Identification of monoclonal antibody drug substances using non-destructive Raman spectroscopy.

Monoclonal antibodies provide highly specific and effective therapies for the treatment of chronic diseases. These protein-based therapeutics, or drug substances, are transported in single used plastic packaging to fill finish sites. According to good manufacturing practice guidelines, each drug substance needs to be identified before manufacturing of the drug product. However, considering their complex structure, it is challenging to correctly identify therapeutic proteins in an efficient manner. Common analytical techniques for therapeutic protein identification are SDS-gel electrophoresis, enzyme linked immunosorbent assays, high performance liquid chromatography and mass spectrometry-based assays. Although effective in correctly identifying the protein therapeutic, most of these techniques need extensive sample preparation and removal of samples from their containers. This step not only risks contamination but the sample taken for the identification is destroyed and cannot be re-used. Moreover, these techniques are often time consuming, sometimes taking several days to process. Here, we address these challenges by developing a rapid and non-destructive identification technique for monoclonal antibody-based drug substances. Raman spectroscopy in combination with chemometrics were used to identify three monoclonal antibody drug substances. This study explored the impact of laser exposure, time out of refrigerator and multiple freeze thaw cycles on the stability of monoclonal antibodies. and demonstrated the potential of using Raman spectroscopy for the identification of protein-based drug substances in the biopharmaceutical industry.

Biochemical and molecular properties of LHCX1, the essential regulator of dynamic photoprotection in diatoms.

Light harvesting is regulated by a process triggered by the acidification of the thylakoid lumen, known as nonphotochemical "energy-dependent quenching" (qE). In diatoms, qE is controlled by the light-harvesting complex (LHC) protein LHCX1, while the LHC stress-related (LHCSR) and photosystem II subunit S proteins are essential for green algae and plants, respectively. Here, we report a biochemical and molecular characterization of LHCX1 to investigate its role in qE. We found that, when grown under intermittent light, Phaeodactylum tricornutum forms very large qE, due to LHCX1 constitutive upregulation. This "super qE" is abolished in LHCX1 knockout mutants. Biochemical and spectroscopic analyses of LHCX1 reveal that this protein might differ in the character of binding pigments relative to the major pool of light-harvesting antenna proteins. The possibility of transient pigment binding or not binding pigments at all is discussed. Targeted mutagenesis of putative protonatable residues (D95 and E205) in transgenic P. tricornutum lines does not alter qE capacity, showing that they are not involved in sensing lumen pH, differently from residues conserved in LHCSR3. Our results suggest functional divergence between LHCX1 and LHCSR3 in qE modulation. We propose that LHCX1 evolved independently to facilitate dynamic tracking of light fluctuations in turbulent waters. The evolution of LHCX(-like) proteins in organisms with secondary red plastids, such as diatoms, might have conferred a selective advantage in the control of dynamic photoprotection, ultimately resulting in their ecological success.

Rapid regulation of photosynthetic light harvesting in the absence of minor antenna and reaction centre complexes.

Plants are subject to dramatic fluctuations in the intensity of sunlight throughout the day. When the photosynthetic machinery is exposed to high light, photons are absorbed in excess, potentially leading to oxidative damage of its delicate membrane components. A photoprotective molecular process called non-photochemical quenching (NPQ) is the fastest response carried out in the thylakoid membranes to harmlessly dissipate excess light energy. Despite having been intensely studied, the site and mechanism of this essential regulatory process are still debated. Here, we show that the main NPQ component called energy-dependent quenching (qE) is present in plants with photosynthetic membranes largely enriched in the major trimeric light-harvesting complex (LHC) II, while being deprived of all minor LHCs and most photosystem core proteins. This fast and reversible quenching depends upon thylakoid lumen acidification (ΔpH). Enhancing ΔpH amplifies the extent of the quenching and restores qE in the membranes lacking PSII subunit S protein (PsbS), whereas the carotenoid zeaxanthin modulates the kinetics and amplitude of the quenching. These findings highlight the self-regulatory properties of the photosynthetic light-harvesting membranes in vivo, where the ability to switch reversibly between the harvesting and dissipative states is an intrinsic property of the major LHCII.

