Advanced Pharmaceutical Bulletin
Volume:5 Issue: 1, Mar 2015

Drug-induced liver injury (DILI) is a major problem for pharmaceutical industry and drug development. Mechanisms of DILI are many and varied. Elucidating the mechanisms of DILI will allow clinicians to prevent liver failure, need for liver transplantation, and death induced by drugs. Methimazole and propylthiouracil (PTU) are two convenient antithyroid agents which their administration is accompanied by hepatotoxicity as a deleterious side effect. Although several cases of antithyroid drugs-induced liver injury are reported, there is no clear idea about the mechanism(s) of hepatotoxicity induced by these medications. Different mechanisms such as reactive metabolites formation, oxidative stress induction, intracellular targets dysfunction, and immune-mediated toxicity are postulated to be involved in antithyroid agents-induced hepatic damage. Due to the idiosyncratic nature of antithyroid drugs-induced hepatotoxicity, it is impossible to draw a specific conclusion about the mechanisms of liver injury. However, it seems that reactive metabolite formation and immune-mediated toxicity have a great role in antithyroids liver toxicity, especially those caused by methimazole. This review attempted to discuss different mechanisms proposed to be involved in the hepatic injury induced by antithyroid drugs.

Crystallization is often used for manufacturing drug substances. Advances of crystallization have achieved control over drug identity and purity, but control over the physical form remains poor. This review discusses the influence of solvents used in crystallization process on crystal habit and agglomeration of crystals with potential implication for dissolution. According to literature it has been known that habit modification of crystals by use of proper solvents may enhance the dissolution properties by changing the size, number and the nature of crystal faces exposed to the dissolution medium. Also, the faster dissolution rate of drug from the agglomerates of crystals compared with the single crystals may be related to porous structure of the agglomerates and consequently their better wettability.It is concluded from this review that in-depth understanding of role of the solvents in crystallization process can be applied to engineering of crystal habit or crystal agglomeration, and predictably dissolution improvement in poorly soluble drugs.

Due to the vast and inappropriate use of the antibiotics, microorganisms have begun to develop resistance to the commonly used antimicrobial agents. So therefore, development of the new and effective antimicrobial agents seems to be necessary. According to some recent reports, carbon-based nanomaterials such as fullerenes, carbon nanotubes (CNTs) (especially single-walled carbon nanotubes (SWCNTs)) and graphene oxide (GO) nanoparticles show potent antimicrobial properties. In present review, we have briefly summarized the antimicrobial activity of carbon-based nanoparticles together with their mechanism of action. Reviewed literature show that the size of carbon nanoparticles plays an important role in the inactivation of the microorganisms. As major mechanism, direct contact of microorganisms with carbon nanostructures seriously affects their cellular membrane integrity, metabolic processes and morphology. The antimicrobial activity of carbon-based nanostructures may interestingly be investigated in the near future owing to their high surface/volume ratio, large inner volume and other unique chemical and physical properties. In addition, application of functionalized carbon nanomaterials as carriers for the ordinary antibiotics possibly will decrease the associated resistance, enhance their bioavailability and provide their targeted delivery.

Lipid suspensions as drug carriers, including conventional liposomes, ethosomes, transferosomes, proniosomes, niosomes, PEG-PPG-PEG niosomes and stratum corneum liposomes (cerosomes), were formulated and compared. Lipid vesicles were formulated and assessed with regards to enhancement of skin permeation of diclofenac and stability profiles of the formulations. Formulation-induced changes of the biophysical structure of excised human skin were monitored using the Fourier transform infrared spectroscopy. The stability profiles of these suspensions over 12 weeks did not show any significant drug leakage from the vesicles of interest (p > 0.05). FTIR observations indicated that the vesicles increased stratum corneum (SC) lipid fluidization and altered protein conformation. Skin permeability experiments showed that the free unencapsulated drug in the cerosomal formulations caused significant increase in drug permeation across the skin (p < 0.01). Low skin permeability of drug from the other lipid suspensions could be due to the entrapment of diclofenac within these vesicles which decreased the solubility of the hydrophilic drug in the skin lipids and the partition coefficient of the drug from these vesicles into the SC. Optimal drug entrapment in vesicles or alteration of the skin structure may not necessarily enhance the permeation of hydrophilic drugs across the human skin. These lipid vesicles may be further developed into carriers of both hydrophilic and hydrophobic drugs for topical and transdermal delivery, respectively.

