Development and in vitro/in vivo evaluation of artemether and lumefantrine co-loaded nanoliposomes for parenteral delivery

Combination therapy of artemether (ART) and lumefantrine (LUM) is well-established for the treatment of uncomplicated malaria worldwide. Nanoliposomes (NLs) encapsulating both drugs were prepared and freeze-dried. The lyophilized nanoliposomes exhibited high entrapment efficiency of artemether (66.18%), relatively low entrapment efficiency of lumefantrine (53.46%), low average size diameter (125.3?nm) and found to be stable at 4?°C for 60 days without significant change in mean particle diameter and drug entrapment efficiencies. In vitro drug release study has shown initial burst effect and then sustained release pattern over a time period of 30?h. In vivo toxicity study was examined by liver and kidney function test as well as histopathological examination. Nanoliposomes showed lower hemolytic potential (～10%) compared to all the components when studied individually. There was no significant change (p?>?0.05) in biochemical parametes between control and treated group of animals. Pharmacokinetic data of ART?+?LUM NLs showed higher the area under the plasma concentration–time curve (AUC) values and prolonged residence time of drug in the blood circulation compared with ART?+?LUM solution. The tissue distribution demonstrated high uptake of ART?+?LUM-NLs in RES organs particularly in liver and spleen. Biocompatibility was confirmed by hepato- and nephrotoxicity analysis showed no sign of fibrosis, fatty infiltration, centrilobular necrosis and lymphocyte infiltration confirmed the suitability of developed formulation for treatment of malaria.     

Development and validation of a chiral liquid chromatographic method, based on Chiralpak to quantify enantiomers of (+/-)-DRF 2725 in rat plasma: lack of inversion of ragaglitazar (S(-)-DRF 2725) to its antipode in plasma

A selective, accurate and reproducible high-performance liquid chromatographic (HPLC) method for the separation of individual enantiomers of DRF 2725 [R(+)-DRF 2725 and S(-)-DRF 2725 or ragaglitazar] was obtained on a chiral HPLC column (Chiralpak). During method optimization, the separation of enantiomers of DRF 2725 was investigated to determine whether mobile phase composition, flow-rate and column temperature could be varied to yield the base line separation of the enantiomers. Following liquid-liquid extraction, separation of enantiomers of DRF 2725 and internal standard (I.S., desmethyl diazepam) was achieved using an amylose based chiral column (Chiralpak AD) with the mobile phase, n-hexane-propanol-ethanol-trifluoro acetic acid (TFA) in the ratio of 89.5:4:6:0.5 (v/v). Baseline separation of DRF 2725 enantiomers and I.S., free from endogenous interferences, was achieved in less than 25 min. The eluate was monitored using an UV detector set at 240 nm. Ratio of peak area of each enantiomer to I.S. was used for quantification of plasma samples. Nominal retention times of R(+)-DRF 2725, S(-)-DRF 2725 and I.S. were 15.8, 17.7 and 22.4 min, respectively. The standard curves for DRF 2725 enantiomers were linear (R(2) > 0.999) in the concentration range 0.3-50 microg/ml for each enantiomer. Absolute recovery, when compared to neat standards, was 70-85% for DRF 2725 enantiomers and 96% for I.S. from rat plasma. The lower limit of quantification (LLOQ) for each enantiomers of DRF 2725 was 0.3 microg/ml. The inter-day precisions were in the range of 1.71-4.60% and 3.77-5.91% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. The intra-day precisions were in the range of 1.06-11.5% and 0.58-12.7% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. Accuracy in the measurement of quality control (QC) samples was in the range 83.4-113% and 83.3-113% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. Both enantiomers and I.S. were stable in the battery of stability studies viz., bench-top (up to 6 h), auto-sampler (up to 12 h) and freeze/thaw cycles (n = 3). Stability of DRF 2725 enantiomers was established for 15 days at -20 degrees C. The application of the assay to a pharmacokinetic study of ragaglitazar [S(-)-DRF 2725] in rats is described. It was unequivocally demonstrated that ragaglitazar does not undergo chiral inversion to its antipode in vivo in rat plasma.     