A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching.

Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment-protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.

Novel Tetrahydroquinazolinamines as Selective Histamine 3 Receptor Antagonists for the Treatment of Obesity.

The histamine 3 receptor (H3R) is a presynaptic receptor, which modulates several neurotransmitters including histamine and various essential physiological processes, such as feeding, arousal, cognition, and pain. The H3R is considered as a drug target for the treatment of several central nervous system disorders. We have synthesized and identified a novel series of 4-aryl-6-methyl-5,6,7,8-tetrahydroquinazolinamines that act as selective H3R antagonists. Among all the synthesized compounds, in vitro and docking studies suggested that the 4-methoxy-phenyl-substituted tetrahydroquinazolinamine compound 4c has potent and selective H3R antagonist activity (IC50 < 0.04 µM). Compound 4c did not exhibit any activity on the hERG ion channel and pan-assay interference compounds liability. Pharmacokinetic studies showed that 4c crosses the blood brain barrier, and in vivo studies demonstrated that 4c induces anorexia and weight loss in obese, but not in lean mice. These data reveal the therapeutic potential of 4c as an anti-obesity candidate drug via antagonizing the H3R.

Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein.

Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved "Arg patch" on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.

Plant and algal chlorophyll synthases function in Synechocystis and interact with the YidC/Alb3 membrane insertase.

In the model cyanobacterium Synechocystis sp. PCC 6803, the terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), forms a complex with high light-inducible proteins, the photosystem II assembly factor Ycf39 and the YidC/Alb3/OxaI membrane insertase, co-ordinating chlorophyll delivery with cotranslational insertion of nascent photosystem polypeptides into the membrane. To gain insight into the ubiquity of this assembly complex in higher photosynthetic organisms, we produced functional foreign chlorophyll synthases in a cyanobacterial host. Synthesis of algal and plant chlorophyll synthases allowed deletion of the otherwise essential native cyanobacterial gene. Analysis of purified protein complexes shows that the interaction with YidC is maintained for both eukaryotic enzymes, indicating that a ChlG-YidC/Alb3 complex may be evolutionarily conserved in algae and plants.

Molecular Origin of Photoprotection in Cyanobacteria Probed by Watermarked Femtosecond Stimulated Raman Spectroscopy.

Photoprotection is fundamental in photosynthesis to avoid oxidative photodamage upon excess light exposure. Excited chlorophylls (Chl) are quenched by carotenoids, but the precise molecular origin remains controversial. The cyanobacterial HliC protein belongs to the Hlip family ancestral to plant light-harvesting complexes, and binds Chl a and ß-carotene in 2:1 ratio. We analyzed HliC by watermarked femtosecond stimulated Raman spectroscopy to follow the time evolution of its vibrational modes. We observed a 2 ps rise of the C═C stretch band of the 2Ag- (S1) state of ß-carotene upon Chl a excitation, demonstrating energy transfer quenching and fast excess-energy dissipation. We detected two distinct ß-carotene conformers by the C═C stretch frequency of the 2Ag- (S1) state, but only the ß-carotene whose 2Ag- energy level is significantly lowered and has a lower C═C stretch frequency is involved in quenching. It implies that the low carotenoid S1 energy that results from specific pigment-protein or pigment-pigment interactions is the key property for creating a dissipative energy channel. We conclude that watermarked femtosecond stimulated Raman spectroscopy constitutes a promising experimental method to assess energy transfer and quenching mechanisms in oxygenic photosynthesis.

Liquid chromatography-tandem mass spectrometry method for the quantification of a potent H3 receptor antagonist conessine in serum and its application to pharmacokinetic studies.

