Mahendra Shukla

Dried blood spots: concepts, present status, and future perspectives in bioanalysis.

Over the past several years, dried blood spot (DBS) sampling technique has emerged as a pertinent method in both qualitative and quantitative bioanalysis context. In the DBS method, the blood sample is directly soaked on to a paper (with or without treatment). After drying it can be analyzed by modern analytical, immunological, or genomic detection systems. Several advantages of the DBS technique such as low blood volume requirement, transportation and storage without special treatment, better analytes stability, enhanced clinical cooperation in clinical trials, and reduced unforeseeable exposure of analysts to biohazards, make it the most appropriate blood sampling technique. This review illustrates the information available on the DBS method which may serve as a single window for investigators in the field of bioanalysis. Also, it explores the proficiency and appliance of the DBS method in pharmacokinetic (PK), therapeutic drug monitoring (TDM), toxicokinetic (TK), metabolomic, and disease diagnosis.

Novel pre-clinical methodologies for pharmacokinetic drug-drug interaction studies: spotlight on "humanized" animal models.

Abstract Poly-therapy is common due to co-occurrence of several ailments in patients, leading to the elevated possibility of drug–drug interactions (DDI). Pharmacokinetic DDI often accounts for severe adverse drug reactions in patients resulting in withdrawal of drug from the market. Hence, the prediction of DDI is necessary at pre-clinical stage of drug development. Several human tissue and cell line-based in vitro systems are routinely used for screening metabolic and transporter pathways of investigational drugs and for predicting their clinical DDI potentials. However, ample constraints are associated with the in vitro systems and sometimes in vitro–in vivo extrapolation (IVIVE) fail to assess the risk of DDI in clinic. In vitro–in vivo correlation model in animals combined with human in vitro studies may be helpful in better prediction of clinical outcome. Native animal models vary remarkably from humans in drug metabolizing enzymes and transporters, hence, the interpretation of results from animal DDI studies is difficult. With the advent of modern molecular biology and engineering tools, novel pre-clinical animal models, namely, knockout rat/mouse, transgenic rat/mouse with humanized drug metabolizing enzymes and/or transporters and chimeric rat/mouse with humanized liver are developed. These models nearly simulate human-like drug metabolism and help to validate the in vivo relevance of the in vitro human DDI data. This review briefly discusses the application of such novel pre-clinical models for screening various type of DDI along with their advantages and limitations.

PEGylated chitosan nanoparticles potentiate repurposing of ormeloxifene in breast cancer therapy.

AIM Development and optimization of ormeloxifene-loaded PEGylated chitosan nanoparticles (CNPs) for enhancing its literature profound therapeutic activity against breast cancer. METHODS CNPs were prepared by ionotropic gelation method and characterized. RESULTS Optimized formulation (CNPs10) had average 304 nm particle size with 0.247 polydispersity index and spherical shape with +31 mV surface charge. CNPs10 had 88.37% entrapment efficiency and 20.93% loading efficiency. CNPs10 demonstrated dose-dependent enhancement in cytotoxicity, cellular uptake, apoptosis, disruption of mitochondrial membrane potential and activation of caspase-3 in breast cancer MDA-MB-231 and MCF-7 cells over free ormeloxifene. In vivo studies divulged improved pharmacokinetic parameters, reduced toxicity, suppressed tumor burden and increased survival in CNPs10-treated female Sprague-Dawley rats. CONCLUSION PEGylated CNPs enhanced anticancer activity of ormeloxifene.

Role of enterohepatic recirculation in drug disposition: cooperation and complications

Abstract Enterohepatic recirculation (EHC) concerns many physiological processes and notably affects pharmacokinetic parameters such as plasma half-life and AUC as well as estimates of bioavailability of drugs. Also, EHC plays a detrimental role as the compounds/drugs are allowed to recycle. An in-depth comprehension of this phenomenon and its consequences on the pharmacological effects of affected drugs is important and decisive in the design and development of new candidate drugs. EHC of a compound/drug occurs by biliary excretion and intestinal reabsorption, sometimes with hepatic conjugation and intestinal deconjugation. EHC leads to prolonged elimination half-life of the drugs, altered pharmacokinetics and pharmacodynamics. Study of the EHC of any drug is complicated due to unavailability of the apposite model, sophisticated procedures and ethical concerns. Different in vitro and in vivo methods for studies in experimental animals and humans have been devised, each having its own merits and demerits. Involvement of the different transporters in biliary excretion, intra- and inter-species, pathological and biochemical variabilities obscure the study of the phenomenon. Modeling of drugs undergoing EHC has always been intricate and exigent models have been exploited to interpret the pharmacokinetic profiles of drugs witnessing multiple peaks due to EHC. Here, we critically appraise the mechanisms of bile formation, factors affecting biliary drug elimination, methods to estimate biliary excretion of drugs, EHC, multiple peak phenomenon and its modeling.

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

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

Abstract 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.

