid chromatography purifications yielded the product in approximately 20%-40% decay-corrected radiochemical yield to provide (18)F-nifrolidine specific activities of approximately 111-185 GBq/mumol. In vitro autoradiography in rat brain slices revealed selective binding of (18)F-nifrolidine to the anteroventral thalamic nucleus, ventral posteriomedial thalamus, dorsolateral geniculate, and, to a lesser extent, cortex and striata, which are known to contain alpha(4)beta(2) sites. This specific binding was completely abolished by 300 mumol/L nicotine. Ex vivo rat brain distribution studies indicated selective binding in the thalamus with a maximal thalamus-to-cerebellum ratio of approximately 3. The PET study revealed selective maximal uptake (0.01% injected dose/mL) in regions of the thalamus (anteroventral and anteromedial thalamus, ventrolateral thalamus) and extrathalamic regions such as cingulate gyrus, lateral geniculate, temporal cortex, and frontal cortex. CONCLUSION: Binding of (18)F-nifrolidine to alpha(4)beta(2) receptor-rich regions in rats and monkeys indicates promise as a PET agent. Additionally, the thalamus-to-cerebellum ratio approached a plateau of 1.7 in 120 min, indicating relatively faster kinetics compared with previously reported imaging agents.     

Practice of and attitudes towards family planning among South Asian American immigrants

Background

Previous studies performed outside of the US that examined contraceptive knowledge and beliefs in South Asian women identified significant barriers. Our study aimed to further understand these practices in this population residing in the US.

Methods

This cross-sectional study surveyed women of all ethnicities receiving health care from either of two ambulatory practices in New Jersey from October 2011 to November 2012. Using chi-squared testing, the frequencies of contraceptive outcomes between South Asians and non-South Asians were compared.

Study Design

There were 42 South Asian respondents and 143 non-South Asian respondents. Our results show that South Asians are statistically significantly less likely to routinely use contraception and that gaps in contraceptive knowledge appear to stem from multiple barriers, including family opposition and pressure from spouse or in-laws to have children, cultural prohibitions, fear of side effects and misinformation, lack of education/knowledge about contraceptives and difficulty in accessing contraception. These gaps in contraceptive knowledge were also found to be multigenerational, despite higher levels of education generally seen in the South Asian population.

Conclusions

Clinicians caring for South Asian women should acknowledge both the barriers and the lack of contraceptive knowledge in this population and provide culturally competent family planning information to them during all women's health encounters.     

Nuts as a replacement for carbohydrates in the diabetic diet: a reanalysis of a randomised controlled trial

Aims/hypothesis

In line with current advice, we assessed the effect of replacing carbohydrate consumption with mixed nut consumption, as a source of unsaturated fat, on cardiovascular risk factors and HbA_1c in type 2 diabetes. The data presented here are from a paper that was retracted at the authors’ request ( https://doi.org/10.2337/dc16-rt02) owing to lack of adjustment for repeated measures in the same individual. Our aim, therefore, was to fix the error and add new complementary data of interest, including information on clotting factors and LDL particle size.

Methods

A total of 117 men and postmenopausal women with type 2 diabetes who were taking oral glucose-lowering agents and with HbA_1c between 47.5 and 63.9 mmol/mol (6.5–8.0%) were randomised after stratification by sex and baseline HbA_1c in a parallel design to one of three diets for 3 months: (1) ‘full-dose nut diet’ (n?=?40): a diet with 2.0 MJ (477 kcal) per 8.4 MJ (2000 kcal) energy provided as mixed nuts (75 g/day); (2) ‘full-dose muffin diet’ (n?=?39): a diet with 1.97 MJ (471 kcal) per 8.4 MJ (2000 kcal) energy provided as three whole-wheat muffins (188 g/day), with a similar protein content to the nuts, and the same carbohydrate-derived energy content as the monounsaturated fatty acid-derived energy content in the nuts; or (3) ‘half-dose nut diet’ (n?=?38): a diet with 1.98 MJ (474 kcal) per 8.4 MJ (2000 kcal) energy provided as half portions of both the nuts and muffins. The primary outcome was change in HbA_1c. The study was carried out in a hospital clinical research centre and concluded in 2008. Only the statistician, study physicians and analytical technicians could be blinded to the group assessment.

