An in vitro biocompatibility testing system

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
The utility model provides an extracorporeal biocompatibility (BC) detecting system comprising a culture device, a temperature device, an injection device and a detection device. By means of combination of the devices, the system is used for establishing a standard program to detect BC or environment poison of biomedical materials.
means mutual adaptation biocompatible biomaterials and cells, tissues and physiological systems. The methods include: extraction, direct contact and indirect contact method. Wherein the extraction and direct contact disadvantage is not truly simulate Biomaterials contact with tissue condition; indirect contact method is the cells were cultured in three-dimensional matrix containing agar or collagen were more able to simulate the body's physiological environment, but the drawback is the need for testing at elevated temperatures or join enzyme to liquefy the matrix, resulting in damage and affect the reliability and validity analysis of cells. Since the polyether polyol F127 (PluiOnic F127) are FDA approved for use in the polymer medicine, its solution has a unique thermo-reversible gelation phenomenon, when the concentration of 15-20% (w / w) temperature to physiological temperature 37 ° C will form a gel, and reduced to a liquid at 4 ° C, its other advantage is that no additional crosslinking agent. The utility model is the use of this colloid in a simple and accurate measurement methods to enhance the efficiency and the wider use of in vitro biocompatibility analysis and biocompatibility replace traditional detection methods.
The utility model is an in vitro biocompatibility testing system, comprising: a culture device having an outer layer and an inner layer laminated, the outer layer is a microplate, for placing the sandwich, the sandwich of dish, for placing the inner layer and the inner layer is temperature-dependent aspect of colloid, which is used to give nutrients to the subject and the subject growth; temperature device, which features state-based control sample colloid; injection device The colloid was injected into the sandwich culture systems; and inspection means in the inner test culture apparatus for the growth of test specimen.
This utility model in vitro biocompatibility testing system, the regulation of the temperature range of the temperature of the device is (TC -25 ° c. And culture colloid layer of the device configuration requires maintaining at 0 ° C -4 ° C, and in After configuration subject to sterilization, and then placed inside the injection device. In addition, the culture apparatus further comprises colloidal, cell membrane stabilizers, are Baker's Basal Medium (Dulbecco 's Modified Eagle Medium) and fetal bovine serum (FBS). The colloid Wu is a polyether polyol F127 (Pluronic F127), and the cell membrane stabilizer is corticosterone, tocopherol, coenzyme Q or lecithin, choline.
This utility model in vitro biocompatibility testing system, the test apparatus system detects the subject survival, the test method is 2-methyl mercaptan diphenyl tetrazolium bromine detection (MTT) or trypan blue (Trypan Blue) cell count. And the detection method of the test device (TC _15 ° C liquefied detect which device is provided at the bottom of the sandwich culture pores, the sample liquid can penetrate into the microplate.
The utility model sandwich culture device, which is embedded dish dish.
This utility model in vitro biocompatibility testing system, which may be the subject cells.
This utility model in vitro biocompatibility testing system can be used as biomaterials testing tools.
This utility model in vitro biocompatibility testing system can be used as environmental toxicants detection tools.
The utility of new detection system (101) as shown in Figure 1, includes a culture device (102), a culture device (102) is an enlarged view, please refer to Figure 2, the culture device (102) to trace the outer disk (106), Sandwich embedded dish (107), the inner layer is temperature-dependent aspect colloid (109), which functions as a nutrient to be given specimen (110) and the specimen (110) growth; temperature means (103), Its function is to control colloid (109) aspect. The utility model detection system (101) also includes an injection device (104) as the colloid (109) was injected into the culture dissection device (102); and testing device (105), which means culture apparatus (102) Inside test, test specimen for (110) growth.
The utility model culture device (102) for the preparation of polyether polyols F127 (Pluronic F127) colloidal solution, according to the mixing ratio of DMEM and FBS. In the preparation of the polyether polyol need F127 solution was maintained at 0 ° C_4 ° C, and the solution must first be sterilized, the configuration of the polyether polyol F127 solution was placed in the injection device (104), this step need to 0 ° C-4 ° C, re-use injection device (104), polyether polyols F127 embedded solution was injected into the dish (107), and maintain the temperature of 37 ° C was colloidal state (109), with a polyether polyol F127 After the solution into a Petri dish embedded colloid, the colloid-containing polyether polyol F127 microplate was placed (106).
Example II: a polyether polyol F127 colloid special parts analysis
The utility model is more in 15% (w / w) polyether polyol F127 / are Baker's basal medium with 15% (w / w) polyether polyol F127 / secondary water at different temperatures to viscosity Changes observed polyether polyols F127 gel properties, its preferred embodiment, as shown in Figure 3 is displayed, with the temperature increase, 15% (w / w) polyether polyol F127 / Du Baker's Basal Medium viscosity values are also there is a growing trend, and the strength of colloids between 37~40 ° C reached a peak, and 15% (w / w) polyether 4 yuan alcohol F127 / secondary water viscosity value is not pulled situation.
The utility model specimen (110) directly onto the different concentrations of polyether polyol F127 / are Baker's Basal Medium / fetal bovine serum colloid surface and cultured specimen 24, 36 and 48 hours, and by the testing device (105) Trypan Blue (Trypan Blue) cell count test analysis.
The results are shown in Figure 4, the culture in the specimen 24 hours, due to the osmotic membrane under high concentration of polyether polyol F127 rise while filling the specimen and increased cytotoxicity caused by the survival of a large number of lower specimen; the specimen culture 36 hours, due to the embedded dish having pores (108) so that it has permeability, can reduce the impact of osmotic membrane specimen, polyether polyols F127 concentration 15% (w / w), the inspection can proliferator and maintain 75% specimen viability; its more preferred embodiment, the specimen cultured on polyether polyols F127 in 48 hours, cell survival can still be maintained as high as 80%.
The utility model and the other a better embodiment, as FIG. 8, the specimen embedded in polyether polyol / FBS colloids can be observed cell microencapsulation, the colloid promote inter-specimen and specimen began to gather. Also, the utility model is subject to the preferred embodiment of the cell.
Another embodiment of the utility model as the polyether polyols F127 concentration in 14% (w / w), 15% (w / w) and 16% (w / w), add the cell membrane stabilizer, which is corticosterone (hydrocortisone), coenzyme Q (QlO) and tocopherols, and the specimen cultured on polyether polyols F127 colloid, by the trypan blue cytometry analysis, 5, 6 and 7 show, check cultured for 24 hours, polyether polyols F127 concentration in 14% (w / w)> 15% (w / w) and 16% (w / w), to add to the preferred embodiment of corticosterone concentrations were O. 6 μ Μ, 6 μ M and 60 μ M; concentrations of coenzyme Q I μ Μ, 10 μ M and 100 μ M; tocopherol concentrations were O. 635mM, 6 35mM, 63 5mM and 635mM… And then through the analysis sample toxicity, but because of the embedded the dish with the 0.4μπι micropores (108) so as to have a semi-permeability, therefore the utility model, the sample (test solution) added directly to the culture device (102) and ways to penetrate into the culture within the system, and with the test specimen viability and morphology, known specimen Have cytotoxicity. In addition, the detection method shall be the test apparatus(105) detects 0°C -15°C.
Therefore, the preferred embodiment of the utility model is when you add cell membrane stabilizer may promote cell attachment and proliferation and survival of the subject to maintain higher than controls. And more preferably the concentration of cases in which the cell membrane stabilizer is implemented as corticosterone concentration of 60 μ Μ, coenzyme Q concentration of 100 μ Μ, tocopherol concentration of 635mM.
A person familiar with the art in this field can quickly appreciate the utility model can easily achieve their goals and get the results and advantages mentioned, as well as those which exist in things. The utility model in an in vitro biocompatibility testing system is representative of the preferred embodiment, which is exemplary and is not limited to the utility model in the field. Familiar with the art that modifications will occur in which the Department and other purposes. These modifications are inherent in the spirit of the utility model and define the scope of the patent.
This utility model describes the content and embodiments are disclosed in detail, such that any person skilled in the art can make and use this creation, even if there are at the various alternatives, modifications, and progress of, should still be considered without departing from the spirit and scope of the utility model. All patents and publications mentioned in the specification, and the creation of related fields are in general skills prevail. All patents and publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. The invention herein illustrated as appropriate, may be in the absence of any element, or a plurality of elements, limitation or limitations are not specific for the next case in the embodiment disclosed herein. Terms and expression used is as described in the specification and not of limitation, and there is no intent to exclude any use of such equivalent features shown and described or portions thereof and expression of the term, but need to recognize that, in this there may be various changes within the scope of the patent application creation. Therefore, we should understand that although the present invention will be specifically disclosed in accordance with the characteristics of the preferred embodiment and arbitrary, but known to the art that modifications and changes will continue to reveal the contents therein, modifications and variations of the present invention still like within the scope of patent applications.