Gender-related pharmacokinetics and bioavailability of a novel anticancer chalcone, cardamonin, in rats determined by liquid chromatography tandem mass spectrometry

A reversed phase liquid chromatography tandem mass spectrometry method was developed and validated for quantification of cardamonin, a potential anticancer chalcone, in rat serum. Curcumin was used as an internal standard. Following liquid-liquid extraction using n-hexane and ethyl acetate (60:40, v/v), the processed samples were chromatographed on a C18 column using acetonitrile and ammonium acetate buffer (0.01 M, pH 4.5) (85:15, v/v) as mobile phase at a flow rate of 0.6 mL min(-1). Mass spectrometric detection was performed in the negative electrospray ionization mode by multiple reaction monitoring (m/z 269→122 and 367→217 for cardamonin and curcumin, respectively). The method was validated in terms of selectivity, accuracy, precision, sensitivity, reproducibility, dilution integrity and stability. The linearity was established in the range of 1-200 ng mL(-1) (r≥0.999). The recovery of cardamonin from spiked serum was always >90%. The intra- and inter-day precision (%RSD) and accuracy (%bias) were well within the acceptable limits. The method was applied for single oral and intravenous dose pharmacokinetics in male and female Sprague Dawley rats. Following oral dose, cardamonin showed peak serum concentration that occurred at ∼2 h with very low bioavailability in both male (0.6%) and female (4.8%) rats. Cardamonin exhibited a significant gender influence on pharmacokinetics and bioavailability in rats.

A mechanistic investigation of the bioavailability enhancing potential of lysergol, a novel bioenhancer, using curcumin

Lysergol (LYZ), a novel bioenhancer, has shown potential to enhance the bioavailability of some antibiotics. In the present investigation, the bioavailability enhancing potential of LYZ on curcumin (CUR) has been explored. We initially carried out in vivo pharmacokinetic and in situ permeation studies of CUR with and without LYZ coadministration. In presence of LYZ, the Cmax, AUC and elimination half-life of CUR were significantly increased. A noteworthy decrease in the clearance of CUR was also observed. An enhancement (3.3-fold) in the effective permeability of CUR was observed. To delve into the mechanistic insights, the probable role of LYZ in inhibiting the metabolism of CUR was investigated. In vitro phase I and II metabolic stability studies of CUR following pre-incubation with LYZ using rat liver microsomes were performed. Also, its effect on major efflux transporters using human P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) membrane preparations was examined. The corroboration of results was provided by in situ permeation and in vivo pharmacokinetic study of digoxin (probe P-gp substrate) and sulfasalazine (probe BCRP substrate) in the presence and absence of LYZ. The results were compared with the inhibitory potential of verapamil (for P-gp) and pantoprazole (for BCRP). Furthermore, the studies ruled out the probability of P-gp inhibition but strongly evidenced the involvement of BCRP inhibition. A remarkable increase in in vitro half-life of CUR and 1.4-fold enhancement in intestinal permeation of sulfasalazine in presence of LYZ clearly revealed that bioavailability enhancing potential is attributed to the inhibition of metabolic enzymes and BCRP efflux transporters.

LC-coupled ESI MS for quantification of miltefosine in human and hamster plasma

BACKGROUND Leishmaniasis, a fatal parasitic disease, is the second largest parasitic killer in the world and miltefosine is the first and only oral drug available for its treatment. RESULTS A rapid, sensitive and simple LC-MS/MS method for miltefosine quantification in hamster and human plasma was developed and validated over the range of 2.5-400 ng/ml. The mass spectrometric detection of the drug was carried out in multiple reaction monitoring mode (m/z 408.1→125.1) using an electrospray positive ionization. The protein precipitation method was employed for sample (50 µl) cleanup. CONCLUSION The proposed method provided accurate and precise high-throughput quantification of miltefosine in plasma and was successfully applied to its oral PK study in Golden Syrian hamsters.

Rapid quantitative analysis of ormeloxifene and its active metabolite, 7-desmethyl ormeloxifene, in rat plasma using liquid chromatography-tandem mass spectrometry.

Ormeloxifene (ORM) is a non-steroidal, selective estrogen receptor modulator used as a once a week oral contraceptive and is intended for long-term clinical use by females. Therefore, a simple, sensitive and rapid LC-MS/MS method was developed and validated for simultaneous quantification of ORM and its active metabolite (7-desmethyl ormeloxifene, 7-DMO) in rat plasma. Also, the method was partially validated in human plasma. Chromatographic separation was achieved on Discovery HS C-18 column (5μm, 50×4.6mm) with mobile phase [acetonitrile: aqueous ammonium acetate (0.01M) buffer (85: 15, %v/v)] at a flow rate of 0.8mL/min. The calibration curve was linear (r≥0.99) for a concentration range of 0.78-100ng/mL for both the analytes. The precision (%RSD; ORM, 1.3 to 13.4; 7-DMO, 3.1 to 15.0) and accuracy (%bias; ORM, -13.8 to 12.5; 7-DMO, -10.6 to 6.8) in both rat and human plasma were within the acceptable limits. The recovery for ORM and 7-DMO was always >79.1% and >72.9%, respectively. Both the analytes were found stable in rat plasma even after 30 days of storage at -80°C and on being subjected to three freeze-thaw cycles. The method has not only short run time (3.5min) but requires a low plasma sample volume (20μL) and is the first reported LC-MS/MS method for simultaneous quantification of the marketed drug known as centchroman (INN: ormeloxifene) and the metabolite 7-DMO in plasma. The method was applied to evaluate drug-drug interaction of ORM with the commonly prescribed antidepressant drug sertraline using serial sampling in rats.