Evaluation of in vitro Anti-psoriatic Activity of a Novel Polyherbal Formulation by Multiparametric Analysis.

BACKGROUND: We have developed a novel aqueous polyherbal formulation (SIRB-001) consisting of 3 herbs; Rheum palmatum L., Lonicera Japonica and Rehmannia glutinosa Libosch in the ratio 1:1:3. SIRB-001 has demonstrated efficacious effects in psoriasis patients. OBJECTIVE: This study was aimed at scientifically evaluating the in vitro antipsoriatic activity of SIRB-001. METHOD: The in vitro anti-psoriatic properties of SIRB-001 were assessed in human keratinocyte cell line; HaCaT. Anti-proliferative effect was studied using MTT assay. Apoptosis was examined by flow cytometry and colorimetric methods. Inflammatory markers and VEGF were determined by ELISA. IL-17/IL-23 secretion was assessed in immune cells. Signaling markers (kinases) by enzymatic assay and Topoisomerase-II activity by Kinetoplast DNA Cleavage assay was tested. RESULTS: SIRB-001 significantly inhibited (p<0.01) proliferation of HaCaT cells and induced apoptosis. Significant (p<0.01) downregulation of pro-inflammatory markers (TNF- α, IFN-γ, IL-6, NO, sPLA2) and VEGF was observed. IL-17/IL-23 secretion was significantly (p<0.01) alleviated in immune cells (RAW264.7 and THP-1). Inhibition of signaling markers (AKT1, FLT3, MAPK1, PRKCA, MAP2K) was observed. SIRB-001 demonstrated inhibition of Topoisomerase-II activity. High Performance Liquid Chromatography (HPLC) analysis of SIRB-001 was carried out using standard marker compounds chlorogenic acid (tR=13.98min), Acteoside (tR=24.22 min) and Rhein (tR=53.76 min). CONCLUSION: The in vitro results substantiate the anti-psoriatic effect of SIRB-001 in patients. SIRB-001 exerted anti-psoriatic effects at cellular level via multiple arms (antiproliferative, pro-apoptotic, anti-inflammatory, anti-angiogenic). This study provides insight into mechanism of action of SIRB-001 and highlights its promising potential for development as a herbal therapeutic agent for psoriasis, emphasizing the need of further pharmacological evaluation and toxicological studies.

Antidiabetic potential of polyherbal formulation DB14201: Preclinical development, safety and efficacy studies.

ETHNOPHARMACOLOGICAL RELEVANCE: The poly-herbal formulation DB14201 is a new combination of ayurvedic ingredients for treatment of diabetes. The aim of present study was to investigate safety and in vivo efficacy of DB14201 extract. Further this work was aimed to develop, characterize and standardize DB14201 extract and develop it as a botanical drug. MATERIALS AND METHODS: The polyherbal extract was standardized using four chemical markers. The LC-MS/MS method was developed for identification and quantification of mangiferin, berberine, kaempferol and curcumin. The extract was standardized for heavy metal content, aflotoxins, and microbial tests. The mechanism of action of DB14201 extract was explored through glucose uptake by adipocytes, TNF-α production and free fatty acid release, in vitro, was studied using murine adipocytes (3T3-L1). The effect of extract on insulin release was evaluated using murine pancreatic beta cell (ß TC-6). The safety and in vivo efficacy of extract was studied using suitable animal model. Hematology and blood biochemistry parameters were also assessed. RESULTS: In vitro studies of DB14201 in murine adipocytes and murine pancreatic beta cells demonstrated the plausible mechanism of action of DB14201 could be through increase in glucose uptake and by stimulation of insulin release by RIN-5f cells. The microbial load, heavy metals were found to be within the AYUSH permissible limits and aflotoxins were absent. Preclinical efficacy studies in animal models proved the anti-diabetic potential of the extract. The preclinical acute dose toxicity study and 90-days repeated dose toxicity study of DB14201 extract in wistar rats by oral route indicated that the extract is safe up to 1000mg/kg dose. Hematology and blood biochemistry parameters were within the normal range. CONCLUSIONS: The data presented herein demonstrated anti-diabetic potential of developed DB14201 extract and this study will serve as the benchmark for the further research on this polyherbal formulation.