Conessine, a steroidal alkaloid obtained from the bark and seeds of the plant species of Apocynaceae family, elicits a histamine antagonistic action, selectively for the H3 histaminergic receptors. This alkaloid is used mainly for the treatment of dysentery and helminthic disorders. For the quantification of conessine in serum, a liquid chromatography-tandem mass spectrometry method was developed. Chromatographic separation was achieved on a Zorbax SB-CN column (100 × 4.6 mm, 3.5 µm), and a mobile phase consisting of 90% methanol in aqueous ammonium acetate buffer (pH 3.5) with 0.1% (v/v) formic acid at an isocratic flow rate of 0.6 ml/min at 40℃ provides efficiency in separation. A volume of 40 µl was injected each time and the run time for each sample was 5 min. Phenacetin (internal standard) was added to 50 µl of serum sample prior to liquid-liquid extraction using 3% isopropanol in n-hexane. The detection was performed on a 5500 QTRAP mass spectrometer by multiple reaction monitoring mode via electrospray ionization source. The multiple reaction monitoring of conessine and IS was m/ z 357.4 to m/ z 312.1 and m/ z 180.1 to m/ z 138.1, respectively. The method that showed selectivity and linearity in the range of 1-200 ng/ml was validated in terms of sensitivity, accuracy, precision and stability. The detection and quantitation limits were recognized at 0.1 and 1 ng/ml, respectively. The intra- and inter-day precision and accuracy fulfils the acceptance criteria. Applying the method to the pharmacokinetic studies in rats, conessine showed a peak serum concentration at 2 h post oral dose with a good bioavailability of 71.28 ± 4.65%.

Substituted carbamothioic amine-1-carbothioic thioanhydrides as novel trichomonicidal fungicides: Design, synthesis, and biology.

Sexually transmitted diseases like trichomoniasis along with opportunistic fungal infections like candidiasis are major global health burden in female reproductive health. In this context a novel non-nitroimidazole class of substituted carbamothioic amine-1-carbothioic thioanhydride series was designed, synthesized, evaluated for trichomonacidal and fungicidal activities, and was found to be more active than the standard drug Metronidazole (MTZ). Compounds were trichomonicidal in the MIC ranges of 4.77-294.1 µM and 32.46-735.20 µM against MTZ-susceptible and -resistant strains, respectively. Further, compounds inhibited the growth of at least two out of ten fungal strains tested at MIC of 7.50-240.38 µM. The most active compound (20) of this series was 3.8 and 9.5 fold more active than the MTZ against the two Trichomonas strains tested. Compound 20 also significantly inhibited the sulfhydryl groups present over Trichomonas vaginalis and was found to be more active than the MTZ in vivo. Further, a docking analysis carried out with cysteine proteases supported their thiol inhibiting ability and preliminary pharmacokinetic study has shown good distribution and systemic clearance.

Binding of pigments to the cyanobacterial high-light-inducible protein HliC.

Cyanobacteria possess a family of one-helix high-light-inducible proteins (HLIPs) that are widely viewed as ancestors of the light-harvesting antenna of plants and algae. HLIPs are essential for viability under various stress conditions, although their exact role is not fully understood. The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains four HLIPs named HliA-D, and HliD has recently been isolated in a small protein complex and shown to bind chlorophyll and ß-carotene. However, no HLIP has been isolated and characterized in a pure form up to now. We have developed a protocol to purify large quantities of His-tagged HliC from an engineered Synechocystis strain. Purified His-HliC is a pigmented homo-oligomer and is associated with chlorophyll and ß-carotene with a 2:1 ratio. This differs from the 3:1 ratio reported for HliD. Comparison of these two HLIPs by resonance Raman spectroscopy revealed a similar conformation for their bound ß-carotenes, but clear differences in their chlorophylls. We present and discuss a structural model of HliC, in which a dimeric protein binds four chlorophyll molecules and two ß-carotenes.

Model based population PK-PD analysis of furosemide for BP lowering effect: A comparative study in primary and secondary hypertension.