Amlodipine is a long acting, dihydropyridine type calcium channel blocker frequently used in the treatment of hypertension and coronary insufficiency. The calcium channel blocking activity resides primarily in the S-amlodipine enantiomer, while R-amlodipine is a potent inhibitor of smooth muscle cell migration. In this study capillary electrophoresis was applied for the enantiomeric separation of amlodipine using different native and derivatized; neutral and charged cyclodextrines as chiral selectors. The effects of pH and composition of the background electrolyte, concentration and type of chiral selector, capillary temperature, running voltage and injection parameters have been investigated. Stereoselective interactions were observed when using α-CD, β-CD, HP-β-CD, RAMEB, CM-β-CD and SBE-β-CD. Optimized separation conditions consisted on a 50 mM phosphate buffer, pH – 3.0, 20 mM RAMEB as chiral selector, + 25 kV applied voltage, 15°C temperature and UV detection at 238 nm. Using the optimized electrophoretic conditions we succeeded the chiral separation of amlodipine enantiomers in approximately 6 minute, the order of migration being R-amlodipine followed by S-amlodipine. The method was successfully applied for the determination of amlodipine enantiomers from commercially available pharmaceuticals. The linearity range, limits of detection and quantification, precision and accuracy were determined and the results obtained confirmed that the method was suitable for this purpose. It can be concluded that the proposed capillary electrophoresis methods can be useful for routine pharmaceutical applications with benefits of its effectivity, simplicity, short analysis time and low consumption of analytes, solvents and chiral selectors.

Breast cancer is the second leading cancer type among people of advanced countries. Various methods have been used for cancer treatment such as chemotherapy and radiotherapy. In the present study we have designed and synthesized a new group of drug delivery systems (DDS) containing a new class of Cell Penetrating Peptides (CPPs) named Peptide Amphiphiles (PAs). Two PAs and anionic peptides were synthesized using solid phase peptide synthesis (SPPS), namely [KW]4, [KW]5, E4 and E8. Then nano-peptides were synthesized by non-covalent binding between PAs and poly anions as [KW]4-E4, [KW]4- 8, [KW]5-E4 and [KW]5-E8. Flow cytometry studies showed that increased chain length of PAs with a higher ratio between hydrophobicity and net charge results in increased intracellular uptake by MCF7 cells after 2h incubation. Moreover, nano-peptides showed greater intracellular uptake compared to PAs. Anti-proliferative assay revealed that by increasing chain length of PAs, the toxicity effect on MCF7 cells is reduced, however nano-peptides did not show significant toxicity on MCF7 cells even at high concentration levels. These data suggest that due to the lack of toxicity effect at high concentration levels and also high cellular uptake, nano-peptides are more suitable carrier compared to PAs for drug delivery.

Polycaprolactone (PCL) is a biodegradable polyester and has attracted attention as a suitable carrier for development of controlled drug delivery due to its non-toxicity and biocompatibility. It has been reported that the biodegradability of PCL can be enhanced by copolymerization with PEG. Molecular weight (Mw) and CL block lengths optimization in a series of synthesized PCEC copolymers was the main purpose of this study. The composition of copolymers was designed using full factorial methodology. Molecular weight of used PEG (4 levels) and weight ratio of epsilon-caprolactone/PEG (3 levels) were selected as independent variables. The PCEC copolymers were synthesized by ring opening polymerization. Formation of copolymers was confirmed by FT-IR spectroscopy as well as H-NMR. The Mn of PCEC copolymers was calculated from HNMR spectra. The thermal behavior of copolymers was characterized on differential scanning calorimeter. Molecular weight of twelve synthesized copolymers was ranged from 1782 to 9264. In order to evaluate the effect of selected variables on the copolymers composition and Mw, a mathematical model for each response parameter with p-value less than 0.001were obtained. Average percent error for prediction of total Mn of copolymers and Mn of CL blocks were 13.81% and 14.88% respectively. In conclusion, the proposed model is significantly valid due to obtained low percent error in Mn prediction of test sets.

