The invention relates to the production of hepatocyte cell lines useful in toxicology screens

Medicilon's pharmacokinetics department offers the clients a broad spectrum of high quality of services in the areas of in vitro ADME, in vivo pharmacokinetics and bioanalysis services, ranging from small molecules to large molecules, such as protein and antibody. The animal species involved in our services are non-human primate, canine, mice, rat, rabbit and hamster. Meanwhile, non-human primate experimental platform and isotope platform for protein/antibody are certified by the Shanghai Government. Email:[email protected] Web:www.medicilon.com
The invention relates to the production of hepatocyte cell lines useful in toxicology screens or therapy. A mammalian hepatocyte comprises, as a single polypeptide, a fusion protein comprising a c-myc protein and an oestrogen receptor, or functional fragments thereof.
This invention relates to conditionally-immortalised hepatocyte cells that can be scaled up for clinical and commercial application. Background to the Invention
The liver is responsible for the detoxification of drugs and poisons from the body. The most common cell type in the liver are hepatocytes; each hepatocyte has the capacity to perform each task required of the whole organ. Due to the metabolism of drugs in the liver, Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME/Tox) testing of drug candidates is performed on liver tissue to assess potential toxicity in vivo.
Traditionally ADME/Tox tests have been performed late in the process of developing new drugs, but the number of new drugs that fail because of toxicity is about 30%. There is therefore great interest in the development of high-throughput ADME/Tox screening that can be applied early in the drug development process, thereby saving time and money.
Many in vitro ADME/Tox studies focus on the liver's main metabolic enzymes, the cytochrome P450 enzyme family, although studies on P-glycoprotein and absorption studies with the CaCo-2 cell line are also commonly used.
Several biological assay systems are used to evaluate cytochrome P450 enzyme isoforms, but they all have some major disadvantages in their ability to predict in vivo toxicity. The main disadvantage of current assays is that only a limited number of liver enzymes can be studied at once, limiting the similarity between the assay and the situation in the liver itself1'2.
Human hepatocytes are the closest in vitro model of the human liver. Primary hepatocyte cultures are often used in ADME/Tox studies, as these express a «normal» hepatocyte phenotype. However, the availability of these cells is low and the expression of markers varies between batches. Foetal hepatocytes proliferate readily when dissociated and plated in cell culture under the right culture conditions, but the expression levels of many mature enzymes differ in foetal hepatocytes. For example, the level of cyp3A4, one of the main metabolic enzymes, is much lower in foetal than mature hepatocytes3. Adult hepatocytes also prove problematic in culture. With a low proliferation capacity, it is difficult to induce them to proliferate more then once in vitro, even with stimulation by growth factors4.
The problems associated with primary hepatocytes led to the development of hepatocyte cell lines. Several human hepatocyte cell lines are currently available, some derived from adult liver and some from foetal liver, either immortalized with virus or simply cultured without immortalisation5'6. However, no cell line expresses a «normal» human hepatocyte phenotype, mainly because the expression of many hepatocyte markers, including cytochrome P450, decreases rapidly or completely disappears in cell culture7. There are seven classes of hepatocyte mRNA markers, as indicated in table 1, together with examples in each class. Table 1 Liver mRNA markers for phenotype assessment.
It is possible to induce and upregulate the expression of some liver markers by adding substances such as 3-methylcholantrene and oncostatin M8'9 to the media.
However.it is not currently possible to upregulate the levels of all or most hepatocyte enzymes to a «normal» level by adding substrates to the media or altering culture conditions10. Several hepatocyte lines are available from other species, including rats and primates11'12, but these also have the problem of low expression of markers and the additional problem of interspecies differences.
There is therefore the need for a human hepatocyte cell line expressing normal liver functions and markers. Such a cell line, that could be used to study most phase I and Il drug metabolism enzymes including the study of cell-cell contact, would be an unlimited cell source, could be used in a high throughput system, give highly reproducible data, have no inter-species differences, and would be cost effective1.