Though numerous reports have demonstrated multiple mechanisms by which furosemide can exert its anti-hypertensive response. However, lack of studies describing PK-PD relationship for furosemide featuring its anti-hypertensive property has limited its usage as a blood pressure (BP) lowering agent. Serum concentrations and mean arterial BP were monitored following 40 and 80mgkg-1 multiple oral dose of furosemide in spontaneously hypertensive rats (SHR) and DOCA-salt induced hypertensive (DOCA-salt) rats. A simultaneous population PK-PD relationship using Emax model with effect compartment was developed to compare the anti-hypertensive efficacy of furosemide in these rat models. A two-compartment PK model with Weibull-type absorption and first-order elimination best described the serum concentration-time profile of furosemide. In the present study, post dose serum concentrations of furosemide were found to be lower than the EC50. The EC50 predicted in DOCA-salt rats was found to be lower (4.5-fold), whereas the tolerance development was higher than that in SHR model. The PK-PD parameter estimates, particularly lower values of EC50, Ke and Q in DOCA-salt rats as compared to SHR, pinpointed the higher BP lowering efficacy of furosemide in volume overload induced hypertensive conditions. Insignificantly altered serum creatinine and electrolyte levels indicated a favorable side effect profile of furosemide. In conclusion, the final PK-PD model described the data well and provides detailed insights into the use of furosemide as an anti-hypertensive agent.

Effect of arteether and pyrimethamine coadministration on the pharmacokinetic and pharmacodynamic profile of ormeloxifene.

The study was intended to investigate the effect of concomitant administration of antimalarial drug (pyrimethamine or arteether) on pharmacokinetic and post coitus contraceptive efficacy of ormeloxifene in female Sprague-Dawley rats. A serial sampling technique coupled with LC-MS/MS detection was utilized for quantification of ormeloxifene in plasma samples collected from female rats treated with ormeloxifene only and ormeloxifene with pyrimethamine or arteether. Coitus-proven female rats were utilized to investigate the effect of pyrimethamine or arteether coadministration on contraceptive efficacy of ormeloxifene by investigating the presence or absence of implantations and status of corpora lutea on day 10 post coitum. None of the sperm-positive rats treated with ormeloxifene with or without coadministration of pyrimethamine or arteether showed any sign of pregnancy, confirming that concomitant administration of antimalarial drugs (pyrimethamine or arteether) did not affect the pharmacodynamic profile of ormeloxifene. Although there was no sign of pharmacodynamic interaction, the volume of distribution of ormeloxifene increased significantly on cotreatment with pyrimethamine. However, coadministration of arteether did not affect any of the pharmacokinetic parameters of ormeloxifene. The compiled results of preliminary study in female rats support that pyrimethamine or arteether can be prescribed with ormeloxifene.

Novel aryl piperazines for alleviation of 'andropause' associated prostatic disorders and depression.

A series of seventeen piperazine derivatives have been synthesized and biologically evaluated for the management of andropause-associated prostatic disorders and depression. Five compounds 16, 19, 20, 21 and 22 significantly inhibited proliferation of androgen-sensitive LNCaP prostatic cell line with EC50 values of 12.4 µM, 15.6 µM, 11.8 µM, 10.4 µM, 12.2 µM respectively and decreased Ca2+ entry through adrenergic-receptor α1A blocking activity. Anti-androgenic behaviour of compound 19 and 22 was evident by decreased luciferase activity. The high EC50 value in AR-negative cells PC3 and DU145 suggested that the cytotoxicity of compounds was due to AR down regulation. Compound 19 reduced the prostate weight of rats by 53.8%. Further, forced-swimming and tail-suspension tests revealed antidepressant-like activity of compound 19, lacking effects on neuromuscular co-ordination. In silico ADMET predictions revealed that the compound 19 had good oral absorption, aqueous solubility, non-hepatotoxic and good affinity for plasma protein binding. Pharmacokinetic and tissue uptake of 19 at 10 mg/kg demonstrated an oral bioavailability of 35.4%. In silico docking studies predicted similar binding pattern of compound 19 on androgen receptor as hydroxyflutamide. Compound 19 appears to be a unique scaffold with promising activities against androgen associated prostatic disorders in males like prostate cancer and BPH and associated depression.

Simultaneous LC-MS-MS Determination of Lopinavir and Rifabutin in Human Plasma.