The present study is aimed to select the suitable method for preparation of camptothecin loaded polymeric nanoparticles by utilizing the multi-criteria decision making method. Novel approaches of drug delivery by formulation using nanotechnology are revolutionizing the future of medicine. Recent years have witnessed unprecedented growth of research and application in the area of nanotechnology. Nanoparticles have become an important area of research in the field of drug delivery because they have the ability to deliver a wide range of drug to varying areas of body. Despite of extensive research and development, polymeric nanoparticles are frequently used to improve the therapeutic effect of drugs. A number of techniques are available for the preparation of polymeric nanoparticles. The Analytical Hierarchy Process (AHP) is a method for decision making, which are derived from individual judgements for qualitative factors, using the pair-wise comparison matrix. In AHP, a decision hierarchy is constructed with a goal, criteria and alternatives. The model uses three main criteria 1) Instrument, 2) Process and Output and 3) Cost. In addition, there are eight sub-criteria’s as well as eight alternatives. Pair-wise comparison matrixes are used to obtain the overall priority weight and ranking for the selection of suitable method. Nanoprecipitation technique is the most suitable method for the preparation of camptothecin loaded polymeric nanoparticles with the highest overall priority weight of 0.297 In particular, the result indicates that the priority weights obtained from AHP could be defined as a multiple output for finding out the most suitable method for preparation of camptothecin loaded polymeric nanoparticles.

Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH 3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH 3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems.

In this study the intensity and duration of analgesic effect of diclofenac Na - Eudragit® RS100 solid dispersion and nanoparticles were evaluated by using formalin test in the rats. The animals received different formulations of diclofenac Na and subsequently 50 μl of formalin solution (2.5%) was injected subcutaneously in the right paws after 1 h, 2 h and 3 h. The paw licking behavior was then evaluated in two phases. A dose of 20 mg/kg of pure diclofenac Na powder was determined as effective dose. In the first phase, in term of reduced paw licking time, no significant differences were found in any of the groups compared to the control group. However, in the second phase, the animals which received pure drug powder and the physical mixture of diclofenac Na with Eudragit® RS100 showed significant differences at the first and second hours. In the animals received the nanoparticles and solid dispersion, significant differences were observed in the third hour compared to the control group. The analgesic effect of diclofenac Na could be improved by formulating its nanoparticles and solid dispersion with Eudragit® RS100. However, the nanoparticles revealed significantly higher analgesic effect than solid dispersion.

The purpose of this study was to evaluate the effect of intrahippocampal injection of vitamin C and progesterone, alone or in combination, on passive avoidance learning (PAL) in multiple sclerosis. Sixty- three male wistar rats were divided into nine groups (n=7) as following: control (saline), lesion, vitamin C (0.2, 1, 5 mg/kg), progesterone (0.01, 0.1, 1 μg/μl) and combination therapy. Lesion was induced by intrahippocampal injection of ethidium bromide. In combination therapy, animals were treated with vitamin C (5 mg/kg) plus progesterone (0.01 mg/kg). Animals in experimental groups received different treatments for 7 days, and then all groups were tested for step through latency (STL). Our results showed that intrahippocampal injection of ethidium bromide destroys PAL significantly (p<0.001). Treatment with vitamin C (5mg/kg) significantly (p<0.05) improved PAL. Lower doses of progesterone did not affect latency but dose of 1 μg/μl significantly (p<0.05) increased STL. In combination therapy group STL was significantly (p<0.05) more than in the lesion group, although it was not significantly different from the vitamin C group. Based on our results, we concluded that intrahippocampal injection of vitamin C improves memory for PAL, but progesterone alone or in combination with vitamin C had no improving effects on memory.

A protective effect for estrogens against cardiovascular problems has long been known. The aim of this study was to investigate the vasorelaxant effect of 17α Ethynylestradiol (17α-EE) on human saphenous vein. The veins were suspended horizontally between two triangular stainless steel hooks for the measurement of isometric tension in individual organ baths containing 10ml Krebs solution, at 37°C and gassed with carbogen under 3gr optimum tension. The effect of different concentrations of 17α-EE (2-40 μM) on vascular tone was investigated in veins precontracted with PGF2α. Relaxation was measured after 40min and expressed as the percent decrease of initial contraction. To determine the involvement of potassium channels, endothelium, nitric oxide synthase, guanylylcyclase and prostaglandins in the vasorelaxant effect of estrogen, the veins were incubated with tetraethyl ammonium, N-nitro-L-arginine methyl ester, methylene blue or indomethacin, respectively for 20min prior to experimentation. Responses to 17α-EE were directly compared to those obtained in the same tissues in the absence of the inhibitors. The mean relaxations induced by 17α-EE with concentrations of 2, 5, 10, 20 and 40μM in tissues precontracted with PGF2α were 19.8 ±5.5%, 26.1±10.8%, 32.2±7.4%, 48.6±10.8%and56±7.6%, respectively. The results of the inhibition of potassium channels, nitric oxide synthase, guanylylcyclase, cyclooxygenase and removing endothelium in relaxation induced by 17α-EE on precontracted veins with PGF2α proved no significant differences. This study showed that 17α-EE has significant vasorelaxant effect on human saphenous vein in a concentration-dependent manner. This effect is probably independent of potassium channels, nitric oxide synthase, guanylylcyclase, prostaglandin synthesis and endothelium functions.

Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori.

The purpose of our study was to evaluate the effect of oral taurine on the incidence of febrile episodes during chemotherapy in young adults with acute lymphoblastic leukemia. Forty young adults with acute lymphoblastic leukemia, at the beginning of maintenance course of their chemotherapy, were eligible for this study. The study population was randomized in a double blind manner to receive either taurine or placebo (2 gram per day orally). Life quality and side effects including febrile episodes were assessed using questionnaire. Data were analyzed using Pearson’s Chi square test. Of total forty participants, 43.8% were female and 56.3 % were male. The mean age was 19.16±1.95 years (ranges: 16-23 years). The results indicated that the levels of white blood cells are significantly (P<0.05) increased in taurine treated group. There was no elevation in blasts count. A total of 70 febrile episodes were observed during study, febrile episodes were significantly (P<0.05) lower in taurine patients in comparison to the control ones. The overall incidence of febrile episodes and infectious complications in acute lymphoblastic leukemia patients receiving taurine was lower than placebo group. Taurine’s ability to increase leukocyte count may result in lower febrile episodes.

Mouse IgG subclasses containing IgG1, IgG2a, IgG2b and IgG3 have been defined and described both physiochemically and immunologically. Sepharose beads conjugated with protein A affinity chromatography was used for purification of mouse IgG2b. Sodium citrate buffer (0.1 M, pH: 3.5) was used for separation of mouse IgG2b. Verification of the purified fractions was monitored by SDS-PAGE (polyacrylamide gel electrophoresis) in reducing condition. Immunized rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. After dialysis against tris-phosphate buffer (pH: 8.1) ion exchange chromatography column was used for purification of rabbit anti-mouse IgG2b. The periodate method was performed for conjugation with some variations. After conjugation, direct ELISA was used to determine the titer of HRP conjugated rabbit IgG against mouse IgG2b. The titer of rabbit anti-mouse IgG2b that determined by ELISA was 32000. The purity of rabbit anti-mouse IgG2b was about 95%. The optimum dilution of prepared HRP conjugated IgG was 1:10000. This study showed that ion-exchange chromatography and affinity chromatography could be appropriate techniques for purification of mouse IgG and IgG subclasses respectively. This study showed that affinity chromatography could be an appropriate method for purification of IgG2b antibodies.

This paper evaluates the impact of cross linking strategy on the characteristics of magnetic chitosan nanogels (MCNs) as targeted drug delivery system for doxorubicin. Sodium tripolyphosphate (TPP) and glutaraldehyde were used as physical (electrostatic) and chemical (covalent binding) cross-linker agents, respectively. MCNs were characterized by means of X-ray diffraction (XRD), Scanning electron microscopy (SEM), fourier transform infrared (FT-IR) spectroscopy and vibrating sample magnetometer (VSM). Scanning electron microscopy (SEM) indicated the formation of spherical nanostructures with the final average particle size of around 35-40 nm. The finding proved the superparamagnetic properties of the MCNs with relatively high-magnetization values which indicate that the MCNs were enough sensitive to external magnetic fields as a magnetic drug carrier. To understand the differences between the drug delivery properties of chemically and physically cross linked MCNs, the drug release studies were also conducted. Altogether, the results of this study clearly indicate that, however, both MCNs exhibited sustained drug release behaviour, the chemically cross linked MCNs provided enhanced controlled drug release characteristics in comparison to physically cross linked MCNs. Besides, according to the drug release behaviour of MCNs in buffer solutions in two different medium with the pH values of 5.3 and 7.4, it was clear that both nanoparticles exhibited pH sensitivity where the extent of drug release in the acidic media was significantly higher than neutral media. It can be concluded that chemically cross linked MCNs may serve as an ideal carrier for stimuli-triggered and controlled anticancer drug delivery.