A method for analyzing the ADME/PK properties

Medicilon's pharmacokinetics department offers the clients a broad spectrum of high quality of services in the areas of in vitro ADME, in vivo pharmacokinetics and bioanalysis services, ranging from small molecules to large molecules, such as protein and antibody. The animal species involved in our services are non-human primate, canine, mice, rat, rabbit and hamster. Meanwhile, non-human primate experimental platform and isotope platform for protein/antibody are certified by the Shanghai Government. Email:[email protected] Web:www.medicilon.com
A method for analyzing the ADME/PK properties of a mixture of compounds is (1) perfusing an animal or organ with a perfluorocarbon emulsion blood substitute, (2) administering the mixture of test compounds, (3) withdrawing an aliquot of the perfusate, (4) disrupting the emulsion, and (5) analyzing the aqueous phase of the perfusate for the concentration of test compounds.
This invention relates to the fields of pharmacokinetics and pharmacological research. More specifically, the invention describes the concept of using perfluorocarbon emulsions for examining the ADME/PK (absorption, distribution, metabolism, excretion and pharmacokinetics) properties of chemical mixtures in animals and a method for preparing the emulsions for direct analysis by techniques such as high performance liquid chromatography (HPLC), mass spectrometry and capillary electrophoresis.
BACKGROUND OF THE INVENTION
Drug development begins with the identification of a lead compound, based on the ability of the compound to exhibit a desired biological effect, such as the ability to inhibit bacterial growth, inhibit the activity of a target enzyme, increase or modulate the uptake of neurotransmitters, and the like. Biological activity is typically determined on the basis of in vitro experimentation or assays designed to rapidly identify candidate drugs. Typically, only a small percentage of the compounds tested will demonstrate sufficient activity and selectivity to merit further investigation.
Once a candidate or lead compound has been identified and selected for further development, its ADME/PK characteristics are determined. ADME/PK study concerns the absorption, distribution, metabolism, excretion and pharmacokinetics of drugs in the body. The ADME/PK properties of a drug are critical, and often serve to distinguish pharmaceutical products from mere lead compounds. For example, a drug that is poorly absorbed orally may require intravenous (or other parenteral) administration to be effective, which may be unacceptable for the condition to be treated. A compound effective as an antibiotic may be ineffective to treat bacterial meningitis if its distribution does not carry it to the central nervous system. A compound that is rapidly metabolized and/or excreted may not reside in the body long enough to serve its intended purpose. These properties are all independent of the drug candidate's in vitro activity, and are difficult or impossible to predict based on current information. The complex factors that influence ADME/PK make it hard to model accurately, and necessitate the use of living tissues and research animals before a compound may proceed with clinical trials.
To enhance the speed of drug discovery and reduce the number of animals required, it is desirable to characterize the ADME/PK of mixtures of lead compounds (rather than single compounds) in procedures that involve either living animals (i.e., in vivo), or isolated organs or organ systems from animals.
In in vivo analyses of ADME/PK assay, plasma is generally the biophase used as the analytical endpoint. Measurement of individual drug candidates in plasma typically involves a unique extraction method based on the physicochemical properties of each molecule, in order to separate and quantify the compound from the numerous plasma components. Optimization of one plasma extraction method for all components of a chemical mixture poses a major problem for rapid screening.
SUMMARY OF THE INVENTION
We have now invented a method for improving the ADME/PK analysis of candidate compounds, by replacing the blood of a test animal or tissue with an emulsified blood substitute, administering a test compound, and analyzing the resulting blood substitute. Preferably, the test compound is administered as a mixture of test compounds.
Another aspect of the invention is the method for designing libraries of pharmaceutical candidates based on ADME/PK properties.

Drug design: many tools for a many-faceted problem

Medicilon's structural biology department offers services supporting structure-based drug discovery from determination of novel targets to final structures. Our platform is one of the earliest established structural biology platforms in China and has been certified by the Shanghai Government. Email:[email protected] Web:www.medicilon.com
Computational Biology & Molecular Modeling
— Structural-Based Drug Design (SBDD)
— De Novo Drug Design
— Virtual Drug Screening
— Quantitative Structure-Activity Relationship (QSAR)
— ADMET Property Optimization
In this newsletter, four recent papers describe some of the state-of-the-art techniques being used to design new drugs, to solve the multi-objective problem and to coordinate the efforts of drug design teams for maximum benefit.
As technology and techniques improve it is becoming common to have access to a solved crystal structure of each protein target. In the case of anti-influenza drug design, Du et al.illustrate the impact that structure-based design can have. The availability of protein crystal structures of most influenza proteins has enabled researchers to rationally locate binding sites and successfully identify potential lead molecules through virtual screening. Understanding the structure–function relationship of these proteins has highlighted new routes into inhibition of the influenza virus. Moreover, knowledge of where viral mutations frequently occur enables researchers to design drugs that bind in the conserved regions thus overcoming issues of drug resistance.
Overcoming drug resistance is also the theme of a review from Hao et al. Two main methods of predicting the effect of mutation on drug binding and protein function are described. Once more protein structure proves invaluable and together with molecular dynamics simulation, can be a powerful method for examining the effect of an amino acid mutation. More accurate prediction can be gained from statistical learning techniques, although these require a large training set of known mutations. A combination of these two techniques therefore is most promising. Having identified resistance mutations, structure-based design can be directed to avoid such problems, for example, by targeting interactions with the unchanging backbone atoms, highly conserved residues or through multi-target design.
Of course, affinity for the target protein (and commonly occurring mutations) is but one facet of a drug. Combining this with numerous other pharmaceutically important properties is the subject of a recent article. Nicolaou et al. describe how multi-objective optimization methods are used with statistical modelling, docking, de novo design and library design to achieve simultaneous improvements against multiple parameters. This is especially challenging in non-continuous ‘chemistry-space’ (where small changes can have disproportionately large effects). One benefit of these methods is that they are capable of identifying multiple solution spaces, thus avoiding the ‘tunnel vision’ where drug designers get stuck in a local optimisation solution but remain ignorant of the superior global solution.
Clearly there is a wealth of techniques available to drug designers and numerous experts will contribute to drug design on a given target. One of the problems then encountered is how to integrate all these inputs in an easy and efficient manner, without overlooking a vital contribution. Robb et al. describe a wiki software system for capturing expert inputs, knowledge and design ideas and sharing these in a simple and effective way, promoting discussion and collaboration. Traditional synthesis-led design is supplemented by automatic structure-based and chemoinformatics input. Analysis of how the system was used enabled scientists to remove the bottlenecks of the drug design process and improve turnaround time for the realisation of each design idea.
The field of drug design is constantly evolving, incorporating new techniques and tools. Only by taking up these new techniques and making the maximum use of all available data and knowledge will drug designers continue to find new drugs and make a meaningful difference to patient lives.

