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ABSTRACT: α4β2⁎ neuronal nicotinic acetylcholine receptor are ligand-gated ion channels and widely expressed throughout the central and peripheral nervous system. α4β2⁎ neuronal nicotinic acetylcholine receptor play crucial role in pain signaling via modulation of multiple neurotransmitters like acetylcholine, dopamine, γ-amino butyric acid (GABA) and norepinephrine. Both spinal and supraspinal pathways are involved in the mechanisms by which α4β2⁎ neuronal nicotinic acetylcholine receptor ligands modulate the neuropathic and inflammatory pain. Selective α4β2⁎ neuronal nicotinic acetylcholine receptor ligands are being developed for the treatment of neuropathic and inflammatory pain as they show considerable efficacy in a wide range of preclinical pain models. Agonists/ partial agonists of α4β2⁎ neuronal nicotinic acetylcholine receptor show efficacy in animal models of pain and their anti-nociceptive properties are blocked by nicotinic antagonists. Positive allosteric modulators are being developed with the aim to increase the potency or therapeutic window of agonists/partial agonists. Accumulating evidences suggest that anti-nociceptive effects of nicotinic acetylcholine receptor ligands may not be mediated solely by α4β2⁎ neuronal nicotinic acetylcholine receptor. We have also reviewed the stage of clinical development of various α4β2⁎ neuronal nicotinic acetylcholine receptor ligands.
European journal of pharmacology 05/2013; · 2.59 Impact Factor
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ABSTRACT: The objective of the current investigation was to develop a simple, rapid method for determining in-vivo 5-hydroxytryptamine type 4 receptor (5-HT4 R) occupancy in rat brain using non-radiolabelled SB207145 as a tracer for accelerating the drug discovery process.
In-vivo tracer optimization studies for tracer dose, survival intervals and brain distribution profile were carried out in rats. The tracer was pharmacologically validated using potent well-characterized 5-HT4 R ligands. The brain regional concentrations of tracer (SB207145); plasma and brain concentrations of 5-HT4 R ligands were quantified using high-performance liquid chromatography coupled with a tandem mass spectrometric detector (LC-MS/MS).
SB207145 showed a higher specific binding in striatum (1.96 ng/g) and lower binding in cerebellum (0.66 ng/g), which is consistent with findings of other published 5-HT4 R expression studies. Pretreatment with potent 5-HT4 ligands dose-dependently reduced striatal SB207145 concentration and the effective dose to achieve 50% receptor occupancy (ED50 ) values were 4.8, 2.0, 7.4, 9.9, 3.8 and 0.02 mg/kg for GR113808, piboserod, prucalopride, RS67333, TD8954 and PF04995274, respectively.
Results from the mass spectrometry approach to determine 5-HT4 R occupancy in rat brain are comparable with those reported using radiolabelled scintillation spectroscopy methods. In conclusion, the LC-MS/MS characterization permits use of tracer at a preclinical stage in high-throughput fashion as well as characterization of target expression.
The Journal of pharmacy and pharmacology. 05/2013; 65(5):704-12.
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ABSTRACT: A sensitive LC-MS/MS method was developed and validated for the quantification of almotriptan in rat brain and blood dialysates. Almotriptan is a 5HT1B/1D receptor agonist used for the treatment of migraine pain. Method consists of rapid gradient elution program with 10mM ammonium formate (pH 3) and acetonitrile on a Xbridge column. The MRM transitions monitored were m/z 336.2-58.1 for almotriptan and m/z 448.2-285.3 for the IS. The assay was linear in the range of 0.1-20ng/ml, with acceptable precision and accuracy along with adequate sensitivity. The between batch accuracy was in the range of 99.0-104.3% with precision in between 0.6% and 5.8%. Microdialysis is an important sampling technique, with the capability of capturing the concentrations of various analytes in different bio fluids, at a single time point. This method was applied to quantify brain and blood dialysate samples obtained from a microdialysis study of rats treated with almotriptan (10mg/kg, p.o.). In vivo recovery experiments were performed to correct the dialysate concentrations into extracellular concentrations. Mean peak dialysate concentrations of almotriptan were found to be 152±78 and 7.4±1.0ng/ml in blood and prefrontal cortex, respectively. The brain penetration of almotriptan is characterized by the AUCbrain/AUCblood found to be 0.07±0.05. The results revealed the importance of measuring the unbound almotriptan concentrations in the brain over the blood for understanding its PK/PD relationship.