Preparation of biocompatible materials by immobilization of apyrase

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
A prosthetic polymer material is made non-thrombogenic by immobilizing apyrase on its surface. Immobilization is preferably carried out by hydrolytically activating the surface of a polyamide polymer or a polyethylene terphthalate polymer, and treating the hydrolyzed polymer with a solution of cross-linking agent and a solution of apyrase. The apyrase converts adenosine diphosphate to adenosine monophosphate and adenosine whereby the formation of thrombi is inhibited.
The present application is a continuation of application Ser. No. 165,708 filed July 3, 1980, now abandoned.
This invention relates to a process for preparing biocompatible polymer and non-polymer materials by immobilising apyrase on their surface, apyrase being an enzyme able to catalyse the conversion of adenosine diphosphate (ADP) to adenosine monophosphate (AMP), followed by the conversion of this latter to adenosine. The invention also relates to the manufactured articles obtained by said process.
The possibility of imparting biocompatible properties to various types of material is of enormous practical importance at the present time. In this respect, materials exist which because of their good mechanical, machining and strength properties and the absence of toxicity would find immediate application in the construction of protheses of medium or long duration for implantation purposes, or in the construction of elements of auxiliary machines for use in extracorporal circulation, such as renal dialysis apparatus or heart-lung machines.
For this purpose, materials could be used lying within a very wide range from aliphatic or aromatic polyamide polymers, polyesters, polycarbonates, polyurethanes and PVC to special metal alloys. Unfortunately, the use of these materials is strongly limited by their generally poor biocompatibility testing. This is because as soon as a foreign material is inserted into the blood circulation, it immediately gives rise to the formation of thrombi by the initiation of an extremely complicated process.
It is believed that there is firstly an adhesion of the blood platelets to the material surface, with the consequent release from the platelets of ADP and serotonin, which then cause platelet aggregation. The platelet aggregation is itself a fundamental stage in the formation of thrombi. This is because it gives rise to the release of phospholipids which are essential in the blood coagulation process, by promoting the conversion of fibrinogen into fibrin.
The role of ADP as a platelet aggregation inducer, and thus as an initiator of the thrombi formation process, is widely known. See for example the fundamental work of A. Gaarder and A. Hellem in Nature 192, 531 (1961). It is therefore apparent that the immobilisation of an enzyme such as apyrase able to convert into AMP and adenosine the ADP produced by the adhesion of the platelets to the surface of the material which is placed in contact with the blood can inhibit subsequent platelet aggregation, so blocking the formation of thrombi. In this respect, it has been found, and forms the subject matter of the present invention, that the immobilisation of apyrase on the surface of thrombogenic materials gives these latter satisfactory biocompatible properties.
These properties do not depend on the system used for immobilising the enzyme. This can be done by adsorption and subsequent cross-linkage on the surface of polymer materials, or even on metal surfaces, for example on the surface of needles used for the arterio-venous connections in extracorporal circulation.
Alternatively, the apyrase can be bonded covalently to functional groups present on the surface of materials of which the biocompatibility testing is to be increased. It is indeed possible, if necessary, to previously activate the material so as to release reactive groups on to its surface which can be used for immobilising the apyrase.
For example, the enzyme can be attached covalently to the amino (or carboxyl) groups of aliphatic or aromatic polyamides subjected to mild surface hydrolysis. In the same manner, carboxyl groups of surface-hydrolysed polyesters can be used.
The invention is described in detail by the following examples, which however are not to be considered limiting.

As a technique for performing compound screening by using the aforementioned apparatus