Ligand Based Drug Design Biology Essay

Medicilon's structural biology department offers services supporting structure-based drug discovery from determination of novel targets to final structures. Our platform is one of the earliest established structural biology platforms in China and has been certified by the Shanghai Government. Email:[email protected] Web:www.medicilon.com
Computational Biology & Molecular Modeling
— Structural-Based Drug Design (SBDD)
— De Novo Drug Design
— Virtual Drug Screening
— Quantitative Structure-Activity Relationship (QSAR)
— ADMET Property Optimization
In practice, drugs were found by synthesizing the variety of compounds in taking a long time as well as many step processes against in vivo biological screens and additional examine is required for their pharmacokinetic properties, metabolic studies and possible toxicity studies. Such pre determined development process has resulted in higher success rates. This type of systematic development process which reduces the various failures such as poor pharmacokinetic studies, lack of efficacy, animal toxicity, adverse effects in humans and various miscellaneous factors.
The process of drug discovery has been cause the major change with the arrival of genomics, bioinformatics, proteomics, and effective technologies like, combinatorial chemistry, virtual screening, high throughput screening (HTS), de novo drug design, in vitro studies and in silico studies for pharmacokinetic screening and also for the structure-based drug design.
The In silico procedures are very useful in identifying drug targets via bioinformatics tools such as computer software programs. Further it is used to examine the lead structures for potential binding or active sites, produce structurally similar molecules, verifying for its drug likeness properties, dock these active molecules (ligands) with the target enzyme, arrange them according to their binding attractions, and finally optimize the lead molecules for to enhance its binding properties.
Nowadays the computers and various computational methods are developed for following reasons such as,
To reduce the complexity
Time consuming
Accurate results
Reproducibility
Lower cost
Novel target identification
Various facilities which brings the drug discovery process in a very simplest way those are,
High performance computing
Data management software
Internet and etc.,
Major advantages of computation in the drug development process as follows,
Virtual screening and de novo drug design
In silico pharmaco kinetic properties prediction
Improved methods for to determine protein-ligand binding.
Currently various protein targets are available through many newer techniques such as
Crystallography
NMR
Bio informatic methods,
Nowadays the demand is increased for computational methods that can encounter and examine active sites of the lead molecules and propose its possible drug molecules that can bind particularly in these binding sites. Usage of computers at early steps is concurrently reducing the cost and time need for the drug discovery and development process.

New report examines the global protein purification market

Medicilon's protein scientists have been working on protein expression and purification for many years. We can start your project even you have nothing in hand but the name of your protein. In Medicilon's laboratories, protein purification is performed in scales from micrograms and milligrams. All Protein Purification Services start with the analysis of physico-chemical and biological properties of a target protein resulting in the development of tailored procedures for its extraction, purification and characterization.Email:[email protected] Web:www.medicilon.com
The reports have also focused on the markets restraint facts which can actually slow down the demand of global Protein Purification market and hamper the regional economy. Whereas, the opportunity for its future growth is also mentioned for the customers so that they can easily understand the market scenario.
Furthermore, the Protein Separation Systems market research report gives an in-depth information about the overall market and various product segments and their growth trends. The future market forecasts about the global Protein Separation Systems market are also covered in the research report.
In addition, the overall market potential is further described in the report along with different countries around the globe.
The latest and the newest trends of the Protein Separation Systems industry are also included in this report. Moreover, overall global market size, the market size by product segment, growth rates of the global market along with and different product segments of the market, and various product segments with their value and volumes evaluation are also included in the research report.
The Global Protein Purification Industry 2017 Market Research Report is a professional and in-depth study on the current state of the Protein Purification industry.
The report provides a basic overview of the industry including definitions and classifications. The Protein Purification market analysis is provided for the international markets including development trends, competitive landscape analysis, and key regions development status.
Development policies and plans are discussed as well as manufacturing processes and cost structures are also analyzed. This report also states import/export consumption, supply and demand Figures, cost, price, revenue and gross margins.