Journal of pharmaceutical and biomedical analysis 04/2013; 81-82C:160-167. · 2.45 Impact Factor
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ABSTRACT: Background: Epidemiologic studies and clinical assessment of schizophrenic population have revealed a high incidence of overlap between schizophrenia and addictive disorders. Objective: The aim of the present investigation was to study the effect of aripiprazole in a preclinical animal model of chronic alcohol self-administration (CASA) and also to evaluate the influence of CASA on plasma pharmacokinetics and dopamine D(2) receptor (D(2)R) occupancy in rats. Methods: The effect of oral administration of aripiprazole (1, 3, and 10 mg/kg) on 4% alcohol intake in CASA was studied for a period of 45 min after a post-dosing interval of 60 min. Brain penetration, pharmacokinetics, and D(2)R occupancy of aripiprazole were evaluated in normal and CASA rats. Results: Aripiprazole reduced alcohol consumption in CASA rats by 13, 28, and 86% at 1, 3, and 10 mg/kg, respectively, and the effect reached statistical significance at 10 mg/kg (p < .01). At this behavioral effective dose, a decrease (75%) in total plasma apparent clearance and an increase in oral area under the concentration-time curve (3.98-fold) and bioavailability (3.50-fold) of aripiprazole was observed in CASA rats. Striatal D(2)R occupancy and brain exposure of aripiprazole were significantly higher (∼twofold) in CASA rats when compared to normal rats (p < .01). Conclusion: Chronic alcohol intake results in a significant increase in exposure of aripiprazole in plasma and brain and striatal D2R occupancy. Scientific significance: Chronic alcohol intake would increase aripiprazole exposure, thus aripiprazole dose might have to be decreased (assuming this same phenomenon occurs in humans).
The American Journal of Drug and Alcohol Abuse 03/2013; 39(2):72-9. · 1.55 Impact Factor
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ABSTRACT: A simple and rapid LC-MS/MS method was developed and validated for the quantification of epalrestat, an aldose reductase inhibitor for the treatment of diabetic neuropathy. Following protein precipitation epalrestat and IS were eluted with 10mM ammonium acetate and acetonitrile using a rapid gradient program on reverse phase column. Multiple reaction monitoring mode was used to monitor the transitions of m/z 318→58 for epalrestat and m/z 410→348 for the IS. The assay exhibited a linear dynamic range of 2-5000ng/mL for epalrestat in rat plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The within batch accuracy was in the range of 101.3-108.0% with precision in the range of 3.0-12.3%. All the other validation parameters were within the acceptable limits. Validated method was applied to analyze rat plasma samples obtained from a pharmacokinetic study. After oral administration of epalrestat at 10mg/kg to wistar rats (n=3) mean C(max), AUC(0-24) (ngh/mL) and t(1/2) were found to be 4077±1327ng/mL, 8989±1590ngh/mL and 2.9±1.4h, respectively. Bioavailability was found to be 90±14% for epalrestat in male wistar rats.
Journal of pharmaceutical and biomedical analysis 02/2013; 74:227-34. · 2.45 Impact Factor
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Journal of Pharmacy and Pharmacology 01/2013; · 2.17 Impact Factor
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Ramakrishna Nirogi,
Anil Shinde,
Anand Daulatabad,
Ramasastri Kambhampati,
Parandhama Gudla,
Mohammad Shaik,
Muralimohan Gampa,
Suresh Balasubramaniam,
Pamuletinarasimhareddy Gangadasari,
Veena Reballi,
Rajeshkumar Badange,
Kumar Bojja,
Ramkumar Subramanian,
Gopinadh Bhyrapuneni,
Nageshwararao Muddana,
Pradeep Jayarajan
Journal of Medicinal Chemistry 10/2012; · 5.25 Impact Factor
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ABSTRACT: A rapid and sensitive liquid chromatography tandem mass spectrometry method for simultaneous quantification of catecholamine neurotransmitters in microdialysates was developed. The catecholamine neurotransmitters dopamine (DA) and norepinephrine (NE) were pre-column derivatized with dansyl chloride and analyzed. A gradient elution method was used to separate the analytes from the interferences on an Agilent Poroshell 120 EC-C18 outer porous micro particulate column. The method was robust and sensitive to determine with the lower limit of quantification value of 0.068pmol/mL and 0.059pmol/mL for DA and NE, respectively. It has acceptable precision and accuracy for concentrations over the standard curve range. The method was successfully applied for simultaneous quantitation of DA and NE in the prefrontal cortex (PFC) dialysates of rats obtained from a microdialysis study dosed with vehicle and atomoxetine through intra peritoneal (i.p.) route at a dose of 3mg/kg to monitor the change in extracellular concentrations. Thus, accomplishment of this method would facilitate the neurochemical monitoring for discovery of new chemical entities targeted for the treatment of attention deficit hyperactivity disorder (ADHD).
