Sridhar Veeraraghavan

Simultaneous quantification of lenalidomide, ibrutinib and its active metabolite PCI-45227 in rat plasma by LC–MS/MS: Application to a pharmacokinetic study

Efficacy assessments using a combination of ibrutinib and lenalidomide necessitate the development of an analytical method for determination of both drugs in plasma with precision. A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of lenalidomide, ibrutinib, and its active metabolite PCI45227 in rat plasma. Extraction of lenalidomide, ibrutinib, PCI45227 and tolbutamide (internal standard; IS) from 50 μl rat plasma was carried out by liquid-liquid extraction with ethyl acetate:dichloromethane (90:10) ratio. Chromatographic separation of analytes was performed on YMC pack ODS AM (150 mm × 4.6 mm, 5 μm) column under gradient conditions with acetonitrile:0.1% formic acid buffer as the mobile phases at a flow rate of 1 ml/min. Precursor ion and product ion transition for analytes and IS were monitored on a triple quadrupole mass spectrometer, operated in the selective reaction monitoring with positive ionization mode. Method was validated over a concentration range of 0.72-183.20 ng/ml for ibrutinib, 0.76-194.33 ng/ml for PCI-45227 and 1.87-479.16 ng/ml for lenalidomide. Mean extraction recovery for ibrutinib, PCI-45227, lenalidomide and IS of 75.2%, 84.5%, 97.3% and 92.3% were consistent across low, medium, and high QC levels. Precision and accuracy at low, medium and high quality control levels were less than 15% across analytes. Bench top, wet, freeze-thaw and long term stability was evaluated for all the analytes. The analytical method was applied to support a pharmacokinetic study of simultaneous estimation of lenalidomide, ibrutinib, and its active metabolite PCI-45227 in Wistar rat. Assay reproducibility was demonstrated by re-analysis of 18 incurred samples.

Design, Synthesis, and Identification of Silicon Incorporated Oxazolidinone Antibiotics with Improved Brain Exposure

Therapeutic options for brain infections caused by pathogens with a reduced sensitivity to drugs are limited. Recent reports on the potential use of linezolid in treating brain infections prompted us to design novel compounds around this scaffold. Herein, we describe the design and synthesis of various oxazolidinone antibiotics with the incorporation of silicon. Our findings in preclinical species suggest that silicon incorporation is highly useful in improving brain exposures. Interestingly, three compounds from this series demonstrated up to a 30-fold higher brain/plasma ratio when compared to linezolid thereby indicating their therapeutic potential in brain associated disorders.

Simultaneous Determination of Celecoxib, Erlotinib, and its Metabolite Desmethyl-Erlotinib (OSI-420) in Rat Plasma by Liquid chromatography/Tandem Mass Spectrometry with Positive/Negative Ion-Switching Electrospray Ionisation.

A new method for the simultaneous determination of celecoxib, erlotinib, and its active metabolite desmethyl-erlotinib (OSI-420) in rat plasma, by liquid chromatography/tandem mass spectrometry with positive/negative ion-switching electrospray ionization mode, was developed and validated. Protein precipitation with methanol was selected as the method for preparing the samples. The analytes were separated on a reverse-phase C18 column (50mm×4.6mm i.d., 3μ) using methanol: 2 mM ammonium acetate buffer, and pH 4.0 as the mobile phase at a flow rate 0.8 mL/min. Sitagliptin and Efervirenz were used as the internal standards for quantification. The determination was carried out on a Theremo Finnigan Quantam ultra triple-quadrupole mass spectrometer, operated in selected reaction monitoring (SRM) mode using the following transitions monitored simultaneously: positive m/z 394.5→278.1 for erlotinib, m/z 380.3→278.1 for desmethyl erlotinib (OSI-420), and negative m/z −380.1→ −316.3 for celecoxib. The limits of quantification (LOQs) were 1.5 ng/mL for Celecoxib, erlotinib, and OSI-420. Within- and between-day accuracy and precision of the validated method were within the acceptable limits of < 15% at all concentrations. The quantitation method was successfully applied for the simultaneous estimation of celecoxib, erlotinib, and desmethyl erlotinib in a pharmacokinetic study in Wistar rats.