Protein Assays Market expected to reach USD 2.41 billion in 2022

Medicilon's protein scientists have been working on protein expression and purification for many years. We can start your project even you have nothing in hand but the name of your protein. In Medicilon's laboratories, protein purification is performed in scales from micrograms and milligrams. All Protein Purification Services start with the analysis of physico-chemical and biological properties of a target protein resulting in the development of tailored procedures for its extraction, purification and characterization.Email:[email protected] Web:www.medicilon.com
The protein assays market is expected to reach USD 1.42 billion by 2017 and is projected to grow at a CAGR of 11.1% between 2017 and 2022, to reach USD 2.41 billion in 2022. Protein assays are used in life science research to determine the total protein concentration. Estimation of protein concentration is necessary for protein purification, electrophoresis, cell biology, molecular biology, and research applications. The protein assays market is witnessing high growth due to factors such as increasing pharmaceutical & biotech R&D expenditure and favorable government funding scenario for proteomics research.
The global protein assays market is segmented based on type, product, technology, application, end user, and region. The protein assays market by product is segmented into reagents, kits, and instruments & accessories. The reagents segment is expected to witness the highest growth during the forecast period, primarily due to the repeated purchases of reagents, unlike instruments that are considered a one-time investment. The market by type is segmented into dye-binding assays, copper-ion-based assays, test strip-based assays, and other protein assays. The dye-binding assays segment is expected to grow at the highest CAGR during the forecast period. This growth can be attributed to the increasing usage of dye-binding assays in disease diagnosis.
On the basis application, the market is segmented into disease diagnosis, drug discovery and development, and other applications (protein purification; electrophoresis; cell biology; molecular biology; host cell protein assays; and protein identification with blood groups, cell surface markers, drugs, and toxins).
The disease diagnosis segment is projected to be the fastest-growing market during the forecast year. This can be attributed to the increasing adoption of protein assays used to diagnose cancer; immune system diseases; and liver, kidney, and bone marrow diseases.
Key players operating in the protein assays market include Thermo Fisher Scientific (U.S.), Bio-Rad Laboratories, Inc. (U.S.), Merck KGaA (Germany), Cell Signaling Technology, Inc. (U.S.), Abcam plc (U.S.), General Electric Company (U.S.), and PerkinElmer Inc. (U.S.).
Research Coverage:
In this report, the global protein assays market is segmented based on product, type, application, technology end user, and region. In addition to comprehensive geographic and product analysis and market sizing, the report also provides a competitive landscape that covers the growth strategies adopted by industry players over the last three years. The company profiles in this report comprise the product portfolios, developments, and strategies adopted by players to maintain and increase their shares in the market. The above mentioned market research data, current market size, and forecast of future trends will help key market players and new entrants to make the necessary decisions regarding product offerings, geographic focus, change in strategic approach, and levels of output in order to remain successful in the protein assays market.
Reasons to Buy the Report:
This report will enable both established firms as well as new entrants/smaller firms to gauge the pulse of the market, which in turn will help these firms garner greater market shares. Firms purchasing the report can use any one or a combination of the below-mentioned five strategies (market penetration, product development/innovation, market development, market diversification, and competitive assessment) for strengthening their market shares.

A G-quadruplex-binding compound showing anti-tumour activity in an in vivo model for pancreatic cancer