Orotate phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase from yeast: kinetic analysis with chromium (III) pyrophosphate

The reactions catalyzed by orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) from yeast differ in the kinetic mechanisms by which they are activated by divalent metal ions. Moreover, whereas OPRTase is activated specifically by Mg(II) or Mn(II), the reactions catalyzed by HGPRTase can utilize a wider range of divalent metal ions, including Mg(II), Mn(II), Co(II), and Zn(II). In this report we describe the results of a kinetic analysis of the effects of the addition of Cr(III) pyrophosphate (Cr-PPi) to the OPRTase and HGPRTase assay solutions, which delineates further the differences between these enzyme activations by metal ions. (1) Cr-PPi is an effective competitive inhibitor of the OPRTase catalysis, when the steady-state forward velocity of orotidine monophosphate (OMP) formation is examined over a range of phosphoribosyl alpha-pyrophosphate (PRibPP) concentrations, whereas pyrophosphate (PPi) has been reaffirmed to be a noncompetitive product inhibitor under the same conditions. (2) Cr-PPi itself serves as a substrate for the OPRTase-catalyzed reverse pyrophosphorolysis of OMP and does not inhibit the utilization of PPi as substrate during this reaction. (3) In contrast, Cr-PPi, at concentrations as high as 6 mM, has no effect on the HGPRTase-catalyzed formation of inosine monophosphate, whereas the inhibition exhibited by PPi during this reaction is noncompetitive but defined by two sets of lines in the double reciprocal plot of the initial velocity versus 1/PRibPP. (4) Cr-PPi is not a substrate for the HGPRTase-catalyzed pyrophosphorolysis of IMP under the conditions of these assay procedures.     

Structure–Activity Relationship of a Highly Selective Peptidyl Inhibitor of Kv1.3 Voltage-Gated K+-Channel from Scorpion (B. sindicus) Venom

Venomous fauna of the world is a unique source of protein and peptide toxins with a wide range of pharmacological and physiological activities targeting in particular the bio-signaling system. Thus the most widely known source of peptidyl neurotoxins used for callipering different ion channels (e.g. Na⁺, K⁺, Ca⁺⁺ or Cl^? etc.) as well as many medicinally important peptides which have been accepted as drugs or are in clinical trials originate from the venom of scorpions of Buthidae family. In the present study, structure–activity relationship of highly selective short-chain neurotoxin from scorpion Buthus sindicus (a common yellow scorpion of Sindh, Pakistan) has been established. The toxin named as Bs-KTx6 (4,115.4 Da) has been isolated, synthesized and found to be a potent as well as selective inhibitor of voltage gated potassium channel Kv1.3 (IC₅₀ = 7.7 pM). The structural studies on channel selective modulators like Bs-KTx6 may serve as a possible template in establishing libraries of peptidyl toxins for treating diseases such as multiple sclerosis, type-1 diabetes mellitus, rheumatoid arthritis, allergic contact dermatitis, bone resorption due to periodontitis and delayed type hypersensitivity. It is also worth mentioning that the peptide based drugs have excellent safety profiles as well as selectivity compared to other non-protein molecules. In conclusion, identified peptide Bs-KTx6 is a specific as well as very selective blocker of Kv1.3 and provides a starting template for the synthesis of peptidyl drugs to treat autoimmune diseases.     

Methylenetetrahydrofolate reductase C677T genetic polymorphisms and risk of leukaemia among the North Indian population

Background: Leukaemia is a heterogeneous disease in which haematopoietic progenitor cells acquire genetic lesions that lead to a block in differentiation, increased self-renewal, and unregulated proliferation. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR), involved in folate metabolism, plays a crucial role in cells because folate availability is important for DNA integrity. The aim of this case–control study was to evaluate the association of the C677T MTHFR gene polymorphism with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML) and chronic lymphocytic leukaemia (CLL). Materials and methods: A total of 275 leukaemia cases – including AML (n = 112), ALL (n = 81), CML (n = 43), CLL (n = 39) – and 251 age/sex-matched healthy control individuals participated in this study. MTHFR C677T polymorphisms in the cases and controls were evaluated by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Results: The average MTHFR 677CC, 677CT, 677TT genotype frequencies of total leukaemia cases were 68.73%, 19.64%, and 11.64% in cases, and 71.71%, 24.30%, and 3.98% in healthy controls, respectively. The average frequency of the MTHFR 677T allele was 21.45% among the cases compared to 16.13% among the controls. Conclusions: In the present case–control study we have observed a higher frequency of the MTHFR 677TT genotype in cases of leukaemia (AML, ALL, CML and CLL) as compared with controls; this might be due to ethnic and geographic variation. As per our findings, although the frequency of the MTHFR 677T allele is moderately high in AML, ALL and CLL, no statistically significant association was found; on the other hand statistically significant association was found in the context of CML cases.     

