Assessment of Clinical Pharmacokinetic Drug-Drug Interaction of Antimalarial Drugs α/ß-Arteether and Sulfadoxine-Pyrimethamine.

Antimalarial drug combination therapy is now being widely used for the treatment of uncomplicated malaria. The objective of the present study was to investigate the effects of coadministration of intramuscular α/ß-arteether (α/ß-AE) and oral sulfadoxine-pyrimethamine (SP) on the pharmacokinetic properties of each drug as a drug-drug interaction study to support the development of a fixed-dose combination therapy. A single-dose, open-label, crossover clinical trial was conducted in healthy adult Indian male volunteers (18 to 45 years, n = 13) who received a single dose of AE or SP or a combination dose of AE and SP. Blood samples were collected up to 21 days postadministration, and concentrations of α-AE, ß-AE, sulfadoxine, and pyrimethamine were determined by using a validated liquid chromatography-tandem mass spectrometry method. Pharmacokinetic parameters were calculated and statistically analyzed to calculate the geometric mean ratio and confidence interval. Following single-dose coadministration of intramuscular AE and oral SP, the pharmacokinetic properties of α/ß-AE were not significantly affected, and α/ß-AE had no significant effect on the pharmacokinetic properties of SP in these selected groups of healthy volunteers. However, more investigations are needed to explore this further. (This study has been registered in the clinical trial registry of India under approval no. CTRI/2011/11/002155.).

Isoformononetin, a methoxydaidzein present in medicinal plants, reverses bone loss in osteopenic rats and exerts bone anabolic action by preventing osteoblast apoptosis.

PURPOSE: Daidzein (Daid) has been implicated in bone health for its estrogen-'like' effects but low bioavailability, unfavorable metabolism and uterine estrogenicity impede its clinical potential. This study was aimed at assessing isoformononetin (Isoformo), a naturally occurring methoxydaidzein, for bone anabolic effect by overcoming the pitfalls associated with Daid. METHODS: Sprague-Dawley ovariectomized (OVx) rats with established osteopenia were administered Isoformo, 17ß-oestradiol (E2) or human parathyroid hormone. Efficacy was evaluated by bone microarchitecture using microcomputed tomography and determination of new bone formation by fluorescent labeling of bone. Osteoblast apoptosis was measured by co-labeling of bone sections with Runx-2 and TUNEL. Biochemical markers of bone metabolism were measured by ELISA. Plasma and bone marrow levels of Isoformo and Daid were determined by LC-MS-MS. Rat bone marrow stromal cells were harvested to study osteoblastic differentiation by Isoformo and Daid. New born rat pups were injected with Isoformo and Daid to study the effect of the compounds on the expression of osteogenic genes in the calvaria by real time PCR. RESULTS: In osteopenic rats, Isoformo treatment restored trabecular microarchitecture, increased new bone formation, increased the serum osteogenic marker (procollagen N-terminal propeptide), decreased resorptive marker (urinary C-terminal teleopeptide of type I collagen) and diminished osteoblast apoptosis in bone. At the most effective osteogenic dose of Isoformo, plasma and bone marrow levels were comprised of ~90% Isoformo and the rest, Daid. Isoformo at the concentration reaching the bone marrow achieved out of its most effective oral dosing induced stromal cell mineralization and osteogenic gene expression in the calvaria of neonatal rats. Isoformo exhibited uterine safety. CONCLUSIONS: Our study demonstrates that Isoformo reverses established osteopenia in adult OVx rats likely via its pro-survival effect on osteoblasts. Given its bone anabolic and anti-catabolic effects accompanied with safety at uterine level we propose its potential in the management of postmenopausal osteoporosis.

Differential subcellular targeting of recombinant human α1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants.