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
A compound screening apparatus (160) divides measurement data representing the amount of binding between one kind of protein and each of a plurality of kinds of compounds, obtained by a measurement apparatus (110), into groups, each including data obtained in a same measurement condition. The compound screening apparatus (160) obtains a representative value of measurement data that is obtained when the protein and a compound are not bound to each other for each of the groups by using the measurement data of the respective groups, and sets a threshold value for extracting a hit compound for each of the groups by using corrected measurement data, obtained by correcting the measurement data for each of the groups so that the representative value obtained for each of the groups becomes the same value. Then, a hit compound is extracted by comparing the threshold value with the corrected measurement data.
Technical Field The present invention relates to a compound screening method and apparatus. Particularly, the present invention relates to a compound screeningmethod and apparatus for extracting a hit compound, which binds to one kind of protein, from a plurality of kinds of compounds. In the compound screening method and apparatus, the hit compound is extracted based on measurement data representing the amount of binding between the one kind of protein and each of the plurality of kinds of compounds.
Art Generally, many kinds of compounds included in medicines achieve their effects and functions by chemically binding to protein in living bodies. Therefore, in development of a medicine, it is important to know whether a candidate compound for the medicine binds to protein. Ideally, a compound included in the medicine should be bindable only to a protein of interest, and it should not be bindable to the other proteins. That is because if the compound is also bindable to proteins other than the protein of interest, so-called adverse side-effects may occur. Therefore, in development of a medicine, screening is performed to extract a compound that is bindable only to a protein of interest from candidate compounds for the medicine.
Various kinds of apparatuses have been proposed to perform screening on candidate compounds for medicines. For example, a measurement apparatus is well known (please refer to «Journal of
Spectroscopical Research of Japan», Vol. 47, No. 1 (1998)). The measurement apparatus is an apparatus utilizing a phenomenon that when a surface plasmon is generated by total reflection of a light beam at the surface of a metal, an attenuated total-reflection angle changes based on a dielectric constant at the vicinity of the surface of .the metal. The attenuated total-reflection angle is a specific reflection angle at which sharp attenuation (attenuated total reflection) of the intensity of light occurs in the totally-reflected light beam. Further, a similar measurement apparatus, for example, such as a leakage-mode measurement apparatus, which utilizes the attenuated total reflection, is also well known (please refer to «Journal of Spectroscopical Research of Japan», Vol. 47, No. 1 (1998) ).
As the apparatus using the principle of surface plasmon, «BIACORE3000», manufacturedby Biacore KK, or the like is well known, for example (please refer to «Real-Time Analysis Experiment Method of Interactions of Living-Body Substances», Kazuhiro Nagata and Hiroshi Handa, published by Springer-Verlag Tokyo).
As a technique for performing compound screening by using the aforementioned apparatus, a technique for extracting a hit compound, which binds to one kind of protein, is being considered. In the technique, measurement data representing the amount of binding between the one kind of protein and each of a multiplicity of kinds of compounds, for example 10000 kinds of compounds, is obtained. Then, a hit compound is extracted, based on the measurement data, from the 10000 kinds of compounds. In this technique, the hit compound is extracted based on a statistical processing result of 10000 kinds of measurement data obtained by measuring the amount of binding between the one kind of protein and each of the 10000 kinds of compounds. Specifically, for example, a threshold value for extracting the hit compound is set based on an average value of the values 'of the 10000 kinds of measurement data and the standard deviation of the values of the 10000 kinds of measurement data. Then, a compound corresponding to measurement data of which the value exceeds the threshold value is extracted as the hit compound. Generally, in compound screening as described above, approximately 1% of screened compounds, namely 100 kinds of compounds out of 10000 kinds of compounds, are hit compounds. It needs two or three days or even longer period to perform screening on so many kinds of compounds. However, if the measurement data that has been obtained as described above is widely dispersed, a compound that binds to a protein, but of which the amount of binding to the protein is small because of its low reaction speed or the like, is not detected in the dispersed data. Hence, such a compound is not extracted as a hit compound in some cases. Specifically, if the measurement data is widely dispersed, the value of measurement data obtained by using such a low-reaction compound becomes less than or equal to a threshold value for judging a hit compound in some cases. In such cases, the result of screening obtained by spending a plenty of time and expense is not sufficiently utilized. Hence, there are requests for more accurate screening of compounds to identify hit compounds.

Method for screening and producing compound libraries

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
A method for screening compound libraries or portions thereof by one or more bioavailability properties including absorption is provided. Novel compound libraries selected for bioavailability as produced by the method also are provided. The methods involve screening of compounds by absorption properties, and optionally absorption and one or more additional properties. The methods are exemplified by screening of a plurality of compounds of a first compound library and generating an absorption profile for each test sample of interest. Absorption profiles derived from screening the first library are compared and compounds having a desired absorption profile are selected to generate a second library. The absorption profiles are capable of being generated by supplying in vitro bioavailability data to a computer-implemented physiologic-based pharmacokinetic tool of the invention to generate as output a simulated absorption profile for each test sample. The process can be repeated one or more times so as to obtain a convergent library of compounds increasingly optimized for a bioavailability parameter including absorption.
Conventional methods to identify leads for drug development involve primary screening of compound libraries for activity «hits,» followed by secondary screening to reduce the number of primary hits to a congeneric series of optimal leads for drug development. The compound libraries, such as synthetic (e.g., combinatorial) and natural product (e.g., biological preparations and extracts) libraries, vary in size and complexity, ranging from hundreds, thousands, to millions or more of related or diverse compounds. The smaller libraries usually are well defined and each of the compounds frequently are contained in a separate storage or test vessel (e.g., dry or liquid form of the compound residing in a well of a multi-well storage or test plate with other members of the library). Larger libraries often are less well defined and typically contain mixtures or pools of compounds per vessel. For libraries containing pools of compounds, where an activity hit resides in one pool compared to the next, deconvolution and chemical analyses are typically performed in parallel to isolate and characterize the compound(s) responsible for the observed activity. Information gleaned from the initial screening and testing process also is used for subsequent rounds of analog synthesis (analog/focused libraries) and convergent screening and testing of particular analogs (i.e., iterative process). Computer-implemented theoretical or virtual compound libraries also provide a repository from which activity hits are selected for known or predicted structure-activity relationships.
Primary activity screening of compound libraries is based on selection of compounds that directly or indirectly interact with a specific biological receptor(s) (i.e., receptor- dependent activity screening). Isolated receptors and cells expressing single or combinations of receptors chosen to mimic a particular biological system or disease state generally provide the context of an assay for receptor-dependent activity screening. For high-throughput screening of larger libraries, automated systems utilizing multi-well arrays representing isolated receptors or cells that express them are the standard.
The driving force behind receptor-dependent activity screening as the primary approach for sifting through compound libraries is simple. Drugs (pharmacological/toxicological agent) elicit a pharmacological response through interaction with one or more biological receptors (drug/receptor-specific interaction). Thus, compounds that interact with a particular receptor or combination of receptors are presumed to be the most promising candidates for exhibiting some mutual activity in vivo and thus targets for secondary screening. Compounds identified from a primary screen are then subjected to successively more focused and quantitative rounds of screening and validation to eliminate false positives and identify those exhibiting optimal biological activity against a target receptor(s) in an in vivo setting. This typically involves a combination of physiochemical and biological testing, including structural characterization and biological studies using cells, tissues and animals. Compounds with the most promising biological activity are selected as leads for drug development.
Drug development involves scale up and detailed toxicity, pharmacodynamic and pharmacokmetic studies that are performed to characterize pharmacological efficacy. These studies are conducted not only to gauge whether a test compound has activity in an in vivo setting, but also to examine bioavailability to assess possible route of administration, delivery formulations and the amount of a test compound necessary over time to produce a therapeutic effect with little or acceptable side effects. A variety of cell, tissue and animal model assays typically are employed for such studies. A handful of compounds (e.g., 5-10) that pass these tests are then tested in scaled up animal studies for further characterization. A lead drug compound with the most promising results in animal studies is then tested in humans in clinical trials.
Pharmacokinetic studies are conducted to characterize the time-dependent concentration of a test compound in the body, which collectively depends on absorption, distribution, metabolism and elimination (ADME) of the compound following administration. For instance, in order to reach the site of action, a lead drug compound that is administered to a subject must first be absorbed across epithelial barriers, usually by passive diffusion and/or active uptake, into the systemic circulation. In the case of intravascular administration, absorption is instantaneous and complete. However, all other routes of administration involve an absoφtion step with the potential that only a fraction of the administered compound may be absorbed into systemic circulation.
Systemic blood then delivers the compound to cells and tissues in the body, where the likely receptor/site of action resides, but various parallel processes compete for the compound. The compound may reversibly bind with proteins (albumin, al-acid glycoprotein) in plasma, or in some instances with tissue proteins. This is important since an unbound compound is typically the form taken up by cells and tissues. These processes determine distribution of the compound.
In a process referred to as excretion or elimination, organs such as the kidney, lung and liver are able to remove an unchanged lead drug compound from systemic circulation. Alternatively, the compound may be metabolized by enzymes frequently localized in all tissues, but mainly in the liver. Such metabolism produces metabolites that are chemically different from the administered compound and generally are more readily excreted from the body (reduced lipid solubility). Often the pharmacological/toxicological activity of a metabolite is reduced compared to that of the parent compound.
Thus while a lead or collection of lead drug compounds may continue to exhibit promising activity profiles early in the drug development process, most fail to make it as a drug product because of poor bioavailability discovered in animals, or worse poor bioavailability not discovered until human clinical trials (e.g., gancyclovir). This unacceptably high and expensive failure rate can be attributed in large part to the biased nature of activity-based screening to identify primary hits ultimately used as lead drug candidates. For instance, activity screening is pursued from the mindset that the greater and more specific the compound-receptor interaction/activity, the more potent a compound, and thus the smaller the dose required and consequent lower potential for toxic side-effects, as well as cheaper product produced and sold. However, a potent compound exhibiting poor bioavailability might require a higher dose than a less potent compound exhibiting superior bioavailability; this less potent compound also may exhibit reduced dose related toxicity. Therefore, the majority of activity levels do not result in drug products.
Receptor-dependent screening and testing also provides little to no information as to the probable route of administration for an activity hit. As an example, a test compound selected for activity may ultimately require intravenous administration, which is a less preferred route of administration. Here again a different less potent compound overlooked or discarded from an activity screen for lower potency may have been a good candidate for a preferred extravascular form of administration (e.g., oral). An oral form would be cheaper to administer even if administered at a higher dose to compensate for lower potency. The dogmatic process of screening compound libraries first by receptor activity likewise reduces the value of the libraries themselves. Newly obtained or previously screened compounds having true therapeutic potential due to superior bioavailability properties are likely never to make it into the drug development pipeline if they fail to pass the primary activity screening process. Also valuable physical and chemical information from compounds otherwise possessing good bioavailability profiles that are discarded or overlooked for having less than some preferred activity level will be lost and unavailable for future development of structurally related activity leads or synthesis of new libraries.
Accordingly, a need exists for identifying compounds that exhibit desired pharmacokinetic properties before the drug development process, as well as guidance for future synthesis. The present invention provides an unprecedented and counterintuitive approach to address these and other needs.

