Avicenna Journal of Medical Biotechnology
Year:2016 | Volume:8 | Issue:4|Papers Count:9

While preclinical research answers basic questions about a drug’s safety, it is not a substitute for studies of ways the drug will interact with the human body. “Clinical research” refers to studies, or trials, that are done in people1, 2. As the developers design the clinical study, they will consider what they want to accomplish for each of the different Clinical Research Phases and begin the Investigational New Drug Process, a process they must go through before clinical research begins. The ultimate goal of drug development is to bring a new compound with proven therapeutic effect to the market. In this context, the transition from preclinical research to clinical stages marks a critical turning point, as it nears the new medicinal product to the market 3, 4. With the promise of marketing authorization, though far ahead in the road, hanging on the horizon, the approval of a clinical trial usually attracts investors and leads to a respectable rise of the company shares. However, everything comes at a price. Clinical trials are not without risks, and while the perspective of success is encouraging, the crude reality is that most compounds fail before reaching the market. As explained in previous entries, despite higher R& D expenditures, attrition rates are high and, what is worse, on the rise. Data collected between 1990 and 2004 show that the number of unsuccessful clinical trials has been steadily increasing during the last years: from 30% to 50% at Phase 1, from 40% to 70% at Phase 2 and from 20% to nearly 50% at Phase 3. As a result, less than 10% of the drugs that enter clinical trials end up being approved by regulatory agencies. Clinical trials are only a small part of the research that goes into developing a new treatment. Drugs of the future, for example, first have to be discovered or created, purified, described, and tested in labs (in cell and animal studies) before ever reaching human clinical trials. Of all the substances that are tested in these early stages, very few are promising enough to be tested in humans. Drug development is the process of bringing a new pharmaceutical drug to the market once a lead compound has been identified through the process of drug discovery. It includes pre-clinical research on microorganisms and animals, filing for regulatory status, such as via the United States Food and Drug Administration for an investigational new drug to initiate clinical trials on humans, and may include the step of obtaining regulatory approval with a new drug application to market the drug. Indeed, of every 5, 000 cancer molecules identified in the laboratory, about 250 will enter pre-clinical testing. Of this 250, fewer than 10 are tested in clinical trials and on average only one will be approved by regulatory authorities. The process of bringing a new treatment from the research stage (laboratory) to clinic is estimated to take between 10-13 years.

Background: Some antidepressant drugs can promote neuronal cell proliferation in vitroas well as hippocampal neurogenesis in human and animal models. Furthermore, adipose tissue is an available source of adult stem cells with the ability to differentiate in to multiple lineages. Therefore, human Adipose-Derived Stem Cells (hADSCs) may be a suitable source for regenerative medical applications. Since there is no evidence for the effect of Paroxetine as the most commonly prescribed antidepressant drug for neurogenic potential of hADSCs, an attempt was made to determine the effect of Paroxetine on proliferation and neural differentiation of hADSCs.Methods: ADSCs were isolated from human abdominal fat. These cells differentiated to neuron-like cells and were treated with Paroxetine.3- [4, 5-dimethylthiazol-2-yl] -2, 5-diphenyl tetrazolium bromide (MTT) assay and immunofluorescence technique were used for assessment of cell proliferation and neurogenic differentiation potential of induced cells, respectively.Results: MTT assay analysis showed that Paroxetine significantly increased the proliferation rate of induced hADSCs (p<0.05), while immunofluorescent staining indicated that Paroxetine treatment during neurogenic differentiation could enhance the mean percentage of Nestin and MAP2 (Microtubule-associated protein-2) positive cells but the mean percentage of GFAP (Glial acidic fibrillary protein) positive cells significantly decreased relative to control group (p<0.05).Conclusion: Our results provide evidence that Paroxetine can promote proliferation and differentiation rate during neurogenic differentiation of hADSCs. Moreover, Paroxetine can reduce gliogenesis of induced hADSCs during neurogenic differentiation.