Evaluation of herb-drug interaction of a polyherbal Ayurvedic formulation through high throughput cytochrome P450 enzyme inhibition assay.

ETHNOPHARMACOLOGICAL RELEVANCE: Arishtas are Ayurvedic formulation made with decoction of herbs. Arjunarishta formulation is being used in Ayurveda for cardio-protective activity. Ashwagandharishta formulation possesses antioxidant, anti-atherosclerotic and anti-stress properties. Ridayarishta, a novel empirical formulation was prepared using combination of selected ingredients from these two formulations to support healthy heart functions and to reduce stress. AIM OF THE STUDY: Aim of the Study was to investigate herb-drug interaction (HDI) of Ridayarishta formulation through human hepatic cytochrome P450 (CYP450) enzyme inhibition assay. MATERIALS AND METHODS: Ridayarishta formulation was phyto-chemically standardized against arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A using high performance thin layer chromatography (HPTLC) analysis. The formulation was standardized with respect to ethanol by gas chromatographic (GC) analysis. HDI was evaluated with Ridayarishta formulation and amlodipine besilate, atenolol, atorvastatin, metformin, glipizide glimepiride cocktail using high throughput CYP450 enzyme inhibition assay; against CYP1A2, 2C19, 2D6 and 3A4 isozymes. RESULTS: Contents of arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A in Ridayarishta formulation were found to be 1.76±0.12, 1.51±0.09, 1.85±0.05, 3.2±0.12, 1.21±0.08, and 2.16±0.09ppm, respectively. Quantity of ethanol in Ridayarishta was found to be 7.95±0.023% (V/V). Ridayarishta showed significantly higher (P<0.001) IC50 value against CYP1A2 (IC50-13.80±1.96µg/mL), 2C19 (IC50-14.343±2.28µg/mL), 2D6 (IC50-0.897±0.28µg/mL) and 3A4 (IC50-32.057±2.51µg/mL) compared to positive controls such as furafylline, tranylcypromine, quinidine and ketoconazole respectively. Cocktail of herbal formulation and cardio protective, antihypertensive, anti-diabetic drugs showed significantly (P<0.001and P<0.01) less or negligible HDI. CONCLUSION: Ridayarishta formulation alone and cocktail with amlodipine besilate, atenolol, atorvastatin, metformin, glipizide, glimepiride had negligible or insignificant effect on CYP450 inhibition. It may be concluded that consumption of Ridayarishta along with selective cardio protective, antihypertensive and anti-diabetic conventional medicine is safe with negligible or without any significant CYP450 (CYP1A2, 2C19, 2D6 and 3A4) inhibition mediated HDI.

Pharmacokinetic evaluation of C-3 modified 1,8-naphthyridine-3-carboxamide derivatives with potent anticancer activity: lead finding.

To develop naphthyridine derivatives as anticancer candidates, pharmacokinetic (PK) evaluations of 10 novel derivatives of 1,4-dihydro-4-oxo-1-proparagyl-1,8-naphthyridine-3-carboxamide, with potent anticancer activity were done using in vitro ADME (absorption, distribution, metabolism, excretion) and pharmacokinetic--pharmcodynamic (PK/PD) assays. Only derivatives 5, 6, 9 and 10 showed better metabolic stability, solubility, permeability, partition coefficient and cytochrome P450 (CYP) inhibition values. PK of derivatives 5, 6, 9 and 10 in rat showed comparable PK profile for derivative 5 (C0 = 6.98 µg/mL) and 6 (C0 = 6.61 µg/mL) with no detectable plasma levels for derivatives 9 and 10 at 5.0 mg/kg i.v. dose. PK/PD assay of derivatives 5 and 6 in tumor-bearing mice (TBM) showed comparable PK but tumor plasma index (TPI) of derivative 6 (4.02) was better than derivative 5 (2.50), suggesting better tumor uptake of derivative 6. Derivative 6, as lead compound, showed highest tumor growth inhibition (TGI) value of 33.6% in human ovary cancer xenograft model.

Efficiency and mechanism of intracellular paclitaxel delivery by novel nanopolymer-based tumor-targeted delivery system, Nanoxel(TM).