Tuberculosis (TB) with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome represents the most common infectious diseases worldwide. Anti-TB drugs are used concurrently with antiretroviral drug for treatment of TB-HIV co-morbidities. Due to lower risk of interaction with protease inhibitors, rifabutin is preferred over rifampicin in treatment of HIV and TB co-morbidity. A simple and specific liquid chromatography tandem mass spectrometry method was developed for quantification of rifabutin (RBT) and lopinavir (LPV) simultaneously in human plasma. Following extraction using 60% n-hexane in ethyl acetate, the processed samples were chromatographed on a Discovery HS C18 column (5 µm, 50 × 4.6 mm, id) using mobile phase [85% acetonitrile in ammonium acetate buffer (10 mM, pH 4.5)] at a flow rate of 0.7 mL/min. Mass spectrometric detection was performed in positive electrospray ionization mode using multiple reaction monitoring (RBT, m/z 847.7 → 815.4; LPV, m/z 629.6 → 447.4). Raloxifene and phenacetin were used as internal standards for RBT and LPV, respectively. Linearity was established in the range of 1-1,000 ng/mL and 0.5-10 µg/mL (R2 ≥ 0.99) for RBT and LPV, respectively. The recovery of LPV and RBT were always >90 and >50%, respectively. The precisions and accuracies were well within the acceptable limits of variation.

An insight into tetrahydro-ß-carboline-tetrazole hybrids: synthesis and bioevaluation as potent antileishmanial agents.

A series of 2,3,4,9-tetrahydro-ß-carboline tetrazole derivatives (14a-u) have been synthesized utilizing the Ugi multicomponent reaction and were identified as potential antileishmanial chemotypes. Most of the screened derivatives exhibited significant in vitro activity against the promastigote (IC50 from 0.59 ± 0.35 to 31 ± 1.27 µM) and intracellular amastigote forms (IC50 from 1.57 ± 0.12 to 17.6 ± 0.2 µM) of L. donovani, and their activity is comparable with standard drugs miltefosine and sodium stibogluconate. The most active compound 14t was further studied in vivo against the L. donovani/golden hamster model at a dose of 50 mg kg-1 through the intraperitoneal route for 5 consecutive days, which displayed 75.04 ± 7.28% inhibition of splenic parasite burden. Pharmacokinetics of compound 14t was studied in the golden Syrian hamster, and following a 50 mg kg-1 oral dose, the compound was detected in hamster serum for up to 24 h. It exhibited a large volume of distribution (651.8 L kg-1), high clearance (43.2 L h-1 kg-1) and long mean residence time (10 h).

Twisting a ß-Carotene, an Adaptive Trick from Nature for Dissipating Energy during Photoprotection.

Cyanobacteria possess a family of one-helix high light-inducible proteins (Hlips) that are homologous to light-harvesting antenna of plants and algae. An Hlip protein, high light-inducible protein D (HliD) purified as a small complex with the Ycf39 protein is evaluated using resonance Raman spectroscopy. We show that the HliD binds two different ß-carotenes, each present in two non-equivalent binding pockets with different conformations, having their (0,0) absorption maxima at 489 and 522 nm, respectively. Both populations of ß-carotene molecules were in all-trans configuration and the absorption position of the farthest blue-shifted ß-carotene was attributed entirely to the polarizability of the environment in its binding pocket. In contrast, the absorption maximum of the red-shifted ß-carotene was attributed to two different factors: the polarizability of the environment in its binding pocket and, more importantly, to the conformation of its ß-rings. This second ß-carotene has highly twisted ß-rings adopting a flat conformation, which implies that the effective conjugation length N is extended up to 10.5 modifying the energetic levels. This increase in N will also result in a lower S1 energy state, which may provide a permanent energy dissipation channel. Analysis of the carbonyl stretching region for chlorophyll a excitations indicates that the HliD binds six chlorophyll a molecules in five non-equivalent binding sites, with at least one chlorophyll a presenting a slight distortion to its macrocycle. The binding modes and conformations of HliD-bound pigments are discussed with respect to the known structures of LHCII and CP29.