Vivo drug development and delivery systems and methods

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
The present disclosure generally pertains to in vivo drug development and delivery systems and methods. The systems include a hollow tubular assembly with a chamber for receiving and transmitting an ionizing substrate solution, and a structure to transmit non-radioactive ionizing radiation to the ionizing substrate solution. The resulting free radical drug is transferred directly into a patient treatment site through an applicator. The systems and methods described herein provide simple, inexpensive techniques for in vivo production of an optimal chemotherapeutic drug without the use of radioactive radiation and directly injecting the drug into the patient's tissue with very minimal systemic side-effects.
This application is a continuation-in-part of and claims priority to U.S. patent application Ser. No. 13/917,522, entitled, “Drug Delivery and Treatment Systems and Methods” and filed on Jun. 13, 2013, which is incorporated herein by reference. This application also claims priority to U.S. Provisional Patent Application No. 61/912,110, entitled “In Vivo Drug Development and Treatment Systems and Methods,” and filed on Dec. 5, 2013, which is incorporated herein by reference, and U.S. Provisional Patent Application No. 61/918,515, entitled “In Vivo Drug Development and Treatment Systems and Methods,” and filed on Dec. 19, 2013, which is incorporated herein by reference. U.S. patent application Ser. No. 13/917,522 claims priority to U.S. Patent Provisional Application 61/659,077, entitled “Drug Delivery and Treatment Systems and Methods,” and filed on Jun. 13, 2012, which is incorporated herein by reference.
Cancer is an insidious and complex disease requiring multiple modality options. The three prevailing treatment options include surgery, chemotherapy, and nuclear or radioactive radiation. Surgical procedures, such as debulking, remove a portion of a malignant tumor, but it is often difficult to eliminate all of the diseased tissue such that the tumor returns. Radiotherapy includes irradiation, radiation therapy, or radiation oncology and is defined as the use of ionizing radioactive radiation to treat disease, kill cancer cells, or shrink tumors. Chemotherapy is the use of chemicals to treat disease, which is not limited to cancer. All three procedures have advantages as well as serious systemic consequences. There are many different types of cancers, as well as other diseases, each requiring individual treatment options utilizing a combination of the above-described therapies.
Many cancer patients receive at least one form of radiotherapy during their treatment cycle. Traditionally, radiotherapy is conducted in specialized facilities at a significant cost, sometimes on the order of hundreds of thousands of dollars per patient. Ionizing radiation is produced when a particle, such as a photon, acquires enough energy to remove an electron from an atom or molecule. Ionizing radiation is a biological and environmental hazard. Direct ionizing radiation describes charged particles (electrons, protons, and alpha particles) with sufficient energy to produce ionization by collision. Indirect ionizing radiation generally refers to the use of uncharged particles (neutrons and photons) to liberate particles by direct ionization. Radiotherapy generally involves the use of indirect radiation for the generation of free radicals, such as hydroxyl radicals, which then damage cancerous or diseased cells.
The energy level of an electromagnetic particle is indirectly proportional to its wavelength. For example, gamma rays with wavelengths of 50 fm have an energy level of about 25 MeV; x-rays with 50 pm wavelength yield about 25 keV; ultraviolet light with a wavelength of 100 nm yields about 12 eV; visible light with a wavelength of 550 nm yields about 2 eV; and microwaves with 1 cm wavelength exhibit roughly 120 μeV.
One major obstacle in the treatment of aggressive cancers is the fact that these cancers require chemotherapeutic or radiotherapeutic doses that are harmful or fatal to the patient. Treatment of cancer is systemic, where the cytotoxic drugs or radiation attack both malignant cells and healthy tissues. Selectively targeting the diseased cells is very difficult. In addition, radiation or chemotherapy treatment suppresses the immune system and therefore makes the patient susceptible to a host of other diseases. An additional complication is the fact that the patient's body adapts to the treatment and becomes resistant to further therapy.
Glioblastoma multiforme (GBM) tumors are the most common and aggressive malignant brain tumors in humans and are classified by the World Health Organization (WHO) as Grade IV tumors. Most GBM tumors originate in the deep white matter of the brain and quickly infiltrate other areas of the brain and the body. GBM tumors may grow very large before symptoms become apparent. GBM tumors are one of the most aggressive, resulting in a typical survival rate of less than a year after diagnosis. Treatment of these types of tumors is generally palliative, i.e., focusing on relieving and preventing the suffering of the patient, as there is no cure currently available. Recurrent tumors usually occur within 2 cm of the original tumor post treatment, which generally involves surgery followed by radiation and chemotherapy. GBM tumors are very resistant to chemotherapy. Aggressive radiation or chemotherapy treatment of recurrent tumors is difficult because the health of the patient is compromised and further procedures will shorten survival time. Patients suffering from GBM tumors and other cancers, such as pancreatic cancer, typically have poor prognosis as the available treatment options become too toxic and ineffective for continued treatment.
What is needed in the art, therefore, is a targeted, localized, minimally invasive cancer treatment which is readily available, causes fewer side effects for the patient and can be periodically repeated as needed to prevent the reoccurrence of the cancer.