INTRODUCTION: An increasing research interest has been directed toward nanoparticle-based drug delivery systems for their advantages. The appropriate amalgamation of pH sensitivity and tumor targeting is a promising strategy to fabricate drug delivery systems with high efficiency, high selectivity and low toxicity. MATERIALS AND METHODS: A novel pH sensitive Cremophor-free paclitaxel formulation, Nanoxel(TM), was developed in which the drug is delivered as nanomicelles using a polymeric carrier that specifically targets tumors. The efficiency and mechanism of intracellular paclitaxel delivery by Nanoxel(TM) was compared with two other commercially available paclitaxel formulations: Abraxane(TM) and Intaxel(TM), using different cell lines representing target cancers [breast, ovary and non-small cell lung carcinoma (NSCLC)] by transmission electron microscopy and quantitative intracellular paclitaxel measurements by high performance liquid chromatography. RESULTS: The data obtained from the present study revealed that the uptake of nanoparticle-based formulations Nanoxel(TM) and Abraxane(TM) is mediated by the process of endocytosis and the uptake of paclitaxel was remarkably superior to Intaxel(TM) in all cell lines tested. Moreover, the intracellular uptake of paclitaxel in Nanoxel(TM)- and Abraxane(TM)-treated groups was comparable. Hence, the nanoparticle-based formulations of paclitaxel (Nanoxel(TM) and Abraxane(TM)) are endowed with higher efficiency to deliver the drug to target cells as compared to the conventional Cremophor-based formulation. CONCLUSION: Nanoxel(TM) appears to be of great promise in tumor targeting and may provide an advantage for paclitaxel delivery into cancer cells.

Application of a liquid chromatography-electrospray mass spectrometry (LC/MS) method to the biodistribution and excretion studies of novel 5'-chloro-2, 3-didehydroindolo (2', 3': 2, 3) betulinic acid (DRF-4012) in tumour-bearing mice.

Novel betulinic acid derivative 5'-chloro-2, 3-didehydroindolo [2', 3': 2, 3] betulinic acid (DRF-4012) is a new effective lupane type triterpenes with greater anticancer activity and efficacy than betulinic acid and currently under advanced preclinical investigation phase. In this study, a sensitive and rapid liquid chromatography-electrospray mass spectrometric (LC/MS) method has been developed for the determination of DRF-4012 in tumour-bearing mice plasma, urine, feces and tissues (liver, brain, lungs, heart, spleen, stomach, thigh muscle, kidneys, urinary bladder, small intestine and tumour). Biodistribution and excretion studies were performed for DRF-4012 nanoparticle (30 mg/kg body weight) after intravenous (i.v.) injection in tumour-bearing mice. DRF-4012 rapidly distributed throughout the body. After 0.5 h, tumour showed the second highest concentration, which was nearly half of the liver. After 4 and 24 h, the highest concentration of DRF-4012 was found in tumour indicating its retention in tumour site for a longer time. Excretion studies revealed that very low amount of unchanged DRF-4012 was observed in urine and primarily excreted through fecal route. This study may be useful to explain the manner in which DRF-4012 can inhibit tumour growth without apparent toxicity and preclinical/clinical evaluation of this potential antitumour agent.

Non-toxic melanoma therapy by a novel tubulin-binding agent.

(S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquino-lin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (EM011) is a tubulin-binding agent with significant anticancer activity. Here we show that EM011 modulates microtubule dynamics at concentrations that do not alter the total polymer mass of tubulin. In particular, EM011 decreases the transition frequencies between growth and shortening phases and increases the duration microtubules spend in an idle 'pause' state. Using B16LS9 murine melanoma cells, we show that EM011 briefly arrests cell-cycle progression at the G2/M phase by formation of multiple aster spindles. An aberrant mitotic exit without cytokinesis then occurs, leading to the accumulation of abnormal multinucleated cells prior to apoptosis. Our pharmacokinetic studies conformed to a linear dose-response relationship upto 150 mg/kg. However, non-linearity was observed at 300 mg/kg. In a syngeneic murine model of subcutaneous melanoma, better antitumor responses were seen at 150 mg/kg compared to 300 mg/kg of EM011. Unlike currently available chemotherapeutics, EM011 is non-toxic to normal tissues and most importantly, does not cause any immunosuppression and neurotoxicity. Our data thus warrant a clinical evaluation of EM011 for melanoma therapy.