The response of protein accumulation site on yield, biological activity and in planta stability of therapeutic recombinant human proteinase inhibitor (α1-PI) was analyzed via targeting to different subcellular locations, like endoplasmic reticulum (ER), apoplast, vacuole and cytosol in leaves of transgenic tomato plants. In situ localization of the recombinant α1-PI protein in transgenic plant cells was monitored by immunohistochemical staining. Maximum accumulation of recombinant α1-PI in T0 and T1 transgenic tomato plants was achieved from 1.5 to 3.2% of total soluble protein (TSP) by retention in ER lumen, followed by vacuole and apoplast, whereas cytosolic targeting resulted into degradation of the protein. The plant-derived recombinant α1-PI showed biological activity for elastase inhibition, as monitored by residual porcine pancreatic elastase (PPE) activity assay and band-shift assay. Recombinant α1-PI was purified from transgenic tomato plants with high yield, homogeneity and biological activity. Purified protein appeared as a single band of ∼48-50 kDa on SDS-PAGE with pI value ranging between 5.1 and 5.3. Results of mass spectrometry and optical spectroscopy of purified recombinant α1-PI revealed the structural integrity of the recombinant protein comparable to native serum α1-PI. Enzymatic deglycosylation and lectin-binding assays with the purified recombinant α1-PI showed compartment-specific N-glycosylation of the protein targeted to ER, apoplast and vacuole. Conformational studies based on urea-induced denaturation and circular dichroism (CD) spectroscopy revealed relatively lower stability of the recombinant α1-PI protein, compared to its serum counterpart. Pharmacokinetic evaluation of plant derived recombinant and human plasma-purified α1-PI in rat, by intravenous route, revealed significantly faster plasma clearance and lower area under curve (AUC) of recombinant protein. Our data suggested significance of protein sorting sequences and feasibility to use transgenic plants for the production of stable, glycosylated and biologically active recombinant α1-PI for further therapeutic applications.

Simultaneous estimation of 16α-hydroxycleroda-3,13(14) Z-dien-15,16-olide from Polyalthia longifolia and its metabolite in hamster plasma: application to pharmacokinetic study.

A selective and sensitive LC-MS-MS method was developed and validated for simultaneous estimation and pharmacokinetic studies of 16α-hydroxycleroda-3,13(14) Z-dien-15,16-olide (K-09) obtained from Polyalthia longifolia and its metabolite (K-9T), a novel antidyslipidemic agent. Sample clean-up involved liquid-liquid extraction of both the analytes and internal standard (rosuvastatin) from 200 µL of hamster plasma. The analytes were chromatographically separated on a Symmetry-Shield C18 (5 µm, 4.6 × 150 mm) column, using acetonitrile-0.1% aqueous formic acid (92:08, v/v) as the mobile phase. Detection was performed using negative ion electrospray ionization in multiple reaction monitoring mode. The MS/MS response was linear over the concentration range 1.56-200 ng/mL, with a correlation coefficient (r²) of 0.998 or better. The within- and between-batch precisions (relative standard deviation, %RSD) and the accuracy (percentage bias) were within acceptable limits as per FDA guidelines. The validated method was successfully applied to reveal the pharmacokinetic parameters of K-09 and metabolite after oral administration. This method will therefore be highly useful for future studies of K-09 and metabolite K-9T pharmacokinetics in preclinical and clinical studies.

Simultaneous estimation of E- and Z-isomers of guggulsterone in rabbit plasma using liquid chromatography tandem mass spectrometry and its application to pharmacokinetic study.

A sensitive and selective liquid chromatography/tandem mass spectrometric method was developed for simultaneous determination of E- and Z-guggulsterone isomers (antihyperlipidemic drug) in rabbit plasma. Both the isomers were resolved on a Symmetry-Shield C(18) (5 µm, 4.6 × 150 mm) column, using gradient elution comprising a mobile phase of methanol, 0.5% v/v formic acid and acetonitrile. With dexamethasone as internal standard, plasma samples were extracted by an automated solid-phase extraction method using C(18) cartridges. Detection was performed by electrospray ionization in multiple reaction monitoring (MRM) in positive mode. The calibration curve was linear over the concentration range of 1.56-200 ng/mL (r(2) ≥ 0.998) for both analytes. The intra-day and inter-day accuracy and precision were within -0.96 to 4.12 (%bias) and 2.73 to 8.00 (%RSD) respectively. The analytes were stable after three freeze-thaw cycles. The method was successfully applied to study steriospecific pharmacokinetics of E- and Z-guggulsterone in NZ rabbit.

Bioanalytical method development and validation of natamycin in rabbit tears and its application to ocular pharmacokinetic studies.