Results

A total of 108 participants had post-intervention data available for analysis (full-dose nut group, n?=?40; full-dose muffin group, n?=?35; half-dose nut group, n?=?33). Compared with the full-dose muffin diet, the full-dose nut diet provided 9.2% (95% CI 7.1, 11.3) greater total energy intake from monounsaturated fat. The full-dose nut diet (median intake, 75 g/day) also reduced HbA_1c compared with the full-dose muffin diet by ?2.0 mmol/mol (95% CI ?3.8, ?0.3 mmol/mol) (?0.19% [95% CI ?0.35%, ?0.02%]), (p?=?0.026). Estimated cholesterol levels in LDL particles with a diameter <255 ångström [LDL-c_<255Å]) and apolipoprotein B were also significantly decreased after the full-dose nut diet compared with the full-dose muffin diet. According to the dose response, the full-dose nut diet is predicted to reduce HbA_1c (?2.0 mmol/mol [?0.18%]; p?=?0.044), cholesterol (?0.25 mmol/l; p?=?0.022), LDL-cholesterol (?0.23 mmol/l; p?=?0.019), non-HDL-cholesterol (?0.26 mmol/l; p?=?0.020), apolipoprotein B (?0.06 g/l, p?=?0.013) and LDL-c<_255Å (?0.42 mmol/l; p?<?0.001). No serious study-related adverse events occurred, but one participant on the half-dose nut diet was hospitalised for atrial fibrillation after shovelling snow.

Conclusions/interpretation

Nut intake as a replacement for carbohydrate consumption improves glycaemic control and lipid risk factors in individuals with type 2 diabetes.

Trial registration

ClinicalTrials.gov NCT00410722

Funding

The study was funded by the International Tree Nut Council Nutrition Research and Education Foundation, the Peanut Institute, Loblaw Companies and the Canada Research Chairs Program of the Government of Canada

     

Artificial intelligence in drug development: present status and future prospects

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Excess of heme induces tissue factor-dependent activation of coagulation in mice

An excess of free heme is present in the blood during many types of hemolytic anemia. This has been linked to organ damage caused by heme-mediated oxidative stress and vascular inflammation. We investigated the mechanism of heme-induced coagulation activation in vivo. Heme caused coagulation activation in wild-type mice that was attenuated by an anti-tissue factor antibody and in mice expressing low levels of tissue factor. In contrast, neither factor XI deletion nor inhibition of factor XIIa-mediated factor XI activation reduced heme-induced coagulation activation, suggesting that the intrinsic coagulation pathway is not involved. We investigated the source of tissue factor in heme-induced coagulation activation. Heme increased the procoagulant activity of mouse macrophages and human PBMCs. Tissue factor-positive staining was observed on leukocytes isolated from the blood of heme-treated mice but not on endothelial cells in the lungs. Furthermore, heme increased vascular permeability in the mouse lungs, kidney and heart. Deletion of tissue factor from either myeloid cells, hematopoietic or endothelial cells, or inhibition of tissue factor expressed by non-hematopoietic cells did not reduce heme-induced coagulation activation. However, heme-induced activation of coagulation was abolished when both non-hematopoietic and hematopoietic cell tissue factor was inhibited. Finally, we demonstrated that coagulation activation was partially attenuated in sickle cell mice treated with recombinant hemopexin to neutralize free heme. Our results indicate that heme promotes tissue factor-dependent coagulation activation and induces tissue factor expression on leukocytes in vivo. We also demonstrated that free heme may contribute to thrombin generation in a mouse model of sickle cell disease.     