Oral bioavailability enhancement of exemestane from self-microemulsifying drug delivery system (SMEDDS).

Limited aqueous solubility of exemestane leads to high variability in absorption after oral administration. To improve the solubility and bioavailability of exemestane, the self-microemulsifying drug delivery system (SMEDDS) was developed. SMEDDS comprises of isotropic mixture of natural or synthetic oil, surfactant, and cosurfactant, which, upon dilution with aqueous media, spontaneously form fine o/w microemulsion with less than 100 nm in droplet size. Solubility of exemestane were determined in various vehicles. Ternary phase diagrams were plotted to identify the efficient self-emulsification region. Dilution studies, droplet size, and zeta potential of the formulations were investigated. The release of exemestane from SMEDDS capsules was studied using USP dissolution apparatus in different dissolution media and compared the release of exemestane from a conventional tablet. Oral pharmacokinetic study was performed in female Wistar rats (n = 8) at the dose of 30 mg kg(-1). The absorption of exemestane from SMEDDS form resulted in about 2.9-fold increase in bioavailability compared with the suspension. Our studies illustrated the potential use of SMEDDS for the delivery of hydrophobic compounds, such as exemestane by the oral route.

High performance liquid chromatography method for the pharmacokinetic study of bicalutamide SMEDDS and suspension formulations after oral administration to rats.

Bicalutamide is a non-steroidal antiandrogen and is an oral medication that is used for treating prostate cancer. To evaluate the bioavailability of bicalutamide from bicalutamide self-microemulsifying drug delivery systems (SMEDDS) and bicalutamide suspension formulations, a sensitive, specific reversed-phase high performance liquid chromatographic (HPLC) method using ultraviolet detection was developed and validated for the analysis of bicalutamide (BCT) in rat blood plasma. Letrozole (LZ) was used as the internal standard. The chromatographic separation was achieved on C18 column at 35 degrees C, with a mobile phase consisting of water: acetonitrile (adjusted to pH 3.0 with 20% o-phosphoric acid) (60:40), at a flow rate of 1.0 mL min(-1). Bicalutamide and letrozole were well separated with retention times of 10.9+/-0.2 and 5.7+/-0.2 min, respectively. The method was successfully used to determine pharmacokinetics of bicalutamide, following oral administration of bicalutamide suspension and bicalutamide SMEDDS to wistar rats. Significant difference was observed in main pharmacokinetic parameters of tmax, Cmax and AUC(0 --> infinity) between SMEDDS and suspension, and a two fold increase in the relative bioavailability of bicalutamide was observed with the SMEDDS compared with suspension formulation. It was concluded that the absorption of bicalutamide from SMEDDS was enhanced.

Development and validation of reversed phase liquid chromatographic method utilizing ultraviolet detection for quantification of irinotecan (CPT-11) and its active metabolite, SN-38, in rat plasma and bile samples: application to pharmacokinetic studies.

A new, simple, sensitive and specific reversed-phase high performance liquid chromatographic (HPLC) method using ultraviolet detection was developed and validated for the analysis of CPT-11 (lambda(max)=254 nm, 365 nm) and its major active metabolite, SN-38 (lambda(max)=380 nm) in rat plasma and bile. The sample pre-treatment from plasma involved a single protein precipitation step with cold acetonitrile. In case of bile, liquid-liquid extraction with dichloromethane: tert-butyl methyl ether (3:7) was carried out. Topotecan, a structurally related camptothecin, was used as an internal standard. An aliquot of 50 microL was injected onto a C-18 column. The chromatographic separation was achieved by gradient elution consisting of acetonitrile and water (pH 3.0 adjusted with 20% o-phosphoric acid) at a flow rate of 1.0 ml/min. Total run time for each sample was 30 min. All the analytes viz. topotecan, CPT-11, SN-38 were well separated with retention times of 11.4, 13.4 and 15.5 min, respectively. Method was found to be selective, linear (R(2) approximately 0.999), accurate (recovery+/-15%) and precise (<5% C.V.) in the selected concentration ranges for both the analytes. The quantification limit for CPT-11 was 40 ngml(-1) and for SN-38 was 25 ngml(-1). The percent extraction efficiency was approximately 97% for CPT-11 and SN-38 from plasma while extraction recovery of CPT-11 and SN-38 from bile was approximately 70% and approximately 60%, respectively. The method was successfully used to determine plasma and biliary excretion time profiles of CPT-11 and SN-38, following oral and intravenous CPT-11 administration in rats. In the present study, irinotecan showed an absolute bioavailability of 30% as calculated from the pharmacokinetic data.