A new selective and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of natamycin in rabbit tears using amphotericin B as internal standard (IS). Chromatographic separation was achieved on a Luna Cyano column (100 mm × 2 mm, 3 µm) using ammonium acetate buffer (pH 4; 3.5mM): methanol (10:90, v/v) as the mobile phase. The run time was 5 min. Detection was performed by negative ion electrospray ionization in multiple reaction monitoring (MRM) mode. The calibration curve was linear over the concentration range from 25 to 800 ng/ml, and lower limit of detection of 12.5 ng/ml. The accuracy and precision of the method were within the acceptable limit of ± 20% at the lower limit of quantitation and ± 15% at other concentrations. Natamycin was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days storage in a freezer at -70 ± 10 °C. The method was successfully applied to the ocular pharmacokinetic studies of natamycin eye drops in New Zealand rabbit tears.

Bioanalytical method development and validation of novel antithrombotic agent S002-333 by LC-MS/MS and its application to pharmacokinetic studies.

A rapid, sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using an electrospray ionization (ECI) source for the quantification of novel anti-thrombotic agent S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-ß-carboxylic acid amide] in rabbit plasma was developed and validated. The extraction from plasma was carried out by simple protein precipitation extraction method. The chromatographic separation was performed on an Ultramex Cyno, (150 x 4.6 mm, 5 µm) with a guard column, using acetonitrile-water (75:25,v/v) with flow rate of 0.6 mL/min as the mobile phase. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z transitions 386.4/215.4 for S002-333 and m/z 393.4/171 for the internal standard dexamethasone, using positive ion mode. The MS/MS response was linear over the concentration range from 1.56 to 200 ng/mL, with a lower limit of detection of 0.78 ng/mL. The accuracy and precision of the method were within the acceptable limit of ±20% at the lower limit of quantitation and ±15% at other concentrations and showed no significant matrix effect. The validated method can be used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic studies.

Nano-vectors for the Ocular Delivery of Nucleic Acid-based Therapeutics.

Nucleic acid-based therapeutics have gained a lot of interest for the treatment of diverse ophthalmic pathologies. The first to enter in clinic has been an oligonucleotide, Vitravene(®) for the treatment of cytomegalovirus infection. More recently, research on aptamers for the treatment of age related macular degeneration has led to the development of Macugen(®). Despite intense potential, effective ocular delivery of nucleic acids is a major challenge since therapeutic targets for nucleic acid-based drugs are mainly located in the posterior eye segment, requiring repeated invasive administration. Of late, nanotechnology-based nano-vectors have been developed in order to overcome the drawbacks of viral and other non-viral vectors. The diversity of nano-vectors allows for ease of use, flexibility in application, low-cost of production, higher transfection efficiency and enhanced genomic safety. Using nano-vector strategies, nucleic acids can be delivered either encapsulated or complexed with cationic lipids, polymers or peptides forming sustained release systems, which can be tailored according to the ocular tissue being targeted. The present review focuses on developments and advances in various nano-vectors for the ocular delivery of nucleic acid-based therapeutics, the barriers that such delivery systems face and methods to overcome them.

Wound healing in guinea pigs after topical application of starfish Pentaceraster regulus extract.

OBJECTIVE: To investigate the healing efficacy of Pentaceraster regulus (starfish) aqueous-methanol extract on cutaneous wounds in guinea pigs. METHOD: Freshly collected starfish were washed with distilled water and soaked in methanol for transportation. After filtering, soaking and concentration, the extract was fractionated into chloroform soluble (5g), 50% aqueous-methanol soluble (20g) and insoluble fractions (25g). Primary screening demonstrated moderate wound-healing activity in male Swiss-strain guinea pigs, so further fractionation into chloroform, 50% aqueous-methanol and insoluble fractions was undertaken.Wound-healing activity was concentrated only in the aqueous-methanol fraction, so this was used for the study. Animals received either 1% aqueous-methanol extract, the vehicle alone or 5% providone-iodine. The following were measured: wound area,wound tensile strength, DNA, total protein and hydroxyproline levels in excised granulation tissue. Histological changes were observed under microscope. RESULTS: Extract-treated wounds healed faster, indicated by a significant contraction in wound area (42%). Cellular proliferation and collagen synthesis at the wound site increased, demonstrated by increase in DNA (33%), protein (29%) and hydroxyproline (37%) content when compared with the controls and povidone-iodine-treated animals (standard care). These findings were confirmed by histological examination. Proper folding of collagen was demonstrated by a significant increase in tensile strength (34%). CONCLUSION: The results suggest that the aqueous-methanol extract of starfish P. regulus promotes wound-healing activity.