Nicotinic α4β2 receptor imaging agents. Part IV. Synthesis and Biological Evaluation of 3-(2-(S)-3,4-dehydropyrrolinyl methoxy)-5-(3′-18F-Fluoropropyl)pyridine (18F-Nifrolene) using PET

Imaging agents for nicotinic α4β2 receptors in the brain have been under way for studying various CNS disorders. Previous studies from our laboratories have reported the successful development of agonist, ¹⁸F-nifene. In attempts to develop potential antagonists, ¹⁸F-nifrolidine and ¹⁸F-nifzetidine were previously reported. Further optimization of these fluoropropyl derivatives has now been carried out resulting in 3-(2-(S)-3,4-dehydropyrrolinylmethoxy)-5-(3′-Fluoropropyl)pyridine (nifrolene) as a new high affinity agent for nicotinic α4β2 receptors. Nifrolene in rat brain homogenate assays – labeled with ³H-cytisine – exhibited a binding affinity of 0.36 nM. The fluorine-18 analog, ¹⁸F-nifrolene, was synthesized in approximately 10%–20% yield and specific activity was estimated to be > 2000 Ci/mmol. Rat brain slices indicated selective binding to anterior thalamic nuclei, thalamus, subiculum, striata, cortex and other regions consistent with α4β2 receptor distribution. This selective binding was displaced > 90% by 300 μM nicotine. Thalamus to cerebellum ratio (> 10) was the highest for ¹⁸F-nifrolene with several other regions showing selective binding. In vivo rat PET studies exhibited rapid uptake of ¹⁸F-nifrolene in the brain with specific retention in the thalamus and other brain regions while clearing out from the cerebellum. Thalamus to cerebellum ratio value in the rat was > 4. Administration of nicotine caused a rapid decline in the thalamic ¹⁸F-nifrolene suggesting reversible binding to nicotinic receptors. PET imaging studies of ¹⁸F-nifrolene in anesthetized rhesus monkey revealed highest binding in the thalamus followed by regions of the lateral cingulated and temporal cortex. Cerebellum showed the least binding. Thalamus to cerebellum ratio in the monkey brain was > 3 at 120 min. These ratios of ¹⁸F-nifrolene are higher than measured for ¹⁸F-nifrolidine and ¹⁸F-nifzetidine. ¹⁸F-Nifrolene thus shows promise as a new PET imaging agent for α4β2 nAChR.     

Nicotinic alpha4beta2 receptor imaging agents: part II. Synthesis and biological evaluation of 2-[18F]fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (18F-nifene) in rodents and imaging by PET in nonhuman primate

The alpha4beta2 nicotinic acetylcholine receptor (nAChR) has been implicated in various neurodegenerative diseases. Optimal positron emission tomography (PET) imaging agents are therefore highly desired for this receptor. We report here the development and initial evaluation of 2-fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (nifene). In vitro binding affinity of nifene in rat brain homogenate using 3H-cytisine exhibited a K(i) = 0.50 nM for the alpha4beta2 sites. The radiosynthesis of 2-18F-fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (18F-nifene) was accomplished in 2.5 h with an overall radiochemical yield of 40-50%, decay corrected. The specific activity was estimated to be approx. 37-185 GBq/micromol. In vitro autoradiography in rat brain slices indicated selective binding of 18F-nifene to anteroventral thalamic (AVT) nucleus, thalamus, subiculum, striata, cortex and other regions consistent with alpha4beta2 receptor distribution. Rat cerebellum showed some binding, whereas regions in the hippocampus had the lowest binding. The highest ratio of >13 between AVT and cerebellum was measured for 18F-nifene in rat brain slices. The specific binding was reduced (>95%) by 300 microM nicotine in these brain regions. Positron emission tomography imaging study of 18F-nifene (130 MBq) in anesthetized rhesus monkey was carried out using an ECAT EXACT HR+ scanner. PET study showed selective maximal uptake in the regions of the anterior medial thalamus, ventro-lateral thalamus, lateral geniculate, cingulate gyrus, temporal cortex including the subiculum. The cerebellum in the monkeys showed lower binding than the other regions. Thalamus-to-cerebellum ratio peaked at 30-35 min postinjection to a value of 2.2 and subsequently reduced. The faster binding profile of 18F-nifene indicates promise as a PET imaging agent and thus needs further evaluation.