Background: It has been reported that secreted frizzled-related protein-4 known as an antagonist of Wnt signaling pathway plays a role in luteinization process of rodent granulosa cells. The purpose of this study was twofold: 1) to determine whether recombinant human secreted frizzled-related protein-4 (rhSFRP-4) could directly induce terminal differentiation of rat Granulosa Cells (GCs) and 2) to understand how the modulation of b-catenin and Protein Kinase B (PKB) /AKT activity by exogenous SFRP-4 could be involved in steroidogenesis.Methods: GCs were firstly stimulated with Follicle-Stimulating Hormone (FSH) named as FSH-primed cells then were treated with luteinizing hormone (LH). Then estradiol (E2) and progesterone (P4) production levels were assessed in the absence or presence of rhSFRP-4 treatment. The expression levels of activatedb-catenin, pAKTser473, pGSK3 b ser9 were assessed by western blot or immuno-fluoresence.Results: In the presence of rhSFRP-4, there was 38% decreased E2 levels compared to untreated FSH-primed cells (p<0.05), and P4 production subsequently decreased.However, in GCs pre-treated with rhSFRP-4 prior to addition of FSH, P4 levels increased 2-fold compared with untreated cells (p<0.05). Unexpectedly, treatment with rhSFRP-4 prior to LH stimulation inhibited LH-induced P4 secretion. Treatment with low (0.5ng/ml) but not high (50 ng/ml) concentrations of rhSFRP-4 led to significantly increased levels of pGSK3 b ser9 (1.6-fold) and nuclear active b -catenin (2.8-fold) in GCs compared with untreated cells. Interestingly, pre-treating GCs with rhsFPR4 prior to LH stimulation resulted in a 38% decrease in pAKTser473 levels compared with those in LH-treated cells (p<0.05).Conclusion: Taken together, our results showed that rhSFRP-4 could directly induce terminal differentiation in GCs via the modulation of b-catenin and PKB/AKT pathways and that it does so in a dose-dependent manner.

Background: Antibiotic resistant bacteria can be considered as a main problem in infection management. Zinc oxide nanoparticles (ZnO NPs), individually or in combination with antibiotics, can be considered as good candidates for struggling against drug resistant bacteria.Methods: In this study, Zinc oxide nanoparticles were synthesized using sol-gel method in low temperature as a cost effective procedure and characterized by X-ray diffraction and Scanning Electron Microscopy. Antibacterial activity of 9 new combinations of Zinc oxide nanoparticles and ceftazidime was assessed against standards and new clinically isolated multi drug resistant Pseudomonas aeruginosa (P. aeruginosa), in order to evaluate enhancement effect of synthesized Zinc oxide nanoparticles on antibacterial activity of ceftazidime.Results: The results indicated that desirable effects can be seen at 6 and 7 mM of Zinc oxide nanoparticles (60 to 100% inhibition). Moreover, after evaluation of 9 new combinations with various concentrations of both components, it was demonstrated that Zinc oxide nanoparticles can enhance the antibacterial activity of ceftazidime, against some bacterial strains of P. aeruginosa. The highest activity was observed with the concentration of 20mg/ml ceftazidime in the presence of 5, 6 or 7 mM of Zinc oxide nanoparticles.Conclusion: Zinc oxide nanoparticles in appropriate concentrations can be proposed as new and promising candidates for overcoming bacterial resistance.

Background: As a drug target and an antigenic agent, HIV-1 protease (HIV-1 PR) is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in E. coli and investigation of its immunoreactivity.Methods: Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning. Protease coding frame was isolated by PCR and cloned in pET102/D. TOPO expression vector and cloned protease was expressed in Escherichia coli (E. coli) BL21. Produced recombinant protein was purified by affinity Ni-NTA column and protein concentration was checked by BCA protein assay kit. Subsequently, immunoreactivity of recombinant protease (rPR) was assayed by Western blotting and ELISA.Results: Cloning of the HIV protease by TOPO cloning system in pET102/D.TOPO was confirmed with PCR and sequencing. The concentration range of purified recombinant protein was 85 to 100mg/ml. Immunogenicity of rPR was confirmed by Western blotting and ELISA.Conclusion: Soluble production of recombinant HIV-1 protease (HIV-1 rPR) was performed successfully. This recombinant protein disclosed 86% specificity and 90% sensitivity in immunoassay tests.

