Why pharmaceutical company needs CRO?

For most pharmaceutical companies, entrusting some or all of their preclinical services to CRO companies is the inevitable choice of modern professional division of labor because it is more professional and more effective.
CRO companies have their own advantages, including integrated outsourcing services, international qualification standards and advanced R & D operation technology, service management process.
Medicilon is the first CRO Company in China to offer a full set of new drugs in line with China's GLP and US GLP standards for preclinical research and reporting;
Medicilon has been recognized as one of the top drug discovery contract research organizations (CRO) in China and is managed by a team of scientists with a wealth of experience in US-based pharmaceutical and biotechnology companies.
As our areas of expertise and service capabilities continue to expand, more and more pharmaceutical and biotechnology companies have taken advantage of our integrated drug discovery and development services.
Medicilon Chemical Department has a comprehensive range of chemical research capabilities and facilities including medicinal chemistry, synthetic chemistry, process research and analytical support, and has a good track record of preclinical research and advances in innovative projects. We can quickly complete the high-efficiency, low-cost way from the compound screening to the new drug clinical research report of new drug research and development projects.
Our team is composed of experienced team of chemists, able to provide customers with high quality and efficient synthesis of various small molecule compounds research services. It is capable to transfer the small amount of compound synthesis method into kilograms of the process line which can ensure the completion of the research project achieved in a very tight time limit.

Adenovirus-based exogenous gene expression in mammalian cells

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Adenoviruses have been used as a model system for understanding gene expression, DNA replication, gene delivery and other molecular biological phenomenon. In this project, adenovirus was used as a model to study exogenous gene expression in mammalian cells. More specifically, several adenoviral components were identified to enhance gene expression together with components needed for viral DNA replication. The adenoviral elements that enhance gene expression were assembled in an expression vector (pEl). These include the viral inverted terminal repeats (ITRs), the El region, the major late promoter (MLP) and the tripartite leader sequence (TPL). The green florescence protein (GFP) was used as a reporter gene. Various aspects of gene expression were examined including DNA delivery and stability inside the cells as well as mRNA transcription and protein expression. First, the effect of DNA quality on its delivery, stability and expression in mammalian cells was studied. Five different conditions of the major DNA contaminants were used in this investigation including ethidium bromide (EtBr), cesium chloride (CsCl), EtBr/CsCl, endotoxins and ethanol. CsCl, EtBr/CsCl and endotoxins affected the delivery process while EtBr affected the expression process but not the delivery. The used EtOH had no significant effect on both. In addition, the effect of all the contaminants was reversible. Next, we looked at the factors that enhance mRNA transcription and translation levels. Three approaches were tested, the first was the co-transfection of pEl and a plasmid that contains adenoviral genes involved in replication (pE2: contains E2 and viral protease). The second was the establishment of a cell line expressing these adenoviral genes involved in replication and the third approach was the super-infection with the wild type adenovirus. The co-transfection did not show any significant increase in gene expression or vector stability. On the other hand, the construction of CHO-E2 cell lines yielded five cell lines but none of them showed protein expression of all the integrated adenoviral E2 genes or enhancement of stability. Adenoviral super-infection enhanced gene expression. CHO cells showed higher enhancement in intensity and time than human embryonic kidney (HEK) 293 cells. In addition, such enhancement was dependent on the multiplicity of infection (MOI). Finally, this study emphasizes the importance of DNA quality on gene expression.

Modulation of indoleamine expression by activated T-cells in breast cancer

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Tumor infiltrating lymphocytes (TILs) secrete cytokines that modulate immune responses at the tumor microenvironment. Tumor suppressor activity of interferon gamma (IFNγ) cytokine also activates protein expression of immune suppressive factors such as IDO and PD-L1 in tumor cells. However, there is still much to learn about how tumor cells counter the immune cells at the gene expression level. In this study, RNA-seq analysis of breast cancer cells after in-vitro co-culture with anti-CD3/CD28 activated human T-cells revealed that the IFNγ induced immune response gene signature is common to both triple negative breast cancer (TNBC) MDA-MB-231 and estrogen receptor positive (ER+) MCF7 cells. However, IDO1 expression was differentially upregulated with significantly higher expression in MDA-MB-231 compared to MCF7 cells. Analysis of the TCGA breast invasive carcinoma dataset revealed subtype specific mRNA expression and IDO1 promoter DNA methylation. We observed that IDO1 mRNA expression and promoter methylation followed inverse correlation. TNBC/Basal subtype was hypomethylated at the IDO1 promoter with higher mRNA expression compared to the ER+ subtype that was hypermethylated with relatively lower IDO1 mRNA expression. The IDO1 promoter methylation was confirmed by pyrosequencing analysis of a panel of breast cancer cell lines and patient tumors. IFNγ treatment of MDA-MB-231 and MCF7 breast cancer cells revealed no difference in terms of upstream signaling and IDO1 mRNA stability. Treatment with demethylating agent, 5-aza deoxycytidine, synergistically up-regulated IDO1 mRNA expression in ER+ MCF7 cells highlighting that CpG methylation controls IDO1 gene expression. We also found a positive correlation between IDO1 and CD8A expression and better relapse free survival in TNBC/basal subtype patients suggesting that IDO1 protein expression is driven by intrinsic immune surveillance of TILs. These findings provide evidence that IDO1 promoter methylation regulates anti-immune responses by tumor cells towards TILs and it could be used as a predictive biomarker for IDO inhibitor-based immunotherapy of breast cancer.