Meidilon's pharmacokinetics department offers the clients a broad spectrum of high quality of services in the areas of in vitro ADME, in vivo pharmacokinetics and bioanalysis services, ranging from small molecules to large molecules, such as protein and antibody. The animal species involved in our services are non-human primate, canine, mice, rat, rabbit and hamster. Meanwhile, non-human primate experimental platform and isotope platform for protein/antibody are certified by the Shanghai Government. Email:[email protected] Web:www.medicilon.com
We report here that a tetra-substituted naphthalene-diimide derivative (MM41) has significant in vivo anti-tumour activity against the MIA PaCa-2 pancreatic cancer xenograft model. IV administration with a twice-weekly 15 mg/kg dose produces ca 80% tumour growth decrease in a group of tumour-bearing animals. Two animals survived tumour-free after 279 days. High levels of MM41 are rapidly transported into cell nuclei and were found to accumulate in the tumour. MM41 is a quadruplex-interactive compound which binds strongly to the quadruplexes encoded in the promoter sequences of the BCL-2 and k-RAS genes, both of which are dis-regulated in many human pancreatic cancers. Levels of BCL-2 were reduced by ca 40% in tumours from MM41-treated animals relative to controls, consistent with BCL-2 being a target for MM41. Molecular modelling suggests that MM41 binds to a BCL-2 quadruplex in a manner resembling that previously observed in co-crystal structures with human telomeric quadruplexes. This supports the concept that MM41 (and by implication other quadruplex-targeting small molecules) can bind to quadruplex-forming promoter regions in a number of genes and down-regulate their transcription. We suggest that quadruplexes within those master genes that are up-regulated drivers for particular cancers, may be selective targets for compounds such as MM41.
MM41 shows anticancer activity in vivo
The maximum tolerated dosage (MTD) for iv administration of MM41 was found to be ca 30mg/kg. A preliminary pharmacokinetic study at 20mg/kg has determined the in vivo half-life to be ca 4hrs. Since the targets (individual genomic DNA quadruplexes) are present in low copy numbers per cell, this half-life value suggests that even with twice-weekly dosing of MM41, there would be sufficient bioavailability to produce transcriptional inhibition of particular genes by means of quadruplex stabilisation. A therapeutic schedule of two doses were explored with the MIA PaCa-2 pancreatic tumour xenograft model, conducted at 10 and 15 mg/kg, each twice weekly, over a period of 5½ weeks (40 days: 12 doses). At this point the mice were sacrificed, apart from two from the 15 mg/kg group, which were maintained for a further 239 days without any further MM41 dosing. These two mice were chosen since their xenografts had completely regressed during the dosage period.
The synthesis and characterisation of MM41 (4,9-bis((3-(4-methylpiperazin-1-yl)propyl)amino)-2,7-bis(3-morpholinopropyl) benzo[lmn][3,8] phenanthroline-1,3,6,8(2H,7H)-tetraone: molecular weight 831.08) has been previously reported23,28. MM41 was analytically pure as shown by lc-ms and NMR methods and was formulated for biological studies as the freely water-soluble formate salt.
FRET studies
FRET DNA melting assays on MM41 were performed using a fluorescence resonance energy transfer (FRET) assay modified as a high-throughput screen in a 96-well format.49 The labelled oligonucleotides had attached the donor fluorophore FAM: 6-carboxyfluorescein and the acceptor fluorophore TAMRA: 6-carboxytetramethyl-rhodamine. The FRET probe sequences were diluted from stock to the correct concentration (400nM) in a 60mM potassium cacodylate buffer (pH 7.4) and then annealed by heating to 95 °C for 10 min, followed by cooling to RT in the heating block (3–3.5hrs). Solutions were prepared using 60 mM potassium cacodylate buffer (pH 7.4). 96-well plates (MJ Research, Waltham, MA) were prepared by aliquoting 50 μl of the annealed DNA into each well, followed by 50 μl of the compound solutions. Measurements were made on a DNA Engine Opticon (MJ Research) with excitation at 450–495nm and detection at 515–545 nm. Fluorescence readings were taken at intervals of 0.5 °C in the range 30–100 °C, with a constant temperature being maintained for 30sec prior to each reading to ensure a stable value. Final analysis of the data was carried out using a script written in the program Origin 7.0. The advanced curve-fitting function in Origin 7.0 was used for calculation of ΔTm values.
Molecular dynamics simulations