Reconstituted tumor microenvironment for anticancer drug development

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
Extracellular matrix bioscaffolds capable of supporting the formation and growth of tumors from tumor cells introduced thereto containing tumor associated macrophages and carcinoma-associated fibroblast-like cells cultured under conditions effective to provide a cellular matrix capable of supporting the formation and growth of tumors from tumor cells introduced to the matrix. Bioscaffold kits and methods for using the bioscaffolds for testing, identifying and drug development of known or novel anticancer therapeutics are also disclosed.
The instant application claims 35 U.S.C. §119(e) priority to U.S. Provisional Patent Application Serial No. 61/212,794 filed April 15, 2009, the disclosure of which is incorporated herein by reference.
The present invention relates, generally, to an extracellular matrix bioscaffold that includes carcinoma-associated fibroblast-like cells and tumor associated macrophages supporting tumor cell growth and tumor formation, which may be used for the testing, identification and development of known or novel anticancer therapeutics.
Despite numerous advances and continuing research efforts, cancer is still one of the leading causes of human death worldwide. Understanding the environment surrounding cancer cell growth and motility is one avenue being explored and could provide useful information for the development of novel therapeutics. Current research suggests that cancer cell growth and invasion may be driven, at least in part, by the interactions between cells and a specific extracellular matrix (ECM). In the case of many epithelial-derived carcinomas, for example, a specialized ECM or basement membrane surrounds the primary tumor and is required for cell growth. With this in mind, many researchers have attempted to develop reconstituted basement membrane matrices that support cell growth in order to study the milieu of such carcinoma cells, as well as identify potential treatment target sites.
One such reconstituted basement membrane is set forth in U.S. Patent Nos. 4,829,000 and 5,158,874. The membrane, or «matrigel,» set forth in these patents is rich in the extracellular matrix proteins laminin, collagen IV, heparan sulfate proteoglycans, entactin, and nidogen. It is formed by first extracting these components from Engelbreth Holm-Swarm (EHS) mouse sacrcomas, then heating and polymerized the extract to form a three dimensional matrix. Such a matrix is embodied within the biological membrane and cell culture reagent BD Matrigel™, available from BD Biosciences. Indeed, this product is widely used by researchers for studying carcinoma cell interaction and identifying putative chemo therapeutic agents.
Many current methods for novel therapeutic evaluation rely on the detection of a change in carcinoma cell growth and motility within or through such a matrix. In particular, methods of assaying the effects of such therapeutics involve measuring a decrease in the number of cells (or the absence of cells) that grow within or penetrate through the matrix upon application of the agent. The use of such models, however, have certain drawbacks. In a large majority of instances, for example, in vitro results using such biological membranes do not correlate well with follow-up tests in vivo. Some researchers surmise that this may be because the murine- based Matrigel™ does not adequately mimic the actual cancer cell microenvironment. Thus, the myriad of cell types, growth factors, chemokines, and other relevant proteins and communication molecules that are involved in tumor cell growth and invasion are almost entirely ignored in the artificial environment. Such a drastic change in the tumor mileu from in vitro to in vivo test conditions could be a major reason why there is such a high incidence of drug failure. Accordingly, there remains a need in the art for more accurately replicating the tumor milieu or microenvironment, both in vitro and in vivo, which would lead to improved testing methodologies for chemo therapeutic compounds.
Beyond just drug screening assays, however, the increased understanding in the genetic variability of cancer cells makes specific and targeted treatment of a particular carcinoma genotype and/or phenotype increasingly possible and more desirable. Again, one current limitation in doing so is the inability to culture cells within an environment that substantially mimics the microenvironment within the patient. To this end, a matrix is also desirable that would replicate such conditions outside of the patient for the purpose of testing and identifying the effects of one or more known or novel agents on those cells. Such a matrix would, in certain instances, be adaptable to current in vitro or in vivo testing methodologies and would ultimately contribute to a personalized therapeutic strategy in treating carcinoma growth and invasion.
The instant invention through its embodiments and examples addresses these needs.
In one embodiment, the instant invention provides an extracellular matrix bioscaffold in which. tumor associated macrophages and carcinoma-associated fibroblast-like cells are cultured under conditions effective to provide a cellular matrix capable of supporting the formation and g &r-1owth of tumors from tumor cells introduced to the matrix.
Carcinoma-associated fibroblast-like cells may include mesenchymal stem cells that are differentiated on a tumor conditioned medium. In a non-limiting embodiment, mesenchymal stem cells are differentiated on tumor conditioned medium for about 1 to 30 days. Resulting cells express stromal-derived factor- 1 or may otherwise be positive for one or more biological markers selected from α-smooth muscle actin, vimentin, and fibroblast surface protein.
Tumor associated macrophages of the instant invention refer to a population of leukocytes exhibiting a macrophage phenotype that promotes tumor cell proliferation, metastasis and/or angiogenesis, or otherwise promotes chemotaxis of MSCs. In one non-limiting embodiment the tumor associated macrophages are phorbol ester differentiated leukocytes, such as HL-60 cells or U937 cells. In further embodiments, the HL-60 cells or U937 cells are differentiated in the presence of 3 nM of TPA for 96 hours.
Tumors capable of being grown in the extracellular matrix of the invention include any tumor cell line provided herein or otherwise known, which may be formed by incubating the tumor cells on the matrix for about one to about four days. In one embodiment, the tumor cells include one or more biological reporter genes. Such biological reporter genes may encode a reporter selected from a green fluorescence protein, luciferase, and combinations thereof. To this end, the biological reporter genes may be provided on one or more expression vectors that are transfected or otherwise expressed within the tumor cells.
In a further embodiment of the instant invention, methods for assaying the efficacy of a chemotherapeutic compound against a tumor cell line are provided, wherein the compound is administered to an extracellular matrix bioscaffold according to the present invention within which tumors are grown from the tumor cell line, and the size of the tumors are measured after.