Distribution of lovastatin to bone and its effect on bone turnover in rats.

Statins, the widely used lipid-lowering drugs, are inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, which catalyses a rate-limiting step in the biosynthesis of cholesterol. Many previous reports show that statins can act both as bone anabolic and as anti-resorptive agents but their beneficial effects on bone turnover are still controversial. Considering their high liver specificity and low oral bioavailability, the distribution of statins to the bone microenvironment is questionable. In this study, the distribution of lovastatin and its active metabolites to bone, with respect to plasma and liver compartments, was examined after oral and intravenous administration in female rats. As compared with oral administration, the distribution of lovastatin to the bone compartment was significantly enhanced after intravenous administration. Further, the effect of lovastatin on bone turnover was studied in-vitro and in-vivo to assess its anti-osteoporotic potential. Lovastatin acid but not lovastatin was found to inhibit parathyroid-hormone-induced bone resorption in an in-vitro chick embryo bone assay. Oral, as well as intravenous, short-term lovastatin treatment significantly reduced the serum total cholesterol, serum total alkaline phosphatase and urinary crosslinks in ovariectomized rats. In accordance with its increased distribution to the bone compartment, intravenously administered lovastatin was more effective in reducing the ovariectomy-induced increase in markers of bone metabolism, especially urinary crosslinks. The findings of this study suggest that statins inhibit bone resorption and that their anti-resorptive efficacy can be increased by administering them by routes other than oral so as to achieve their enhanced concentration in bone.

Anilino-(2-bromophenyl) acetonitrile: a promising orally effective antileishmanial agent.

Visceral leishmaniasis (VL) or kala-azar is a worldwide disseminated intracellular infection caused by the hemoflagellate protozoan parasites Leishmania donovani. Chemotherapeutic scenario presents a deplorable picture and demands an urgent search for a new and safe anti-VL drugs, preferably active by oral route. In search of new antileishmanial agents, a total of 16 compounds belonging to the anilino-(substituted phenyl)-acetonitrile class were tested in vitro in promastigote/macrophase-amastigote systems and in vivo in L. donvoani/hamster model for their antileishmanial activity. Compound 3, anilino-(2-bromophenyl)-acetonitrile, exhibited most promising activity both in vitro at a concentration of 100 microg/ml (82.33 and 94.36% in promastigote and macrophase-amastigote systems, respectively) and in vivo at a dose of 50 mg/kg for 5 days (82.11 and 80% by i.p. and p.o. routes, respectively), hence this compound was investigated in detail. To maximize its bioavailability, dissolution profile, absorption, the compound was also tested in vivo as its soluble form. But no enhancement in activity was observed. From the results of different parameters for example ED(50) and LD(50) etc. compound 3 appears to be a potent orally effective compound which could further be investigated to establish its potential as a candidate molecule of antileishmanial therapy.

A simple and rapid evaluation of methemoglobin toxicity of 8-aminoquinolines and related compounds.

Methemoglobin, a toxic ferric form of hemoglobin, is continuously formed in normal erythrocytes, but during abnormal situations in situ, the level is enhanced. 8-Amino-quinolines and related compounds are causative agents for methemoglobin formation. Employing oxyhemoglobin, methemoglobin toxicity was about six times higher with primaquine compared to CDRI Compound 80/53 at 10(-9) M concentration. Methemoglobin reductase activity was also completely inhibited by primaquine, whereas 24% inhibition was noted in the case of 80/53 at the same concentrations. Mastomys, a rodent animal model, was found to be equally good for comparative evaluation of methemoglobin toxicity. Further, with the use of primaquine transdermal tape on the Mastomys model, a rise in methemoglobin occurred with increase in time. In conclusion, the study presents simple, economical, less time-consuming methods for the evaluation of methemoglobin toxicity, in vitro and in vivo, without employing the conventional Beagle dog model.

Dysobinin, a tetranortriterpenoid.

The planar furan ring in the title compound (6beta-acetoxyazadirone, C(30)H(38)O(6)) is twisted with respect to the steroid D ring. The crystal structure is stabilized by C-H.O hydrogen bonds and van der Waals interactions.

In vitro and in vivo wound healing activity of asiaticoside isolated from Centella asiatica.