A new Bombyx mori larval ovarian cell line highly susceptible to nucleopolyhedrovirus

Lepidopteran cell lines constitute the backbone for studying baculoviral biology in culturo and for baculovirus vector based recombinant protein expression systems. In the present study, we report establishment of a new continuous cell line designated as DZNU-Bm-1 from larval ovaries of the silkworm, Bombyx mori. The cells were grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat inactivated B. mori haemolymph at 25+/-1 degrees C. A large number of attached epithelial-like and round refractive cells migrated from the explants and multiplied in the primary cultures. Both type of cells were subcultured initially for a few passages but after 10 passages the round refractive cells dominated the population, which could be subcultured continuously using MGM-448 medium with 10% FBS. The population doubling time of cell line was about 42h at 25+/-1 degrees C. The cell populations were largely diploids and triploids, while a few tetraploids and hexaploids were also observed. DNA profiles using Inter Simple Sequence Repeat (ISSR)-PCR and Simple Sequence Repeat (SSR) loci established the differences between DZNU-Bm-1 cell line and most widely used BmN cell line and the B. mori W-chromosome specific sequences confirmed the origin of DZNU-Bm-1 cell line to be from female silkworm. When cells were infected with free nonoccluded B. mori nucleopolyhedrovirus (BmNPV), the cell line was found to be highly susceptible with 92-94% of the cells harbouring BmNPV and having an average of 20-23 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV based baculoviral expression system and also for studying in culturo virus replication.     

Sera from Children with Autism Induce Autistic Features Which Can Be Rescued with a CNTF Small Peptide Mimetic in Rats

Autism is a neurodevelopmental disorder characterized clinically by impairments in social interaction and verbal and non-verbal communication skills as well as restricted interests and repetitive behavior. It has been hypothesized that altered brain environment including an imbalance in neurotrophic support during early development contributes to the pathophysiology of autism. Here we report that sera from children with autism which exhibited abnormal levels of various neurotrophic factors induced cell death and oxidative stress in mouse primary cultured cortical neurons. The effects of sera from autistic children were rescued by pre-treatment with a ciliary neurotrophic factor (CNTF) small peptide mimetic, Peptide 6 (P6), which was previously shown to exert its neuroprotective effect by modulating CNTF/JAK/STAT pathway and LIF signaling and by enhancing brain derived neurotrophic factor (BDNF) expression. Similar neurotoxic effects and neuroinflammation were observed in young Wistar rats injected intracerebroventricularly with autism sera within hours after birth. The autism sera injected rats demonstrated developmental delay and deficits in social communication, interaction, and novelty. Both the neurobiological changes and the behavioral autistic phenotype were ameliorated by P6 treatment. These findings implicate the involvement of neurotrophic imbalance during early brain development in the pathophysiology of autism and a proof of principle of P6 as a potential therapeutic strategy for autism.     

Evaluation of Iron in Serum and Urine and their Relation with Thyroid Function in Female Goitrous Patients

In many developing countries, women are at high risk of goiter and iron deficiency anemia (IDA). Iron deficiency adversely affects thyroid metabolism and may decrease the efficiency of thyroid hormones in areas of endemic goiter. The aim of the present study was to compare the level of iron (Fe) in biological samples (serum and urine) and serum thyroid hormones, thyroid stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxin (FT4) of goitrous female patients (GFPs; n = 69) with those of nongoitrous women as control subjects (n = 117), age range 21–45 years. The biological samples were analyzed for Fe concentration using flame atomic absorption spectrophotometer, prior to microwave-assisted wet acid digestion. The validity and accuracy of the method was checked by the certified sample and with those obtained by conventional wet acid digestion method on the same CRM and real samples. The overall recoveries of Fe in serum and urine were found in the range of 97.2–98.6% of certified values. The results of this study showed that the mean values of Fe in serum and urine samples of GFPs were significantly reduced as compared to control subjects (p = 0.002 and p = 0.015, respectively). The mean values of FT3 and FT4 were found to be lower in GFPs than in the age-matched healthy control women; in contrast, high mean values of TSH were detected in GFPs (p = 0.003). There was a positive correlation between serum Fe concentration and TSH (r = 0.85, p = 0.01), FT3 (r = 0.95, p = 0.003), and FT4 levels (r = 0.98, p = 0.007) in GFPs. It was observed that iron deficiency is prevalent in GFPs, so the need of Fe supplementation will be required to improve the efficacy of thyroid metabolism in goitrous women.     

AP-APSE dpol intein: A novel family A DNA polymerase intein domain

Inteins are "protein introns" that remove themselves from their host proteins through an autocatalytic protein-splicing. After their discovery, inteins have been quickly identified in organisms from all three kingdoms of life - eucarya, bacteria and archaea, but their distribution is sporadic. Here we report the identification and bioinformatics characterization of intein in DNA polymerase A gene of bacteriophage APSE (Acyrthosiphon pisum Secondary Endosymbiont bacteriophage) infecting the Aphid secondary endosymbionts of eukaryotic insects such as Acyrthosiphon pisum, Uroleucon rudbeckiae. The insertion site of intein within APSE family A DNA polymerase extein was identified to be dpola. Hence we propose this as a unique intein of family A DNA polymerase (dpola insertion site) and only reported intein in podoviridae family.