System for facilitating drug discovery data and method thereof

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
A drug discovery research system which includes a plurality of computers. The drug discovery research system provides for at least one of the plurality of computers to run a multi-platform object oriented programming code, and at least one of the plurality of computers to store drug discovery related data. The system has a network architecture interconnecting the plurality of computers. The network architecture allows objects to transparently communicate with each other. The drug discovery research system provides for integrating and organizing data to facilitate drug discovery research.
The present invention relates generally to a client/server based collaborative system which allows for the integration of problem specific objects, algorithms and analyses. In particular, the present invention relates to a system which integrates and organizes biological and/or chemical data to facilitate drug discovery and design.
The use of computers for retrieval and analysis of data has become the standard in information-intensive industries such as finance and the sciences. As bodies of information have grown and become distributed into databases, a new set of disciplines (Informatics) aimed at studying the context of this data has been created.
Pharmaceutical and biotechnology companies place a high value on DNA and protein sequence information. For example, in 1996 a major pharmaceutical company earned revenues of $38 million based on subscription fees for the use of its sequence databases. Many pharmaceutical companies have large contracts and/or investments with gene discovery companies. However, since an unanalyzed DNA sequence has limited value, the outcome of gene discovery often hinges on bioinformatics—the application of computer technology to the analysis and management of sequence data.
Computer technology is essential to analyzing data such as DNA sequences, but today users of informatics related software find themselves in a dilemma. On one hand, the complexity of information makes the presentation of results crucial to understanding so the best informatics programs make use of interactive, graphical presentations. The best environment for understanding complex data relationships is a desktop computer running a graphical interface, but the computational demands of DNA sequence analysis require powerful workstations, or supercomputers. Such (often very expensive) computers do not support interactive graphical representations of analyses. At present there is no single program that performs all of the functions necessary for successfully analyzing DNA and protein sequences.
Although Web (e.g., network) technology allows users at a desktop computer to access programs and databases on remote computers, such programs lack a unifying standard. In particular, these programs have their own unique interface—program specific format for input and output. Ease of use is sacrificed, since users must learn to operate many different programs and must jump formidable technical hurdles to exchange data between these programs. As this often involves laborious and tedious manipulation of data files as well as detailed knowledge of the operations of programs and the quirks of each operating system, the chances of error are significant. Currently, scientists either spend unnecessary hours to accomplish tasks with these tools, or simply choose not to try, and potentially miss important observations.
In addition, web technology does not permit sufficient interactions between programs and databases. When a new program or database becomes available, the program or database needs to be first integrated and organized into each computer wishing to assess such new program or database before interactions between any old program or database with the new program or database is permissible. Each program or database originally installed on the computers needs to be told the addition of the new program or database in order to effectively utilize the new program or database. This could potentially be time consuming and costly.
In industrial fields, there are additional information management issues. Oftentimes, several researchers working in different offices in different states or countries have a need to share data results of tests and findings to maximize efficiency. Data management and analysis software, to date, has failed to fulfill this important need set, leaving the user to communicate his findings via post, E-mail, or informal verbal communication. These situations particularly exist in such fields as bioinformatics and chemi-informatics, where users have a strong need for sophisticated manipulation of data, with interactive and accessible output. Additionally, these users have an identifiable need for real-time sharing of pertinent information across multi-functional teams.
One answer to this dilemma is the use of a client/server system, (e.g. software on a personal computer or workstation, running a graphical user interface (GUI), acting as a client of server software running on larger, faster machines). Data is stored on central machines, allowing easy access for everyone on a project team. However, for such a system to function smoothly, the clients and the servers must share communication protocols, so either the software developer must control both the client and server software or a common standard must be adopted. While client-server solutions have become increasingly popular, traditional client/server systems are deficient in several ways which have made them unsuitable as an effective software support for a rapidly changing field like drug discovery.
Conventional client/server systems tend to suffer from inherent inflexibility, due to the tight coupling of the client and server. To operate properly, the client software must “know” on what particular computer the server software runs, and the protocol with which to “talk” to the server. If the server machine is busy or down, the client software is unable to work, even if other machines are available that could process its request. Such software is not very “soft”, as too many decisions are hardwired to it. If well designed, such systems can handle existing needs, but often need to be scrapped and totally rewritten if business needs change. In a rapidly changing field like bioinformatics, for example, the useful life of such software might be measured in months. Conventional client/server systems are, in addition, often very difficult to maintain and upgrade, since any changes made to the server requires complementary changes to the client. This situation is known as the “fat client” problem. For example, in a system that may have hundreds or even thousands of clients, even the slightest improvement in the server may lead to an enormous task for the system administrator in updating the improvement among the clients.
Furthermore, researchers in industry face significant security issues. Sequence data (that may have cost millions of dollars to collect) cannot be sent over the extremely public Internet where anyone might be listening. Consequently, many useful tools for sequence analysis (e.g., those provided over the Internet by the National Center for Biotechnology Information (NCBI), such as BLAST or Entrez) may be undesirable to use for researchers in industry due to the lack of security.
Drug discovery includes an almost parallel situation as mentioned above with respect to chemical data. Like Bioinformatics, there is no system currently available in the area of chemi-informatics which facilitates drug discovery without encountering many of the aforementioned deficiencies of conventional systems.
Thus, in light of the above problems associated with client/server systems and their applicability to Bioinformatics, Chemi-informatics and other data intensive industries, there is a strong need in the art for a system that overcomes these problems. In particular, there is a strong need for a system that provides for integrating and organizing biological and/or chemical data in order to facilitate drug discovery and design.
Moreover, there is a strong need for a system that provides for a secure research environment that can be used by researchers in industry.