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2012; 913-914C:41-47. · 2.78 Impact Factor
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ABSTRACT: Deferasirox is an iron chelating agent for the treatment of transfusional iron over load in patients with chronic anemia. These anemic patients require close monitoring of the deferasirox exposures for ensuring its therapeutic efficacy. Dried blood spot (DBS) sampling methodology has the advantages of low volume of blood withdrawal and ease of transportation and storage over liquid blood methods. A LC-MS/MS based analytical method was developed using reversed phase column with gradient elution program and quantitated in MRM mode. Linearity range for the liquid blood was 1-1000ng/mL and for DBS was 5-5000ng/mL under similar mass spectrometry conditions. The method was validated with respective (M-H)(-) ions, m/z 372→118 for deferasirox and m/z 410→348 for fluvastatin (internal standard). The validated method was applied for the analysis of DBS samples from a rat pharmacokinetic study and results were compared against liquid blood samples from the same animal. The mean C(max) from DBS sample (1121ng/mL) was comparable to mean C(max) found in blood samples (1015ng/mL) at 2h after oral dose of deferasirox. All the other calculated pharmacokinetic parameters were quite comparable for both liquid blood and DBS samples.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2012; 907:65-73. · 2.78 Impact Factor
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Ramakrishna Nirogi,
Ramanatha Saralaya,
Vishwottam Kandikere,
Nageswararao Muddana,
Nilkanth Naik,
Raghava Chowdary Palacharla,
Suraj Tubachi,
Ramkumar Subramanian,
Muddukrishna Chillakur,
Gopinadh Bhyrapuneni,
Mohammed Abdul Rasheed,
Koteshwara Mudigonda,
Prashanth Komarneni
Alzheimer's & dementia: the journal of the Alzheimer's Association 07/2012; 8(4):P400. · 5.90 Impact Factor
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Ramakrishna Nirogi,
Gopinadh Bhyrapuneni,
Vishwottam Kandikere,
Ramanatha Saralaya,
Nageswararao Muddana,
Raghava Chowdary Palacharla,
Vijaya Bhargava Kanamarlapudi,
Ramoji Kosuru,
Nilkanth Naik,
Suraj Tubachi,
Ramkumar Subramanian,
Muddukrishna Chillakur,
Anil Shinde,
Adireddy Dwarampudi
Alzheimer's & dementia: the journal of the Alzheimer's Association 07/2012; 8(4):P712-P713. · 5.90 Impact Factor
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Ramakrishna Nirogi,
Pradeep Jayarajan,
Vishwottam Kandikere,
Medapati Rajesh Babu,
Vijay Benade,
Grandhi V. Ramalingayya,
Lakshmi Narayana Manjunath,
Ranjithkumar Ponnamaneni,
Mohammed Abdul Rasheed,
Mohamad Sadik Mulla,
Suresh Yarlagadda,
Ravella Srinivasa Rao
Alzheimer's & dementia: the journal of the Alzheimer's Association 07/2012; 8(4):P399. · 5.90 Impact Factor
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Alzheimer's & dementia: the journal of the Alzheimer's Association 07/2012; 8(4):P199. · 5.90 Impact Factor
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Ramakrishna Nirogi,
Vijay Benade,
Vishwottam Kandikere,
Ranjithkumar Ponnamaneni,
Lakshmi Narayana Manjunath,
Ramanatha Saralaya,
Gopinadh Bhyrapuneni,
Pradeep Jayarajan,
Renny Abraham,
Ramu Yaramasu,
Saravana Kumar Marimuthu,
Swayam Sampurna Mohanty
Alzheimer's & dementia: the journal of the Alzheimer's Association 07/2012; 8(4):P206. · 5.90 Impact Factor
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Ramakrishna Nirogi,
Pradeep Jayarajan,
Vishwottam Kandikere,
Medapati Rajesh Babu,
Vijay Benade,
Grandhi V. Ramalingayya,
Lakshmi Narayana Manjunath,
Ranjithkumar Ponnamaneni,
Mohammed Abdul Rasheed,
Mohamad Sadik Mulla,
Suresh Yarlagadda,
Ravella Srinivasa Rao
Alzheimer's & Dementia. 07/2012; 8(4):P399.