A combination of complexation and self-nanoemulsifying drug delivery system for enhancing oral bioavailability and anticancer efficacy of curcumin.

OBJECTIVE: Curcumin, the golden spice from Indian saffron, has shown chemoprotective action against many types of cancer including breast cancer. However, poor oral bioavailability is the major hurdle in its clinical application. In the recent years, self-nanoemulsifying drug delivery system (SNEDDS) has emerged as a promising tool to improve the oral absorption and enhancing the bioavailability of poorly water-soluble drugs. In this context, complexation with lipid carriers like phospholipid has also shown the tremendous potential to improve the solubility and therapeutic efficacy of certain drugs with poor oral bioavailability. METHODS: In the present investigation, a systematic combination of both the approaches is utilized to prepare the phospholipid complex of curcumin and facilitate its incorporation into SNEDDS. The combined use of both the approaches has been explored for the first time to enhance the oral bioavailability and in turn increase the anticancer activity of curcumin. RESULTS: As evident from the pharmacokinetic studies and in situ single pass intestinal perfusion studies in Sprague-Dawley rats, the optimized SNEDDS of curcumin-phospholipid complex has shown enhanced oral absorption and bioavailability of curcumin. The cytotoxicity study in metastatic breast carcinoma cell line has shown the enhancement of cytotoxic action by 38.7%. The primary tumor growth reduction by 58.9% as compared with the control group in 4T1 tumor-bearing BALB/c mice further supported the theory of enhancement of anticancer activity of curcumin in SNEDDS. CONCLUSION: The developed formulation can be a potential and safe carrier for the oral delivery of curcumin.

Preclinical pharmacokinetics and ADME characterization of a novel anticancer chalcone, cardamonin.

Cardamonin (CRD), a chalconoid obtained from several medicinal plants of Zingiberaceae family, had shown promising potential in cancer prevention and therapy. For further development and better pharmacological elucidation, we performed a series of in vitro and in vivo studies to characterize its preclinical pharmacokinetics. The study samples were analyzed using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high performance liquid chromatography-ultra violet (HPLC-UV) methods. CRD is partially soluble (<10 µM) and possess high permeability (>0.2 × 10-4 cm/sec). It is moderately bound to plasma proteins (<50%). It shows partitioning in red blood cell (RBC) compartment with the partition coefficient between RBCs and plasma (KRBC/P ) of 0.95 at 0 min to 1.39 at 60 min, indicating significant but slow RBC uptake. In mice, CRD is poorly absorbed after oral administration with 18% oral bioavailability. It possesses high clearance, short mean residence time, and high volume of distribution in mice. It exhibited multiple peak phenomena both after oral and intravenous administration and is excreted both as conjugated and unchanged CRD in bile. It is majorly excreted in faeces and negligibly in urine. The preclinical absorption, distribution, metabolism, and excretion data are expected to succour the future clinical investigations of CRD as a promising anticancer agent. Copyright © 2016 John Wiley & Sons, Ltd.

2-Methyl-4/5-nitroimidazole derivatives potentiated against sexually transmitted Trichomonas: Design, synthesis, biology and 3D-QSAR study.

Trichomoniasis is the most prevalent, non-viral sexually transmitted diseases (STD) caused by amitochondriate protozoan Trichomonas vaginalis. Increased resistance of T. vaginalis to the marketed drug Metronidazole necessitates the development of newer chemical entities. A library of sixty 2-methyl-4/5-nitroimidazole derivatives was synthesized via nucleophilic ring opening reaction of epoxide and the efficacies against drug-susceptible and -resistant Trichomonas vaginalis were evaluated. All the molecules except two were found to be active against both susceptible and resistant strains with MICs ranging 8.55-336.70 µM and 28.80-1445.08 µM, respectively. Most of the compounds were remarkably more effective than the standard Metronidazole. This study analyzes the in vitro and in vivo activities of the new 5-nitroimidazoles, which were found to be safe against human cervical HeLa cells with good selectivity index. The exploration of SAR by the synthesis of four different prototypes and 3D-QSAR study has shown the importance of prototype 1 over other prototypes.