Swati Jaiswal

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.

Development and optimization of polymeric nanoparticles of antitubercular drugs using central composite factorial design.

Objective: The objective of the present study was to develop sustained release biodegradable polymeric nanoparticles (PNs) of two anti-tubercular drugs (ATDs), rifampicin (RIF) and isoniazid (INH) using circumscribed central composite factorial design (CCD) and evaluate in vivo uptake potential using rhodamine labeled PNs (RPNs). Methods: CCD was employed to study the influence of independent formulation factors, drug:polymer ratio (D:P) and surfactant concentration (SC), on dependent physicochemical characteristics, particle size (PS), polydispersity index (PI) and percentage entrapment efficiency (%EE) of the drugs. Optimized PNs prepared using response surface methodology (RSM) were evaluated for in vitro kinetics at endosomal macrophage pH 5.2 and physiological pH 7.4 and in vivo targeting potential in peritoneal macrophages (PMs) by fluorescence microscopy (FM) and confocal laser scanning microscopy (CLSM). Results: Optimized PNs exhibited spherical and porous surface with a mean PS of 202 nm, PI of 0.178, zeta potential of -25.49 mV and %EE of 76.12% and 54.25% for RIF and INH, respectively. Conclusions: Highly hydrophilic INH could be encapsulated with lypophilic RIF with efficiency. In vivo uptake studies of RPNs in PMs suggested endocytosis of RPNs without any surface adsorption phenomenon. Hence, further studies need to be performed for establishing the pharmacokinetic potential of PNs.

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.

Novel, potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that demonstrated activity against both replicating and non-replicating M. tuberculosis.

The mycobacterial F0F1-ATP synthase (ATPase) is a validated target for the development of tuberculosis (TB) therapeutics. Therefore, a series of eighteen novel compounds has been designed, synthesized and evaluated against Mycobacterium smegmatis ATPase. The observed ATPase inhibitory activities (IC50) of these compounds range between 0.36 and 5.45μM. The lead compound 9d [N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,3-dichlorobenzenesulfonamide] with null cytotoxicity (CC50>300μg/mL) and excellent anti-mycobacterial activity and selectivity (mycobacterium ATPase IC50=0.51μM, mammalian ATPase IC50>100μM, and selectivity >200) exhibited a complete growth inhibition of replicating Mycobacterium tuberculosis H37Rv at 3.12μg/mL. In addition, it also exhibited bactericidal effect (approximately 2.4log10 reductions in CFU) in the hypoxic culture of non-replicating M. tuberculosis at 100μg/mL (32-fold of its MIC) as compared to positive control isoniazid [approximately 0.2log10 reduction in CFU at 5μg/mL (50-fold of its MIC)]. The pharmacokinetics of 9d after p.o. and IV administration in male Sprague-Dawley rats indicated its quick absorption, distribution and slow elimination. It exhibited a high volume of distribution (Vss, 0.41L/kg), moderate clearance (0.06L/h/kg), long half-life (4.2h) and low absolute bioavailability (1.72%). In the murine model system of chronic TB, 9d showed 2.12log10 reductions in CFU in both lung and spleen at 173μmol/kg dose as compared to the growth of untreated control group of Balb/C male mice infected with replicating M. tuberculosis H37Rv. The in vivo efficacy of 9d is at least double of the control drug ethambutol. These results suggest 9d as a promising candidate molecule for further preclinical evaluation against resistant TB strains.

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.

Identification of novel phenyl butenonyl C-glycosides with ureidyl and sulfonamidyl moieties as antimalarial agents.

A new series of C-linked phenyl butenonyl glycosides bearing ureidyl(thioureidyl) and sulfonamidyl moieties in the phenyl rings were designed, synthesized, and evaluated for their in vitro antimalarial activities against Plasmodium falciparum 3D7 (CQ sensitive) and K1 (CQ resistant) strains. Among all the compounds screened the C-linked phenyl butenonyl glycosides bearing sulfonamidyl moiety (5a) and ureidyl moiety in the phenyl ring (7d and 8c) showed promising antimalarial activities against both 3D7 and K1 strains with IC50 values in micromolar range and low cytotoxicity offering new HITS for further exploration.

Click biotinylation of PLGA template for biotin receptor oriented delivery of doxorubicin hydrochloride in 4T1 cell induced breast cancer

PLGA was functionalized with PEG and biotin using click chemistry to generate a biotin receptor targeted copolymer (biotinylated-PEG-PLGA) which in turn was used to fabricate ultrafine nanoparticles (BPNP) of doxorubicin hydrochloride (DOX) for effective delivery in 4T1 cell induced breast cancer. However, adequate entrapment of a hydrophilic bioactive like DOX in a hydrophobic polymer system made of PLGA is not usually possible. We therefore modified a conventional W/O/W emulsion method by utilizing NH4Cl in the external phase to constrain DOX in dissolved polymer phase by suppressing DOXs inherent aqueous solubility as per common ion effect. This resulted in over 8-fold enhancement in entrapment efficiency of DOX inside BPNP, which otherwise is highly susceptible to leakage due to its relatively high aqueous solubility. TEM and DLS established BPNP to be sized below 100 nm, storage stability studies showed that BPNP were stable for one month at 4 °C, and in vitro release suggested significant control in drug release. Extensive in vitro and in vivo studies were conducted to propound anticancer and antiproliferative activity of BPNP. Plasma and tissue distribution study supplemented by pertinent in vivo fluorescence imaging mapped the exact fate of DOX contained inside BPNP once it was administered intravenously. A comparative safety profile via acute toxicity studies in mice was also generated to out rightly establish usefulness of BPNP. Results suggest that BPNP substantially enhance anticancer activity of DOX while simultaneously mitigating its toxic potential due to altered spatial and temporal presentation of drug and consequently deserve further allometric iteration.