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ABSTRACT: Measurement of unbound test compound concentrations at the biophase is routinely carried out in the drug discovery. Microdialysis is an established sampling technique for in vivo measurement of endogenous and exogenous compounds and it is commonly used for monitoring true concentrations. Endogenous compounds like neurotransmitters and neuropeptides in the brain are routinely evaluated as a proof of pharmacological activity of test compounds. Although, microdialysis offers several advantages over the conventional techniques for its use in brain pharmacokinetics, the absolute determination of extracellular concentrations of test compound depends on the predictable non-specific binding to the tubing and probe membrane. In the present investigation, we have demonstrated steps to predict non-specific binding and described approaches to reduce while working with compounds having different degree of adsorption properties. Non-specific binding to the tubing was measured in vitro for seven structurally diverse compounds and based on the binding characteristics, changes were adapted in study conditions. In vitro probe extraction efficiency was evaluated by gain and loss, which was further used as a second layer of measurement for non-specific binding. For selected compounds, in vivo probe extraction efficiencies were carried out and brain pharmacokinetics was evaluated in the prefrontal cortex of male Sprague-Dawley rats. Thus, the present approach demonstrates a systematic approach for evaluating and reducing the non-specific binding of test compounds to the microdialysis tubing and probe membranes. The stepwise approach described will strengthen the applicability of microdialysis in brain pharmacokinetics.
Journal of neuroscience methods 06/2012; 209(2):379-87. · 2.30 Impact Factor
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ABSTRACT: During pregnancy, the disposition of various drugs is altered due to changes in physiological condition, maternal gastrointestinal absorption, gastric secretion and motility. A fixed dose combination of antiretrovirals is commonly prescribed for the treatment of HIV infection. There is a need to understand the pharmacokinetics and placental transfer of efavirenz-emtricitabine-tenofovir in fixed dose combination during pregnancy. The pharmacokinetics and placental transfer of efavirenz-emtricitabine-tenofovir fixed dose combination was evaluated in timed pregnant and non-pregnant Sprague-Dawley rats at 30, 10, 15 mg/kg p.o., respectively. The plasma, placental tissue, amniotic fluid and fetal tissue concentrations were measured using high performance liquid chromatography combined with tandem mass spectrometric detector (LC-MS/MS). To summarize, the pharmacokinetic profile of efavirenz remained similar in the pregnant and non-pregnant rats. However, a considerable difference in the pharmacokinetics of emtricitabine and tenofovir was observed in pregnant and non-pregnant rats. Efavirenz and emtricitabine showed appreciable placental, amniotic fluid and fetal exposure compared with tenofovir. The present study suggests that a profound impact on antiretroviral pharmacokinetics was observed during pregnancy and there is a need to monitor the exposure levels of each drug when administered as a fixed dose combination during pregnancy. Further studies to explore the pharmacokinetic parameters of fixed dose antiretrovirals during the preclinical stage in a timed-pregnancy rat model are required. Such studies can help in the development of safe and effective medications with a reduced risk of perinatal transmission of HIV-1 infection.
Biopharmaceutics & Drug Disposition 05/2012; 33(5):265-77. · 2.07 Impact Factor
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ABSTRACT: Reduction of cerebral cortical and hippocampal α7 neuronal nicotinic acetylcholine receptor (nAChR) density was observed in the Alzheimer's disease (AD) and other neurodegenerative diseases. Mapping the subtypes of nAChRs with selective ligand by viable, quick and consistent method in preclinical drug discovery may lead to rapid development of more effective therapeutic agents. The objective of this study was to evaluate the use of methyllycaconitine (MLA) in non-radiolabeled form for mapping α7 nAChRs in rat brain.
MLA pharmacokinetic and brain penetration properties were assessed in male Wistar rats. The tracer properties of MLA were evaluated in rat brain by dose and time dependent differential regional distribution studies. Target specificity was validated after blocking with potent α7 nAChR agonists ABBF, PNU282987 and nicotine. High performance liquid chromatography combined with triple quad mass spectral detector (LC-MS/MS) was used to measure the plasma and brain tissue concentrations of MLA.