Pre-clinical evidence for altered absorption and biliary excretion of irinotecan (CPT-11) in combination with quercetin: possible contribution of P-glycoprotein.

P-glycoprotein (P-gp) is found to play a very significant role in intestinal and biliary transport of irinotecan and its active metabolite, SN-38. This makes P-gp inhibition a logical strategy for improving irinotecan's oral efficacy and reducing its toxicity. The objective of the present study was to identify the most suitable P-gp inhibitor, amongst various commonly used herbal components via in vitro screening; followed by determination of in vivo effects in rats. Caco-2 cell monolayers were used to investigate the influence of various components (quercetin, hesperitin, piperine, curcumin and naringenin) on the transport of irinotecan. The secretory transport (basolateral-to-apical) was significantly decreased by all components (p<0.05) except piperine. In the apical-to-basolateral transport, quercetin showed the highest absorptive permeability enhancement and P-gp interaction potential making it an appropriate candidate for further in vivo studies in female Wistar rats. Quercetin pre-treatment resulted in increased irinotecan C(max) and area under curve (AUC) with a concomitant decrease in t(max), plasma clearance and volume of distribution (p<0.05). The absolute bioavailability (F) of irinotecan control was 33%, which was increased to 43% (1.3 fold) by quercetin administration. The amounts of irinotecan and SN-38 eliminated in bile in control rats, is reduced to almost half when treated with quercetin. Our studies not only propose a safe approach for bioavailability enhancement and reducing toxicity of irinotecan by P-gp inhibition but in another way also reiterate the significance of elucidating herb-drug interactions for future insights.

Preclinical pharmacokinetics and bioavailability of noscapine, a tubulin-binding anticancer agent.

BACKGROUND: Noscapine, a naturally occurring antitussive phthalideisoquinoline alkaloid, is a tubulin-binding agent currently in Phase I/II clinical trials for anticancer therapy. Unlike currently available antimitotics such as taxanes and vincas, noscapine is water-soluble, well tolerated, and shows no detectable toxicity. OBJECTIVE: The goal was to develop a simple, sensitive, quantitative, selective, and less time-consuming high-performance liquid chromatography (HPLC) method for determination of noscapine and to study its pharmacokinetics in mice models. METHOD: Noscapine was extracted from mice plasma using the protein-precipitation method and detected using a reversed-phase C8 column with mobile phase consisting of 35% acetonitrile and 65% ammonium acetate buffer (pH 4.5) at 232 nm wavelength. Pharmacokinetic studies of noscapine were performed in mice following intravenous bolus at 10 mg/kg and oral administrations at 75, 150, and 300 mg/kg. RESULTS: The standard curves for noscapine estimation were linear between 390 and 50,000 ng/ml (lower limit of quantification was 390 ng/ml) and the recovery was approximately 80%. Following 10 mg/kg intravenous dose, mean plasma concentrations of 7.88 microg/ml were achieved at 5 min in mice and declined with undetectable levels at 4 h. The mean total body clearance was 4.78 l/h. The mean volume of distribution (V (d)) was 5.05 l. Non-compartmental analysis yielded the mean area under the plasma concentration-time curve (AUC) for noscapine as 53.42, 64.08, and 198.35 h microg/ml reaching maximum plasma concentrations (C (max)) of 12.74, 23.24, and 46.73 microg/ml at a t (max) of 1.12, 1.50, and 0.46 h at the linearly increasing dose levels. CONCLUSION: A rapid and simple HPLC/UV method for the quantification of noscapine in plasma has been developed to study pharmacokinetics of noscapine at tumor-suppressive doses in the mouse. Since orally available anticancer drugs are rare, therefore, noscapine, an innocuous agent, having a mean oral bioavailability of 31.5% over the studied dose range merits its further advancement in humans for anticancer therapy.