The starting point for the modelling study was the NMR structure of the BCL-2 promoter quadruplex, with mixed parallel/antiparallel G-strands forming the core of the quadruplex29, and a docked MM41 molecule (details of the docking procedure are given in the Supplementary Information). The G-stem bases exhibit both syn and anti-orientations. Previous studies have shown that parmXOL4 (XOL4) modification of the Cornell et al. force field refines the syn-anti balance as it facilitates syn-anti transitions through the 120° X region by decreasing the energy barrier for this transition, increases it through the 350° X region and refines the shape and depth of the syn minimum36. Previous simulations of G-quadruplexes in XOL4 have also shown an improvement in structures with respect to the simulations with earlier force field versions36. The XOL4 refinement has recently been included as DNA default force field in the AMBER code. Therefore, we carried out the present simulations using the parmbsc0 version of the Cornell et al. force-field with the modifications35,36,50,51 Solvation and addition of more ions to the quadruplex were performed with the help of the xleap module of the AMBER12 program. The quadruplex was neutralized using K+ ions and TIP3P water molecules were used for solvation. The system was placed in a periodic box whose boundaries extended at least 10 Å from any solute atom. The parameters for K+ ions were adapted for AMBER from a previous study (radius 1.593Å and well depth 0.4297054kcal mol1)52. The parameters for the MM41 ligand were generated via the Antechamber module of the AMBER software using the GAFF force field.
Standard equilibration protocols were used for initial minimization of the structure. The first round of equilibration with explicit solvent and ions involved 1000 steps of steepest descent, followed by 1000 steps of conjugate gradient energy minimization. A 300ps MD equilibration was performed in which the quadruplex was constrained, whereas the solvent and ions were allowed to equilibrate. The system was gently heated from 0K to 300K with a time step of 0.5 ps. This was followed by subsequent rounds of MD simulation, at constant pressure and 300K for 1ns. The constraints were gradually relaxed, until no constraints were applied to the system. The final MD simulations were carried out for 350ns using ACEMD53. The periodic boundary conditions were defined by the PME algorithm and non-bonded the cut-off was set to 10Å54. Covalent bonds involving hydrogen atoms were constrained using the SHAKE algorithm with a tolerance of 0.0001Å, which allowed the use of an integration time step of 2fs55. All the simulations were carried out at constant pressure of 1atm and constant temperature of 300K. The temperature and pressure was maintained using a Berendsen weak coupling thermostat56. The final production run without restraints was carried out for a continuous 350 ns and the frames were collected every 20ps. Analyses of trajectory were performed using the ptraj module of AMBER57 and the VMD58 and PYMOL programs were used for visualization.
Confocal studies
The human cancer cell line, MIA PaCa-2, was purchased from ATCC. Cells were maintained in DMEM culture medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1:100 dilution of penicillin-streptomycin solution Hybri-MaxTM. The cell line was maintained at 37°C, 5% CO2 and routinely passaged. Cells were seeded on poly-D-lysine coated coverslips in 12-well plates, 24 hours prior to addition of anticancer compound. To prepare the coverslips they were sterilised with 100% ethanol and rinsed with 1X PBS. Coverslips were then coated with 25 μg/ml poly-D-lysine for 1 hr at room temperature, rinsed in 1X PBS, and left to fully dry in the Laminar Flow Cabinet overnight before use. MM41 was added to the cells at a concentration of 333.2nM (equivalent to 20 X IC50) in culture medium. Following a 30 min incubation period with MM41 at 37°C, cells were rinsed with 1 x PBS and then fixed with ice-cold methanol for 15 min at RT. The fixed cells were washed twice with ice cold 1 x PBS. Coverslips were then mounted with a drop of vectashield mounting medium to microscope slides. Coverslips were sealed in place with nail polish. Trans light confocal images were obtained with a Zeiss LSM 710 microscope. Fluorescence from MM41 was collected with a 633 nm HeNe laser. Cells were imaged using a 63 x oil immersion lens.
Pharmacokinetics studies
A single dose of 20 mg/Kg was administered intravenously to mice and 25 μl blood was drawn from the tail vein at time-points 10, 20, 30, 60, 120, 240, 360 and 1440 mins. These samples were mixed immediately with 225 μl phosphate buffered saline and centrifuged for 5 min at 21,000 × g. 200 μl supernatant was transferred to a cryovial and frozen at –70 °C until analysis.
Plasma samples were thawed and the MM41 was extracted using SOLA HRP 10 mg/ml cartridges according to manufacturer’s instructions. Samples were dried under nitrogen and reconstituted in 200 μl mobile phase. High performance liquid chromatography was performed using a C18 reversed phase column with a water (0.1% TFA)/ acetonitrile (0.1% TFA) gradient. The flow rate was 1 ml/min and the column oven was set to 40°C. MM41 was detected by its fluorescence, with an excitation wavelength 280nm and an emission wavelength 660 nm. The standard curve range was 100–10,000 nM.