Simultaneous quantitation of IC87114, roflumilast and its active metabolite roflumilast N‐oxide in plasma by LC‐MS/MS: application for a pharmacokinetic study

A sensitive and reliable high-performance liquid chromatography-mass spectrometry (LC-MS/MS) was developed and validated for simultaneous quantification IC87114, roflumilast (RFM), and its active metabolite roflumilast N-oxide (RFN) using tolbutamide as an internal standard. The analytes were extracted by using liquid-liquid extraction and separated on a reverse phase C(18) column (50 mm × 3 mm i.d., 4.6 µ) using methanol: 2 mM ammonium acetate buffer, pH 4.0 as mobile phase at a flow rate 1 mL/min in gradient mode. Selective reaction monitoring was performed using the transitions m/z 398.3 > 145.9, 403.1 >186.9, 419.1 > 187.0 and 271.1 > 155.0 to quantify quantification IC87114, RFM, RFN and tolbutamide, respectively. The method was validated over the concentration range of 0.1-60 ng.mL(-1) for RFM and RFN and 6 to 2980 ng.mL(-1) for IC87114. Intra- and inter-day accuracy and precision of validated method were within the acceptable limits of <15% at all concentrations. Coefficients of correlation (r(2) ) for the calibration curves were >0.99 for all analytes. The quantitation method was successfully applied for simultaneous estimation of IC87114, RFM and RFN in a pharmacokinetic drug-drug interaction study in Wistar rats.

Simultaneous quantification of idelalisib, fludarabine and lenalidomide in rat plasma by using high-performance liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry

A sensitive and reliable high-performance liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of idelalisib, fludarabine and lenalidomide using tolbutamide as an internal standard. Analytes were recovered by liquid-liquid extraction and separated on a reverse phase C18 column (150mm×4.6mm i.d., 5μm) using methanol:0.1% formic acid buffer (70:30) as mobile phase at a flow rate of 1mL/min in isocratic mode. Selective reaction monitoring was performed using the transitions, i.e. m/z 416.25/176.48, 286.11/154.10, 260.15/149.15, and 271.14/155.06 to quantify idelalisib, fludarabine and lenalidomide and tolbutamide, respectively. The method was validated over the concentration range of 1.15-576.84ng/mL for idelalisib, 0.95-476.25ng/mL for fludarabine and 0.97-486.19ng/mL for lenalidomide. Intra and inter-day accuracy and precision of validated method were within the acceptable limits of <15%. Coefficients of correlation (r(2)) for the calibration curves were >0.998 for all analytes. The method was successfully applied for simultaneous estimation of idelalisib, fludarabine and lenalidomide in a pharmacokinetic study in rats.

Simultaneous quantification of ruxolitinib and nilotinib in rat plasma by LC–MS/MS: Application to a pharmacokinetic study

Efficacy assessments using a combination of ruxolitinib and nilotinib necessitate the development of a high precision analytical method for determination of both drugs in plasma. A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of ruxolitinib and nilotinib in rat plasma. Extraction of ruxolitinib, nilotinib and dasatinib (internal standard; IS) from 50μl rat plasma was carried out by protein precipitation with methanol. Chromatographic separation of analytes was performed on YMC pack ODS AM (150mm×4.6mm, 5μm) column under gradient conditions with acetonitrile:2.0mM ammonium acetate buffer as the mobile phase at a flow rate of 1ml/min. Precursor ion and product ion transition for both analytes and IS were monitored on a triple quadrupole mass spectrometer, operated in the selective reaction monitoring with positive ionization mode. Method was validated over a concentration range of 0.16-247ng/ml for ruxolitinib and 0.86-219ng/ml for nilotinib. Mean extraction recovery for ruxolitinib, nilotinib, and IS of 99.6%, 97.6% and 90.3% were consistent across low, medium, and high QC levels. Precision and accuracy at low, medium and high quality control levels were less than 15% across analytes. Bench top, wet, freeze-thaw and long term stability were evaluated for both analytes. The analytical method was applied to support a pharmacokinetic study of simultaneous estimation of ruxolitinib and nilotinib in Wistar rat. Assay reproducibility was demonstrated by re-analysis of 18 incurred samples.

DDD-028: a potent potential non-opioid, non-cannabinoid analgesic for neuropathic and inflammatory pain.

DDD-028 (4), a novel pentacyclic pyridoindolobenzazepine derivative was evaluated in vitro for receptor binding affinity and in vivo for analgesic activity using rodent models of neuropathic and inflammatory pain. DDD-028 does not bind to opioid, cannabinoid, dopamine, or histamine receptors. DDD-028 is very active even at the low oral dose of 1-5 mg/kg in both neuropathic, (spinal nerve ligation and chronic constriction injury) and inflammatory (Complete Freunds Adjuvant Induced) models of pain. DDD-028 appears to be about 6-fold more potent than pregabalin and indomethacin. Visual observation of all the animals used in these studies indicated that DDD-028 is well tolerated without any sedation. Thus, DDD-028 seems to be a promising candidate for the treatment of neuropathic and inflammatory pain without the possible side effects or abuse potential associated with opioid or cannabinoid activities.

Effect of multidose cilostazol on pharmacokinetic and lipid profile of atorvastatin in male Wistar rats.

Objectives  Atorvastatin (ATV) and cilostazol (CLZ) are often co‐prescribed to treat conditions such as peripheral arterial disease. In the present study, the drug–drug interaction potential of multi‐dose CLZ on both pharmacokinetics and the lipid‐lowering ability of single‐dose ATV is demonstrated.