The mutagenic potency of materials used in dentistry is of great concern. The Ames test is a bacterial reverse mutation assay, which is used to determine the mutagenicity potential of chemicals. In this study, the Ames test was used to compare mutagenic effects of three pulpotomy agents, namely, CEM cement, formocresol and ferric sulfate. TA100 strain of Salmonella typhimurium was used to evaluate mutagenicity of different concentrations of pulpotomy materials in the presence and absence of enzymatic system found in rat liver S9 fraction. Negative controls were 1% dimethyl sulfoxide and water. The positive controls were sodium azide and 2-aminoanthracene. The number of colonies per plate was counted. The material was regarded mutagenic if the number of histidine revertant colonies was twice or more than the spontaneous revertant colonies (Ames mutagenicity ratio). Ferric sulfate was found mutagenic in the concentrations prepared by addition of 50 μL of its 1 in 100 and 1 in 1000 times diluted solutions to the culture medium in the absence of S9 fraction (Ames test ratios of 2.8 and 2.2, respectively). Formocresol showed strong toxicity toward TA100 strain of S. typhimurium up to the concentration as low achieved using 1000 times diluted solution of the original preparation, particularly in the presence of S9 fraction. Ames assay failed to detect significant reverse mutations in all the concentrations of CEM cement. In contrast to formocresol and ferric sulfate, CEM cement is a less toxic and non-mutagenic agent.

The purpose of this study was to investigate the regular moderate exercise effect on the miR-192 expression changes in kidney of Streptozotocin- induced diabetic rats. Forty adult male Wistar rats were divided into four groups of 10, including Sedentary Control group, Healthy 60 days Exercise group, diabetic group and Diabetic 60 days Exercise. Diabetes was induced by injection of 60 mg/kg Streptozotocin and after 4 hour blood glucose levels higher than 250 mg/dl were included to diabetic rats. After 48 hour of induction diabetes, exercise protocol was begun. Animals performed 5 days of consecutive treadmill exercise (60 min/day) with 22 m/min speeds for 60 days. Kidney of the rats has removed and MicroRNA was extracted from kidney using miRCURYTM RNA isolation kit. Exercise upregulated miR-192 expression level significantly in the kidney of diabetic rats in comparison to healthy group. There is not any significant change in miR 192 expression in diabetic 60 days exercise compared to control group. These results may indicate that exercise can help to prevent the progression of diabetic nephropathy.

A validated ultra performance liquid chromatography (UPLC) method has been developed and validated for the simultaneous determination of losartan potassium and chlorthalidone in pharmaceutical preparations. Waters-Acquity UPLC system equipped with Auto Sampler, PDA detector and operated with Empower-2 software was used for the present study. Detection was done at wavelength of 230 nm, HSS C18, 100 mm x 2.1x 1.8 μm column with a reverse phase elution and mobile phase composed of A and B mixed in the ratio 56:44 v/v (Where mobile phase A consists of potassium dihydrogen phosphate buffer of pH 3.0 and Mobile phase B consists of acetonitrile and methanol mixed in the ratio of 90:10 v/v) used at a flow rate of 0.4ml per minute. The retention times for losartan potassium and chlorthalidone were observed at 0.72 and 1.89 minutes. The developed method was validated as per ICH guidelines. Linearity ranges were found to be 12.5-125 μg/ml and 3.125-31.25 μg/ml for losartan potassium and chlorthalidone, respectively. This method is fast, accurate, precise and sensitive hence it can be employed for routine quality control of tablets containing both drugs in industries.

Association between HLA-DR4–DQ8 haplotype and type 1 Diabetes Mellitus (DM-1A) was investigated in children of East Azerbaijan state of Iran because such an association has not been previously studied in this population. HLA-typing was performed by polymerase chain reaction sequence specific priming. For haplotype analysis, the logistic regression model was performed. Of the three investigated alleles, the frequency of DRB1*0401 was significantly higher among patients compared with that in healthy subjects (76.74% vs. 23.26%). The findings of the current study are consistent with those of previous studies and show that DRB1*0401 is associated with DM-1A; the frequencies of the two other alleles were also higher among patients, although the differences were not statistically significant. Two haplotypes associated with these alleles were also surveyed, and DRB1*0401−-DQA1*0301−, and DRB1*0401−-DQA1*0301−-DQB1*0302− were the most frequent haplotypes among the patient group.