The activity of asiaticoside, isolated from Centella asiatica, has been studied in normal as well as delayed-type wound healing. In guinea pig punch wounds topical applications of 0.2% solution of asiaticoside produced 56% increase in hydroxyproline, 57% increase in tensile strength, increased collagen content and better epithelisation. In streptozotocin diabetic rats, where healing is delayed, topical application of 0.4% solution of asiaticoside over punch wounds increased hydroxyproline content, tensile strength, collagen content and epithelisation thereby facilitating the healing. Asiaticoside was active by the oral route also at 1 mg/kg dose in the guinea pig punch wound model. It promoted angiogenesis in the chick chorioallantoic membrane model at 40 microg/disk concentration. These results indicate that asiaticoside exhibits significant wound healing activity in normal as well as delayed healing models and is the main active constituent of Centella asiatica.

A time course study for the development of an immunocompromised wound model, using hydrocortisone.

Although wound healing is essentially a physiologic process, some chronic wounds exhibit considerable delay in healing. Often these do not heal perfectly in individuals with low immune profiles. Thus, the present study was undertaken to develop an excision wound model in the immunocompromised state induced by pretreatment with hydrocortisone (HC) 40 mg/kg intramuscularly in male rats. Wounds of 8-mm diameter were made on the preshaved dorsal surface of rats using an Acuderm biopsy punch, following pretreatment with HC. After 14 days HC-treated animals exhibited atrophy of spleen and adrenal glands and a significant reduction of circulating lymphocytes and increase in neutrophils; these changes are indicative of immunosuppressive state of animals. The cell proliferation was significantly affected as shown by decreases in DNA (23%) and protein (11%). Furthermore, there were also significant reductions in tensile strength (37%) and hydroxyproline (33%) contents. These results were further supported by lack of contraction of wound edges. It is concluded that animals primed with HC 1 week prior to wounding developed prolonged immunosuppression, which significantly impaired the wound healing as compared with other groups. Thus, this can be experimentally employed as an immunocompromised wound model for evaluating compounds as novel wound healers suitable for immunocompromised subjects.

In vitro percutaneous absorption of verapamil.

The in vitro percutaneous absorption of verapamil hydrochloride (VHCl) was investigated in order to assess its feasibility for transdermal development. The experiments were carried across mice and guinea pig skins using Keshary-Chien diffusion cell. The values of diffusion rate (J) and permeability coefficient (Kp) across guinea pig skin were lowered as compared to mouse skin. Increased drug concentration in donor compartment increased value of J but decreased value of Kp. Under similar conditions, values of J and Kp were lowered for dorsal skin as compared to abdominal skin, both for mice and guinea pig. The results indicate that verapamil can be administered transdermally.

In vitro transdermal delivery of atenolol using mouse and guinea pig.

In vitro percutaneous absorption of atenolol was done in order to assess its feasibility for transdermal development across mouse and guinea pig skins using Keshary-Chien type of diffusion cell. Values of diffusion rate (J) and permeability coefficient (Kp) across guinea pig skin were lowered as compared to those in mouse skin. When the concentration of drug in donor compartment was increased a decrease in Kp and increase in J value were observed with both the skins. Under the same conditions, values of J and Kp were lowered for dorsal skin compared to abdominal skin both for mouse and guinea pig. The results suggest that atenolol can be pursued further for transdermal system development.

Spontaneous choledocoduodenal fistula--due to chronic duodenal ulcer.

A case of spontaneous choledocoduodenal fistula due to penetrating posterior duodenal ulcer is reported. The only presenting symptoms were pain and vomiting. There was no fever or recurrent jaundice which is usually expected in such a condition. The radiological findings included barium and air in the biliary tract. Biliary fistula are not uncommon. Although external biliary fistulae are seldom seen in present times, internal biliary fistulae are not a rare entity. Internal biliary fistulae are either spontaneous or due to operations on biliary tract. The common causes for spontaneous internal biliary fistula includes cholelithiasis, peptic ulceration and malignant neoplasm (Shiu) 1967. In a study of 819 cases by Waggoner and Le Mone (1949) 51% of such fistulae were cholecystoduodenal, 21% cholecystocolic, 19% choledocoduodenal, while the rest were choledocogastric and cholecystocholedocal. Most common cause for spontaneous choledocoduodenal fistula is due to gall stones, but, rarely posterior penetrating duodenal ulcer may also cause this condition. The following report concerns a spontaneous biliary fistula of the choledocoduodenal type, due to chronic duodenal ulcer.