ADMEt Evaluation of Anti-tuberculosis Compounds and New Methodologies Development

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Drug metabolism assays were used to study existing anti-tuberculosis (TB) drugs and to extend the half-life of experimental anti-TB compounds in internal drug discovery projects. A new cell model using HepaRG cells was developed and validated to study the induction potential of an experimental compound on liver enzymes. A multiplexed cytotoxicity assay was developed and validated with HepG2 cells to monitor three toxicity markers from the same culture. Finally, research was initiated to develop a novel TB active metabolite assay (TAMA) for monitoring the anti-TB activity of drug metabolites without the need for structural identification and synthesis.
Initial work involved the establishment of a panel of ADMEt assays. This panel assessed microsome stability, protein binding, plasma stability, gastric acid stability, cytochrome (CYP) P450 inhibition and induction, multiplexed cytotoxicity using HepG2 cells and metabolite toxicity using HepaRG cells. All of the assays were developed and validated in 96-well format in order to increase the throughput.
This panel of ADMEt assays was then applied to retrospective drug metabolism studies on existing anti-TB drugs. A bio-analytical method to detect TB drugs was developed and validated, which could simultaneously quantitate 15 existing anti-TB drugs from human serum plasma samples in one run. The method was given a name of ‘detection of multiple TB drugs’ (DMTD). The development and validation of DMTD followed FDA guidelines and appeared to be the first bio-analytical method that could detect more than 10 drugs in one run for anti-tuberculosis drugs. Compared with a previously reported method, which could simultaneously quantitate four anti-TB drugs in one run, DMTD gave an equal, or up to 10-fold lower, limit of quantitation (LOQ). Therefore, DMTD offered higher throughput and greater sensitivity than the previously published method.
DMTD was then applied in the assessment of the in vitro stability for all 15 established and experimental anti-TB drugs in both mouse and human microsomes. Five drugs were relatively unstable in both mouse and human microsomes; four of those drugs had previously been reported to be metabolically unstable and to have active metabolites. An extensive literature search failed to reveal any reports of metabolic stability for LL3858, which is in Phase I clinical trial for the treatment of TB. The experimental data, however, showed that LL3858 was metabolically unstable, and its metabolites were identified. This lays the groundwork for improving the drug-like properties of this series including the determination of the anti-TB activity of the (synthesized) metabolites.
The second series was based on a tetrahydroindazole template with a low micromolar minimum inhibitory concentration (MIC). Similar compounds have been previously reported to have promising anti-TB activity but to be relatively unstable in liver microsomes. However, the metabolites of this type of compound have yet to be identified. Through metabolite identification, the tert-butyl group of tetrahydroindazole was identified as the major metabolism site for hydroxylation. The metabolites were then synthesized but showed little anti-TB activity. Guided by microsomal stability data, a series of compounds were designed to block the metabolism site. A new lead compound, with a trifluoromethoxy group replacing the tert-butyl group, showed the best drug-like profile among all analogs synthesized with a 5-fold increase in microsomal stability, and similar anti-TB activity to the original screening hit. When administered orally to mice, plasma concentrations exceeded the MIC for more than 24 hours. Further research confirmed the compound to be active in inhibiting the growth of intracellular M. tuberculosis and to be effective against strains resistant to individual established TB drugs development.

Rational therapeutics in the biotechnology era

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This dissertation probes the rhetoric and reality of rational drug design in the United States biopharmaceutical industry at the close of the Therapeutic Revolution and the dawn of the Biotechnology Era. Starting in the mid-1970s, molecular biologists, genetic engineers, and biotechnology entrepreneurs imagined a new class of biopharmaceuticals for the twenty-first century. Biomedical scientists boasted that they were learning so much about the body's processes at the molecular level that traditional trial-and-error methods of drug discovery soon would be obsolete. They pursued a reductionist molecular etiology of disease and envisioned molecular manipulation of genetic material as the cure. These claims promised nothing less than a radical shift in biopharmaceutical research, from a pharmacology based in chemistry to one based in biology, and from the laborious, random, compound-screening method of drug discovery to rational, efficient drug design.
Three case studies demonstrate that even if therapeutic compounds could be produced successfully under conditions of rational design in the industrial laboratory, it did not necessarily follow that they could be evaluated under rational conditions in the hospital clinic, executive boardroom, regulatory arena, or pharmaceutical marketplace. Case studies include Immunetech's anti-allergy pentapeptide, Pentyde; Immunex's recombinant DNA granulocyte-macrophage colony-stimulating factor, Leukine; and Centocor's antisepsis monoclonal antibody, Centoxin. The cases cast biopharmaceutical endeavors as a triangular enterprise space bounded by entrepreneurship, technoscientific change, and regulation. Within this conceptual triangle, the cases explore the factors that stymied linear progress and introduced cul-de-sacs or hairpin turns into drug development pathways. And they delve into the technoscientific, sociopolitical, regulatory, and competitive factors that affected the safety, efficacy, and market viability of rationally designed drugs in the last third of the twentieth century.
The contributions of molecular biology to disease understanding, molecular pharmacology to drug discovery, statistical methodologies to clinical drug evaluation, and biotechnologies to drug production processes largely have failed to produce the miracle drugs promised by rational drug design. This dissertation reveals the range of scientific and extra-scientific hurdles to achieving a pharmacopoeia of rationally designed drugs and balances the biotechnology industry's success stories with analyses of some of its failures.