Background: In the recent years, there has been an increasing interest in secretory production of recombinant proteins, due to its various advantages compared with intracellular expression. Signal peptides play a critical role in prosperous secretion of recombinant proteins. Accordingly, different signal peptides have been assessed for their ability to produce secretory proteins by trial-and-error experiments. The aim of this study was to evaluate the effect of L-asparaginase II signal peptide on the recombinant human Growth Hormone (hGH) protein secretion in the Escherichia coli (E. coli) host.Methods: Cloning and expression of a synthetic hGH gene, containing L-asparaginase II signal sequence was performed in E. coli BL21 (DE3) using 0.1mM IPTG as an inducer at 23oC overnight. Periplasmic protein extraction was performed using three methods, including osmotic shock, osmotic shock in the presence of glycine and combined Lysozyme/ EDTA osmotic shock. Afterwards, the hGH expression was determined by SDSPAGE.Results: Based on experimentally obtained results, hGH protein is expressed as inclusion body even in the presence of L-asparaginase II signal peptide.Conclusion: Therefore, this signal peptide is not effective for secretory production of the recombinant hGH.

Background: L-tryptophan is used widespread in the pharmaceutical industry. The majority of L-Trp production depends on microbial processes that produce L-tryptophan from indole and L-serine. These processes are very costly due to the costs of precursors, especially L-serine. Use of inexpensive substitutions as the L-serine source of Ltryptophan production enables us to reach a cost-effective process. In this paper, effect of Triton X-100 on L-Trp production and the ability to use Iranian cane molasses as inexpensive L-serine source was investigated.Methods: Escherichia coli (E. coli) ATCC 11303 cells were grown in 10-L fermenter containing minimal medium supplemented with beet molasses as an inexpensive carbon source and indole as tryptophan synthase inducer. Whole cells of stationary phase were used as biocatalyst for L-Trp production. Triton X-100 addition to the production medium as indole reservoir was investigated. Then, cane molasses was used as LSer source in L-Trp production medium. Amount of L-Tryptophan and theoretical yield of L-Trp production was determined by HPLC and by a colorimetrically method on the basis of remaining indole assay, respectively.Results: As a result, triton X-100 increased L-Trp production three times. Also, the result showed that 0.68mM L-Tryptophan was produced in the presence of cane molasses at 37°C for 8 hr.Conclusion: This result showed that cane molasses of Qazvin sugar factory includes significant amounts of L-Ser that makes it a suitable substitution for L-Ser in L-Trp production. Therefore, it has the potential to be used for cost-effective L-Trp production in industrial scale.

Background: Genetic polymorphisms of drug metabolisms by cytochrome P450 (P450s) could affect drug response, attracting particular interest in the pharmacogenetics.Due to the importance of CYP2C19* 17 allele and its capability of super- fast metabolism and also lack of information about distribution of the alleles in Iranian population, this research aimed to use High Resolution Melting (HRM) method compared to PCR-RFLP for genotyping healthy Iranian population.Methods: Blood samples were collected from 100 healthy Iranian volunteers. DNA was extracted by salting out method. Real-time PCR was used for amplification of the CYP2C19 gene and the alleles were identified by HRM. Sequencing was used to confirm the amplified DNA fragments and data were analyzed using SPSS software ver.18.Results: The frequency of alleles CYP2C19*1/*1, CYP2C19*1/*17 and CYP2C19*17/*17 were estimated as 58.33, 29.1 and 11.1%, respectively. Specificity and sensitivity of HRM method were 90% and 100%, with respect to PCR-RFLP. Also, HRM analysis has been evaluated as a faster and more effective approach.Conclusion: Comparison of our results based on HRM analysis with PCR-RFLP showed that our developed method is rapid, accurate, fast and economic to study the CYP2C19*17 allele and it is appropriate for other similar population genetic studies.

Recurrent Aphthous Stomatitis (RAS) is the most common oral inflammatory disease, which is a painful, ulcerative condition of the oral cavity1 and is characterized by episodic, small, round ulcers with erythematous halos2. Although several factors such as systemic diseases, nutritional factors, psychological stress, local trauma, allergies, smoking and hormonal alterations could be associated with RAS, genetic factors seem to have an important role in predisposition to this condition whereas the exact pathogenesis of the disease has not clearly been understood3.