A-Synuclein Antibodies Enter Phase2, Sans Biomarker

Meidilon's pharmacokinetics department offers the clients a broad spectrum of high quality of services in the areas of in vitro ADME, in vivo pharmacokinetics and bioanalysis services, ranging from small molecules to large molecules, such as protein and antibody. The animal species involved in our services are non-human primate, canine, mice, rat, rabbit and hamster. Meanwhile, non-human primate experimental platform and isotope platform for protein/antibody are certified by the Shanghai Government. Email:[email protected] Web:www.medicilon.com
In the search for ways to slow down the progression of Parkinson’s disease, researchers currently have α-synuclein in their crosshairs. A handful of approaches are in development, and immunotherapy strategies are now advancing through clinical trials. At the 13th International Conference on Alzheimer’s and Parkinson’s Diseases, held March29 to April2 in Vienna, speakers from Prothena and Biogen presented Phase 1 data on their respective α-synuclein antibodies. Prothena discussed plans for Phase2, including smartphone monitoring to collect more detailed clinical data (see related AD/PD story). At the same time, researchers bemoaned the continued absence of good biomarkers to track disease progression and show efficacy of their drugs. What they really want is a PET tracer that detects pathological forms of the protein in living brain. Motivated by a large prize, scientists are working feverishly to find tracers, and in Vienna, Andreas Muhs of AC Immune, Lausanne, Switzerland, showed preclinical data on a candidate that selectively binds aggregated α-synuclein and appears to have suitable pharmacokinetics studies in rodents.
Academic and pharmaceutical researchers at AD/PD said they feel more hopeful than ever for meaningful progress on this disease. “The science has gotten more sophisticated, and we’re taking good bets in the clinical space,” noted Gene Kinney, who leads Prothena. At the same time, Kinney cautioned that many unknowns in these new types of trials make the way forward unclear.
The antibody appeared safe, Jankovic said. Participants did not make anti-drug antibodies. There were no serious adverse events, though some people reported skin reactions such as rash at the infusion site, and others had gastrointestinal complaints, headaches, or peripheral edema. Participants showed no improvement whatsoever on the Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS), though that was not expected in this relatively short study, Jankovic said.
The pharmacokinetic profile was largely typical for an antibody, although the half-life was on the short side at 14 days. As with other antibodies, only 0.3 percent of the amount in blood made it into CSF. This CSF/serum ratio was the same for all dose groups at week nine. Unbound α-synuclein in serum dropped by as much as 97 percent at the highest antibody dose, indicating target engagement in the periphery. Antibody levels rose in CSF in accordance with dose, but the amount of monomeric α-synuclein in CSF did not change. This was expected, since PRX002 targets primarily aggregates, Jankovic noted. The scientists do not have an assay to measure the concentration of α-syn aggregates in CSF before and after treatment.
Based on these findings, Prothena and Roche will begin a Phase 2, year-long efficacy study in 300 PD patients this year, with the primary outcome being change on the MDS-UPDRS.
Meanwhile, Biogen’s antibody BIIB054 is not far behind. Like PRX002, this human monoclonal antibody binds pathological, aggregated α-synuclein while sparing physiological forms. It was not clear whether physiological means the monomer or a postulated tetramer, and Biogen researchers could not be reached for comment. In Vienna, Andreas Weihofen of Biogen, Cambridge, Massachusetts, presented preclinical findings. In cell culture, BIIB054 reduced spreading of aggregated α-synuclein between neurons, and in mice injected with α-synuclein fibrils, BIIB054 slowed down pathology and improved motor function, Weihofen said.
In 2015, Biogen started a Phase 1 single ascending dose study in 48 healthy people between age 40 and 65. At two U.S. sites, volunteers received infusions of either 1, 5, 15, 45, 90, or a whopping 135 mg/kg, reported Biogen’s Miroslaw Brys in Vienna. Participants underwent three MRI scans, at baseline, day three, and week four. They donated CSF samples at baseline, eight hours, 24 hours, and week three. Researchers followed participants for 16 weeks after dosing, doing clinical assessments and electrocardiograms in search of adverse effects.
Doses up to 90 mg/kg were well-tolerated, with similar adverse events on placebo and drug, Brys said. In the 135 mg/kg cohort—which translates to 9.4 grams in a person weighing 70 kg, or 154 pounds—one participant developed asymptomatic ischemia in the right parietal lobe; this dose will not be used further. Some participants complained of headache, dizziness, or pain related to the infusion, and one person developed a skin rash at the infusion site.
The pharmacokinetic studies profile was as expected, with a half-life of 28 days and a CSF/serum ratio of 0.2 percent at all doses. The maximum concentration in blood was proportional to the antibody dose given. The researchers are still analyzing what happens to plasma α-synuclein, Brys noted. This trial is ongoing, aiming to enroll 66 people, but based on the preliminary data, the researchers are already planning to take BIIB054 into Phase 2, Brys said.

New Opportunities for Antineoplastic Drugs

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. Email:[email protected] Web:www.medicilon.com
In recent years, the incidence of malignant tumors increased year by year, anti-tumor drugs has become the fastest growing market areas. There are increased nearly 4 million new cancer patients annual in China, and overall size of anti-tumor drug market has reached nearly 100 billion Yuan, but it is far from meeting the actual needs.
Mostly prices of Antineoplastic drugs are high, not only bring a heavy burden to the family of patients to, but also test the wisdom of pricing for the enterprise. Before the negotiation system, many of the new marketed antineoplastic drugs were expensive, many families and patients had to retreat, or take conservative Chinese medicine treatment, or at risk of purchasing foreign drugs or APIs.
It should be said that China's anti-tumor drug market development is lagging behind and deformed. A large number of cancer-assisted drugs, proprietary Chinese medicine wasted health insurance resources, those who really have the exact effect of drugs, many patients cannot afford due to tender, or because of health insurance adjustment lag and other reasons.
With the development of genetic testing technology, precision medicine has arrived. In the field of anti-tumor, gene-based targeting drugs have become first-line antitumor drugs because of its significant effect and minimal side effect.
Therefore, tumor gene is complex, the existing drug coverage target is not enough, blindly follow the trend, imitation of many enterprises, which all have become the potential factors to restrict the development of targeted drugs. Data show that the treatment of lung cancer gefitinib, for example, CFDA in the approval of the business as many as 40, erlotinib is up to more than 100.
Targeting drugs must be one of the main anti-tumor drugs in the future, like the field of antibiotics as a full range, for different targets have different drugs, but one should be noticed: do not repeat and imitate.
Whether anti-cancer drugs can enter health insurance is not only related to the health of millions of patients, but also related to the success or failure of a product. In the past, China's health insurance system is lagging behind; many cutting-edge, new listing of drugs cannot be included in the health insurance directory, which is also because of backward enterprise education. Enterprises will generally start listing large-scale education to promote after drugs listed. Many clinical experts and medical workers also need some time to understand on the use of drugs, so health care will be more delayed.
Some domestic enterprises should fully study the foreign advanced experience. Some foreign cancer drug production enterprises will be published from time to time to progress, and let doctors and patients look forward to their projects. Once the clinical trial is successful, this projects can be quickly accepted by clinical and patients. Even if there is the possibility of failure, the enterprises establishing a corporate image of responsibility, and is also very favorable for follow-up branding building.
In addition to the government financial health insurance, enterprises should also actively try to build cooperation projects with commercial insurance. Now a lot of insurance companies specifically launched for the insurance products of malignant tumors, the combination of pharmaceutical companies and insurance companies should also be a positive exploration of one of the direction.