Simultaneous Quantification of Baricitinib and Methotrexate in Rat Plasma by LC-MS/MS: Application to a Pharmacokinetic Study

Efficacy assessments using a combination of baricitinib and methotrexate necessitate the development of an analytical method for the determination of both drugs in plasma with precision. A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of baricitinib and methotrexate in rat plasma. Extraction of baricitinib, methotrexate, and tolbutamide (internal standard; IS) from 50 µL of rat plasma was carried out by protein precipitation with methanol. Chromatographic separation of the analytes was performed on the YMC pack ODS AM (150 mm × 4.6 mm, 5 µm) column under gradient conditions with methanol: 2.0 mM ammonium acetate buffer as the mobile phases at a flow rate of 1 mL/min. The precursor ion and product ion transition for both analytes and IS were monitored on a triple quadrupole mass spectrometer, operated with selective reaction monitoring in positive ionization mode. The method was validated over a concentration range of 0.5–250.00 ng/mL for baricitinib and methotrexate. Mean extraction recoveries for baricitinib, methotrexate, and IS of 86.8%, 89.4%, and 91.8% were consistent across low, medium, and high QC levels, respectively. Precision and accuracy at low, medium, and high quality control levels were less than 15% across the analytes. Benchtop, wet, freeze-thaw, and long-term stability were evaluated for both of the analytes. The analytical method was applied to support the pharmacokinetic study of simultaneous estimation of baricitinib and methotrexate in Wistar rats. Assay reproducibility was demonstrated by reanalysis of 18 incurred samples

Abstract B287: Selective targeting of Met-kinase by RP1400 attenuates tumor progression in mouse models of gastric cancer, glioblastoma, and hepatocellular carcinoma.

Background: c-Met is a proto-oncogene that encodes the protein Met with intrinsic tyrosine kinase activity. Aberrant Met kinase activity triggers a series of unwarranted phosphorylation events and signalling processes that ultimately lead to the development of cancer. Alteration of the Met kinase signalling cascade represents an attractive approach aimed at blocking invasion and metastasis of cancer cells. Herein, we describe the biological activity and pharmacokinetic properties of RP1400, a novel, selective, and potent Met kinase inhibitor with scope to be developed as a clinical candidate for cancers mediated by dysregulated Met kinase activity. Methods: Met Kinase activity of RP1400 was determined using an HTRF® KinEASE assay kit (Cisbio, Bedford, MA) with modifications. Met-dependent antiproliferative effect was determined in a host of Met amplified cell lines representative of various cancers. Inhibition of constitutive Met kinase phosphorylation in MKN-45 and NCI-H441 cells was measured in an ELISA assay. Subsequently, effect of the compound on Akt and Stat-5a phosphorylation, downstream markers in the Met signalling cascade, was determined. In vivo efficacy of RP1400 was evaluated in subcutaneous MKN-45 (gastric cancer), U87MG (glioblastoma), and MHCC97H (hepatocellular carcinoma) xenografts using SCID or nude mice. Pharmacokinetic behavior of the compound in plasma after single dose oral administration or IV injection was determined in Balb/c mice. Results: RP1400 demonstrated remarkable potency against the purified Met kinase enzyme (8.9 nM) with >50-fold selectivity against other kinases in a 451-kinase panel. Inhibition of Met kinase activity was accompanied by a significant reduction in constitutive Met phosphorylation in MKN-45 (28.6 nM) and NCI-H441 (1.8 nM) cells. As a consequence, Akt and Stat-5a phosphorylation were inhibited half-maximally in MKN-45 cells at 16.2 nM and 11.2 nM respectively. RP1400 caused a significant inhibition in proliferation of Met amplified cell lines including MKN-45, EBC-1, SNU-5, and MHCC97H with IC50 values ranging from 3-80 nM. Compounded with a favorable pharmacokinetic profile, in vitro potency of RP1400 translated into excellent in vivo efficacy with >80% reduction in tumor growth noticed in MKN-45, U87MG, and MHCC97H xenografts at 100 mg/kg/BID/PO dose. Conclusions: Our findings demonstrate the potency of RP1400, a novel and selective small-molecule inhibitor of Met kinase with efficacy values comparable or superior to existing Met kinase inhibitors in development. On lines with selective inhibitors, the compound displayed antiproliferative effect only in cell types harboring amplification of the Met kinase gene. RP1400 is currently undergoing extensive toxicological evaluation with clinical trials anticipated in H1 2014. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B287. Citation Format: Srikant Viswanadha, Babu G, Sridhar Veeraraghavan, Swaroop Vakkalanka. Selective targeting of Met-kinase by RP1400 attenuates tumor progression in mouse models of gastric cancer, glioblastoma, and hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B287.