Methods and platforms for drug discovery

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
Genetic variations (e.g., polymorphic alleles) within and among human patient populations underlie, to a large extent, differences in individual disposition to diseases, disease manifestation, disease severity, and response to treatment (e.g., to drug treatment). The prevalent animal and cellular models for human disease and drug discovery provide a poor representation of the genotypic/phenotypic spectrum extant in the patient populations to be treated. For example, strains of mice and rats commonly used in drug discovery are highly inbred, and thus only represent a very narrow range of possible genotype/phenotype combinations in mice or rats, let alone humans. Likewise, the relatively small number of human cell lines used for drug screening may reflect the genotypic/phenotypic scope of the individuals from which they were derived, but not that of a genetically diverse population. Further, most human cell lines are quite limited in their capacity to generate or phenocopy specific differentiated cell types (e.g., neurons, cardiomyocytes, and hepatocytes) affected by a particular health condition. Also, the cell lines are not representative of cell populations in a subject, since cell lines have been altered to indefinitely replicate. Importantly, in many cases animal models or genetically modified cell models of disease simply fail to adequately recapitulate the cellular disease phenotypes as they actually occur in a human patient's cells. Thus, typical preclinical drug discovery strategies miss many genotype/phenotypes that are present in the human population and will have a direct impact on the therapeutic efficacy and toxicity of a candidate drug compound. A practical consequence of these facts is that more often than not lead compounds fail in human clinical trials despite successful preclinical testing in animal models and transformed cell line models, as mentioned above. Ideally, drug screening and drug target discovery would be performed in biological models that recapitulate the genetic and phenotypic diversity present in a human patient population and the appropriate disease state at the cellular level, well before the clinical trial stage. These drug discovery paradigms are illustrated schematically in FIG. 1. In the traditional drug discovery model (left), candidate therapeutic agents are selected for clinical trials in patients based on their action on specific drug targets and their efficacy/lack of toxicity in animal models. In an alternative drug discovery model (right) the disease-relevant cells derived from patient iPSC lines, as described herein, are the starting point for identification of lead compounds based on their ability to ameliorate a disease-relevant cellular phenotype in patient derived cells.
Accordingly, the present disclosure describes human induced pluripotent stem cell lines from selected individuals (e.g., patients), genetically diverse panels of such cell lines, differentiated cells derived from such cell lines, and methods for their use in disease modeling, drug discovery, diagnostics, and individualized therapy.
II. Definitions
“Candidate drug compound,” as used herein, refers to any test compound to be assayed for its ability to affect a functional endpoint. Some examples of such functional endpoints are ligand binding to a receptor, receptor antagonism, receptor agonism, protein-protein interactions, enzymatic activities, transcriptional responses, etc.
“Correcting” a phenotype, as used herein, refers to altering a phenotype such that it more closely approximates a normal phenotype.
“iPSC donor,” as used herein, refers to a subject, e.g. a human patient from which one or more induced stem cell lines have been generated. Generally, the genome of an iPSC line corresponds to that of its iPSC donor.
“Phenomic analysis,” as used herein, refers to the analysis of phenotypes (e.g., resting calcium level, gene expression profiles, apoptotic index, electrophysiological properties, sensitivity to free radicals, compound uptake and extrusion, kinase activity, second messenger pathway responses) exhibited by a particular type of cell (e.g., cardiomyocytes).
“Phenome,” as used herein refers to the set of phenotypes that is subject and cell-type specific. For example, the phenome of hepatocytes and cardiomyocytes from the same individual will be quite distinct even though they share the same genome.
An “endogenous allele,” as used herein, refers to a naturally occurring allele that is native to the genome of a cell, i.e., an allele that is not introduced by recombinant methodologies.
An “iPSC-derived cell,” as used herein, refers to a cell that is generated from an iPSC either by proliferation of the iPSC to generate more iPSCs, or by differentiation of the iPSC into a different cell type. iPSC-derived cells include cells not differentiated directly from an iPSC, but from an intermediary cell type, e.g., a glial progenitor cell, a neural stem cell, or a cardiac progenitor cell.
A “normal” phenotype, as used herein, refers to a phenotype (e.g., apoptotic rate, resting calcium level, kinase activity, gene expression level) that falls within a range of phenotypes found in healthy individuals or that are not associated with (e.g., predictive of) a health condition.
III. Induced Stem Cell Lines for Drug Screening and Drug Target Discovery
A. Overview
The present disclosure provides human induced pluripotent stem cell (iPSC) lines, panels of stem cell lines, and methods for their use in drug discovery, diagnostic, and therapeutic methods as described in detail below. The induced pluripotent stem cell lines disclosed herein are characterized by long term self renewal, a normal karyotype, and the developmental potential to differentiate into a wide variety of cell types (e.g., neurons, cardiomyocytes, and hepatocytes). Induced pluripotent stem cell lines can be differentiated into cell lineages of all three germ layers, i.e., ectoderm, mesoderm, and endoderm.
An important nexus exists between a subject (e.g., a patient) and iPSC lines generated from that subject. First, all of the genotypes of iPSC lines and those of the corresponding subject are identical. Thus, genotype-phenotype correlations, uncovered in one are informative for the other, and vice versa. Second, differentiated cells (e.g., neurons) derived ex vivo from an iPSC line will exhibit a complete set of cellular phenotypes (referred to herein as a “phenome”) that are very similar, if not identical, to those of differentiated cells in vivo in the corresponding subject. This point is particularly relevant for developing therapeutics targeted to cells that cannot be routinely obtained from patients (e.g., neurons, cardiomyocytes, hepatocytes, or pancreatic cells). For example, in the case of a patient suffering from a neurodegenerative disease (e.g., parkinson's disease), dopaminergic neurons, which are typically affected by this condition, can be obtained non-invasively by differentiating an iPSC line from the subject, and can then be screened in multiple assays. Thus, iPSC lines provide a renewable source of differentiated cells (e.g., inaccessible differentiated cells) in which pathological cellular phenotypes that are associated with a disease, cell type, and individual may be examined and screened against test compounds. An exemplary, non-limiting embodiment of this approach to disease modeling and drug discovery is schematically illustrated in FIG. 2. iPSC lines and iPSC-derived cells (e.g., motor neurons) are also useful for predicting the efficacy and/or adverse side effects of a candidate drug compound in specific individuals or groups of individuals, as schematically illustrated in FIG. 3. For example, test compounds can be tested for toxicity in hepatocytes differentiated from a genetically diverse panel of induced pluripotent stem cells. Toxicity testing in iPSC-derived hepatocytes can reveal both the overall likelihood of toxicity of a test compound in a target patient population, and the likelihood of toxicity in specific patients within that population.
In effect, iPSC lines and iPSC-derived cells (e.g., pancreatic cells) can serve as “cellular avatars,” that reveal cellular phenotypes that are disease, cell-type, and subject-specific to the extent the phenotypes are determined or predisposed by the genome. Collectively, panels of patient induced stem cell lines will represent a wide range of genotype/phenotype combinations in a patient population. Thus, they are useful for developing therapeutics that are effective and safe across a wide range of the relevant target population, or for determining which individuals can be treated effectively and safely with a given therapeutic agent.
B. Screening and Selection of Subject Samples
Some of the methods described herein utilize induced stem cell lines or panels of induced stem cell lines derived from subjects that meet one or more pre-determined criteria. In some cases subjects and cellular samples from such subjects may be selected for the generation of induced stem cell lines and panels of induced stem cell lines based on one or more of such pre-determined criteria. These include, but are not limited to, the presence or absence of a health condition in a subject, one or more positive diagnostic criteria for a health condition, a family medical history indicating a predisposition or recurrence of a health condition, the presence or absence of a genotype associated with a health condition, or the presence of at least one polymorphic allele that is not already represented in a panel of induced stem cell lines.
In some cases, a panel of induced stem cell lines is generated specifically from individuals diagnosed with a health condition, and from subjects that are free of the health condition. Such health conditions include, without limitation, neurodegenerative disorders; neurological disorders such as cognitive impairment, and mood disorders; auditory disease such as deafness; osteoporosis; cardiovascular diseases; diabetes; metabolic disorders; respiratory diseases; drug sensitivity conditions; eye diseases such as macular degeneration; immunological disorders; hematological diseases; kidney diseases; proliferative disorders; genetic disorders, traumatic injury, stroke, organ failure, or loss of limb.