Purpose and Guidelines for Toxicokinetic Studies within the National Toxicology Program

Toxicokinetics (TK) is generation of kinetic data for systemic exposure and toxicity assessment of the drug. These studies help us to estimate the observed toxicity to that dose. TK evaluation is very important in drug development phase in both regulatory and scientific perspective. There are several guidelines to conduct TK study in animals recommended by regulatory bodies (OECD). TK evaluation is useful in selection of dose, dosing form, alternative dosing route, evaluation of toxicological mechanism, and also used for the setting safe dose level in clinical phases. This TK studies also used to reduces the animal number (replacement, reduction and refinement). On the other hand, TK data are practically used for the purpose of drug discovery such as lead-optimization and candidate-selection. Email:[email protected] Web:www.medicilon.com
The purpose of National Toxicology Program (NTP) toxicology/carcinogenicity studies is to identify toxic/carcinogenic effects resulting from exposure to a particular chemical or agent and to characterize dose-response relationships. In support of the toxicology/carcinogenicity studies, toxicokinetic (TK) studies are conducted to characterize the absorption, distribution, metabolism, and elimination (ADME) of xenobiotic materials and to quantify the influence of exposure on those properties. Toxicokinetic studies are proposed to * help in the design (e.g., dose selection, route of exposure, etc.) of meaningful and useful toxicology studies; * aid in the interpretation of toxicology studies by increasing our understanding of relationships between exposure, time-dependent target organ dosimetry, and adverse effects; and * define the parameters of dose, distribution, metabolism, and elimination that can be used in human risk assessment. Toxicokinetic studies also investigate the effects of sex, species, and age on ADME. Data collected during a toxicokinetic study focus on time profiles of parent chemical and metabolite concentrations in plasma and other tissues and can include rates of chemical absorption, metabolism, and excretion, chemical related changes in blood chemistry, bioavailability, protein binding, and depletion of cofactors. Ultimately, the data gathered during toxicology/carcinogenicity studies are used to estimate the risk to human health from exposure to a chemical. Characterization of relationships between toxic/carcinogenic endpoints and target organ dosimetry aid in the estimation of human risk. A description of a study design well-suited to the support of human risk assessment follows. This comprehensive design aims to collect the data necessary to develop physiologically based pharmacokinetic (PBPK) models incorporating some degree of anatomical realism and including all relevant biochemical and physiological processes. Prior to the determination of toxicological effects, it is impossible to propose a TK study design that is sufficient for all chemicals. The design proposed below contains elements believed to be necessary for the construction of PBPK models. The relative importance of some measurements may change depending on the compound under study. Two of the most difficult but critical problems in risk assessment are defining equivalent human dose and delineating the dose-response curve in the low-dose region. These problems motivate the recommendations to collect data to support the development and validation of PBPK models. If the only information available to the risk assessor is the applied or extemal dose, the estimated human equivalency dose will be based on several assumptions, such as equivalent absorption in laboratory animals and humans. However, uncertainties may exist in the predicted low-dose effects in humans if all of the doses that caused the adverse effect in the animal model occurred above the metabolic saturation exposure, if the chemical is absorbed differently in animals than in humans, or if the chemical is metabolized differently (qualitatively or quantitatively) between species. An objective of toxicokinetic studies is to improve our understanding of the relationship between external exposure and the tissue level of the toxic agent at the target site. By increasing our understanding of the relationships between target organ dosimetry and adverse effects, confidence in low-dose extrapolations of risk to humans is increased. Thus, knowledge of internal dose and factors that influence absorption, distribution, metabolism, and elimination in experimental animals and humans provide a greater scientific rationale for estimating low-dose risk than do estimates that are based solely on external exposure. Exposure- and time-dependent changes in target tissue concentrations of a parent compound and metabolites (especially if the adverse affects of exposure are due to particular metabolites) provide an even stronger scientific foundation for estimating equivalent human dose in various risk assessment models. Toxicokinetic/pharmacokinetic models provide the basis for more accurate low-dose extrapolations to humans. PBPK models can be adapted to different routes of exposure and dosage regimens and can accommodate factors that contribute to interindividual variabilities. Thus, the modeler can also provide valuable information relevant to the distribution of risk in exposed populations. The combination of dosimetry models with mechanistic data (e.g., mutagenicity, altered gene expression) can lead to more realistic biological models that would be useful in predicting site-specific dose responses and assessing human risk.