MLA has shown rapid brain uptake followed by a 3-5 fold higher specific binding in regions containing the α7 nAChRs (hypothalamus - 1.60 ng/g), when compared to non-specific regions (striatum - 0.53 ng/g, hippocampus - 0.46 ng/g, midbrain - 0.37 ng/g, frontal cortex - 0.35 ng/g and cerebellum - 0.30 ng/g). Pretreatment with potent α7 nAChR agonists significantly blocked the MLA uptake in hypothalamus. The non-radiolabeled MLA binding to brain region was comparable with the α7 mRNA localization and receptor distribution reported for [(3)H] MLA in rat brain.
The rat pharmacokinetic, brain penetration and differential brain regional distribution features favor that MLA is suitable to use in preclinical stage for mapping α7 nAChRs. Hence, this approach can be employed as an essential tool for quicker development of novel selective ligand to map variation in the α7 receptor densities, as well as to evaluate potential new chemical entities targeting neurodegenerative diseases.
Journal of pharmacological and toxicological methods 05/2012; 66(1):22-8. · 2.32 Impact Factor
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ABSTRACT: The most commonly used Von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment. In preclinical research, neuropathic pain models of nerve injury with varied etiology like partial sciatic nerve ligation (PNL), chronic constricted injury (CCI) and spinal nerve ligation (SNL) are employed to screen the analgesic drugs to treat symptoms like allodynia and hyperalgesia. The current study was aimed to validate and compare conventionally used Von Frey monofilaments and automated dynamic plantar aesthesiometer using three different pain models.
PNL, CCI and SNL rats were used to compare and validate the assessment of neuropathic pain using Von Frey monofilaments and automated dynamic plantar aesthesiometer.
Mechanical allodynia was assessed at various time points to mimic drug testing conditions in neuropathic pain models and anticipated to observe reliable and reproducible paw withdrawal threshold measurements across these models. Consistent paw withdrawal thresholds were observed in all the three models of neuropathic pain with Von Frey monofilaments, whereas variable paw withdrawal thresholds were noticed in PNL and CCI models but not in SNL model with dynamic plantar aesthesiometer.
Manually used Von Frey filaments can be used in assessment of mechanical allodynia in all the three models, whereas dynamic plantar aesthesiometer is suitable for assessing mechanical allodynia in SNL but not in PNL and CCI models. The reason for variable paw withdrawal thresholds during assessment of mechanical allodynia in PNL and CCI models with dynamic plantar aesthesiometer may be due to the paw deformity and change in foot posture.
Journal of pharmacological and toxicological methods 04/2012; 66(1):8-13. · 2.32 Impact Factor
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ABSTRACT: In vivo brain receptor occupancy has been the key assay in driving preclinical drug discovery program and there is a need to hasten this screening step. Radiolabeled methods, which are time consuming and expensive, are most widely employed to measure receptor occupancy. Thus we sought to develop and validate an alternative novel approach for measuring rat brain α₄β₂ neuronal nicotinic acetylcholine receptor occupancy using high performance liquid chromatography combined with tandem mass spectrometric detector (LC-MS/MS).
Tracer optimization studies like in vivo dose and time dependent brain regional distribution; saturation binding and blocking study with nicotine and atropine were carried out for ZW-104 in rats. Assay validity was tested by pretreatment with potent α₄β₂ ligands; TC-1734, cytisine, ABT-089, ABT-594 and A-366833. Receptor occupancy along with plasma and brain exposure levels of α₄β₂ ligand was measured in the same set of animals.
The regional distribution of ZW-104 in rat was found to be, thalamus>frontal cortex>striatum>hippocampus>cerebellum, and is in accordance with the distribution and regional densities of α₄β₂ nAChRs measured using [¹⁸F]ZW-104 in mice and baboons. Pretreatment with nicotine and α₄β₂ ligands dose dependently reduced the binding of ZW-104 in the thalamus. Non-nicotinic antagonist atropine did not alter the binding of ZW-104 in the thalamus, indicating the tracer specificity. The ED₅₀ values calculated for occupancy were found to be 3.01, 0.83, 14.81, 0.001 and 0.11 mg/kg for TC-1734, cytisine, ABT-089, ABT-594, and A-366833, respectively.
These findings demonstrate that non-radiolabeled ZW-104 is suitable for determining the α₄β₂ receptor occupancy in rat brain. The LC-MS/MS based receptor occupancy assay is a rapid method and allows the generation of occupancy data along with the brain and plasma concentration in the same group of animals.
Journal of pharmacological and toxicological methods 04/2012; 65(3):136-41. · 2.32 Impact Factor