Method and apparatus for elemental analysis of a fluid downhole

Our process analytical sciences group provides support for research, development and commercial production. For more details, please click the link Process Development Service.At Medicilon, the importance of analytical support in pharmaceutical development and manufacturing is well understood. We provide our clients with analytical method development and all other services required to support their regulatory needs.Email:[email protected] Web:www.medicilon.com
The present invention provides a method and apparatus for performing elemental analysis of a formation fluid downhole. The present invention provides elemental analysis of a formation fluid downhole using breakdown spectroscopy. In one aspect of the invention, a method and apparatus are provided for performing laser induced breakdown on a formation fluid sample is provided. In another aspect of the invention a method and apparatus are provided for performing spark induced breakdown spectroscopy. Plasma is induced in a fluid under test downhole. Emissions from the plasma are analyzed to determine the elemental composition of the fluid under test. Emissions include but are not limited to light in the ultraviolet, visible, and near infrared regions of the spectrum. A spectrometer is provided for elemental analysis of a fluid downhole. Elemental analysis yields information about the fluid and the formation from which the fluid originated.
The present invention relates to compositional analysis of a fluid sample downhole. More particularly, the present invention relates to the elemental analysis of samples downhole such that they may be analyzed for their constituent components via laser induced breakdown spectroscopy (LIBS), spark-induced breakdown spectroscopy (SIBS) or some similar technique of plasma generation and optical emission analysis.
There are many situations where it is necessary or desirable to obtain substantially instantaneous and/or immediate major and trace constituent analysis of a sample material. Sample materials may include geological samples, soil samples, powder metallurgy, ceramics, food, pharmaceuticals, and many other materials. There are many reasons why it would be desirable to test these materials for their composition of components. Hydrocarbon production is costly and knowing that production of a particular hydrocarbon bearing formation is not feasible due to content of undesirable elements such as sulfur may deem a formation infeasible. Compartmentalization is also a problem encounter during hydrocarbon production and the existence of such compartmentalization is valuable knowledge affecting production decisions involving millions of dollars in production expense.
There is currently no known method and apparatus for performing elemental analysis downhole. It would be useful to perform elemental analysis downhole on formation fluid to determine the characteristics of a formation fluid sample and the formation from which the fluid originated.
The present invention provides a method and apparatus for performing elemental analysis of a formation fluid downhole. The present invention provides elemental analysis of a formation fluid downhole using breakdown spectroscopy. Plasma is induced in a fluid under test downhole. Emissions from the plasma are analyzed to determine the composition of the fluid under test. Emissions include but are not limited to light in the ultraviolet, visible, and near infrared regions of the spectrum. A spectrometer is provided for elemental and compositional analysis of a fluid downhole. Compositional analysis yields information about the fluid and the formation from which the fluid originated. In one aspect of the invention, a method and apparatus for performing laser induced breakdown on a formation fluid sample is provided. In another aspect of the invention a method and apparatus are provide for performing spark induced breakdown spectroscopy. It is well known how to apply breakdown spectroscopy in air in the laboratory at room pressure. However, applying this technology downhole presents several challenges. First of all, downhole fluids are typically under tremendous pressures of 10-20 kpsi. Therefore, to apply such a technique downhole, sufficient energy must be applied over a small enough volume within a short enough period of time (for example by using a strong enough laser or a spark), so as to raise the temperature high enough (about 10,000 C) that the pressure within the plasma exceeds the pressure within the fluid. In this way, it becomes possible for a small bubble of plasma to form within the high pressure fluid. Secondly, it must be possible to detect the light coming from this bubble of plasma even though this bubble is immersed within a dark fluid (such as crude oil) that strongly absorbs the light which it emits.
The present invention is useful for analysis of formation fluid extracted from a dilled wellbore or for analysis for fluid in a monitoring while drilling operation when deployed from a drill string or coiled tubing. The term fluid is used in this specification to mean a gas, fluid or a multiphase mixture of gas, fluid and condensate or particulate suspended therein. In an alternative embodiment, the present invention may also be deployed in a pipeline for analysis of fluid transported in the pipeline. In each case, a LIBS device is provided to perform elemental analysis of a fluid associated with the deployment environment. Similarly, a spark induced spark spectroscopy (SIBS) device may be used in place of the LIBS device for elemental analysis of the fluid. Elemental analysis enables the present invention to estimate the composition of a fluid and estimate a property of the formation from which the fluid originated.
Spark induced breakdown spectroscopy (SIBS), Laser-Induced Plasma Spectroscopy (LIPS) or, as it is more often known, Laser-Induced Breakdown Spectroscopy (LIBS), is a form of atomic emission spectroscopy in which a pulsed laser is used as the excitation source. The output of a pulsed laser, such as a Q-switched Nd: YAG, is focused into or onto the surface or of the material to be analyzed. For the duration of the laser pulse, which is typically 10-20 nanoseconds, the power density at the surface of the material can exceed 1 Giga watt per cm2 using only a compact laser device and simple focusing lenses.
At these very high power densities, a fraction of a microgram of material is ejected from the surface by a process known as laser ablation and a short-lived but highly luminous plasma with instantaneous temperatures reaching 10,000° C. is formed at the surface of the material. Within this hot plasma, the ejected material is dissociated into excited ionic and atomic species. At the end of the laser pulse, the plasma quickly cools as it expands outwards at supersonic speeds. During this time the excited ions and atoms emit characteristic optical radiation as they revert to lower energy states. Detection and spectral analysis of this optical radiation using a sensitive spectrograph can be used to yield information on the elemental composition of the material.

Kinase and phosphatase assays conducted by elemental analysis

Our process analytical sciences group provides support for research, development and commercial production. For more details, please click the link Process Development Service.At Medicilon, the importance of analytical support in pharmaceutical development and manufacturing is well understood. We provide our clients with analytical method development and all other services required to support their regulatory needs.Email:[email protected] Web:www.medicilon.com
Assay methods and kits for kinase and phosphatase enzymes involved in post-translational modifications of proteins are provided. The methods employ elemental analysis, including inductively coupled plasma mass spectrometry (ICP-MS), in conjunction with metal ion coordination complexes such as titanium dioxide or element-tagged antibodies, both of which may have affinity for phosphate groups. The methods allow for convenient and accurate assays for enzymes involved in post-translational modifications of substrates.
This invention pertains to the determination of post-translational modification of proteins such as phosphorylation and dephosphorylation, including methods and kits, employing elemental analysis.
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyrights whatsoever.
This invention pertains to the determination of post-translational modification of proteins such as phosphorylation and dephosphorylation, including methods and kits, employing elemental analysis.
The methods described facilitate high-throughput assays through multiplexing assays that until now have largely been performed individually. The principal, but not exclusive, target of the method is to provide for the evaluation of agonists and antagonists to phosphorylation (kinase) and dephosphorylation (phosphatase), as these are targets for pharmaceutical drug discovery applications.
Overall there are no less than 20 platform technologies available (for example, Radioactivity, Fluorescence Polarization, Time Resolved Fluorescence, Fluorescence Resonance Energy Transfer', etc.), however most display important limitations for the development of a coherent screening-profiling platform. Well known drawbacks include those related to heterogeneous assay systems, limitations in ATP concentration, compound interferences and limitations of substrate size and charge. As well these methods have low level of sensitivity and are difficult to multiplex2, i.e. assay dozens of different kinases/phosphatases simultaneously. Thus there are many needs unfulfilled by the prior art, including but not limited to a need for a sensitive, robust and quantitative assay for protein post-translational modification. Further, there is a need for a multiplexed enzymatic assay that enables high throughput operation.
Among the many advantages offered by the applicant's teaching are the following: I.
The assay can be applied to any type of protein kinase; II. The assay can be applied to any type of protein phosphatase; Ill. The assay does not exclusively rely on the use of antibodies (although some embodiments might include antibodies); IV. The methods can be used to detect and study protein kinase antagonists and agonists; V. The methods can be used to study protein kinase signal transduction cascades; VI. The methods can be used with a number of different protein kinase buffers; VII. The assay can be supplied as a kit; VIII.
The assay can be used to measure activity of multiple kinases/phosphatases in cell free systems; IX. The assay can be used to determine activity of multiple kinases/phosphatases in cellular lysates; X. The assay =
can be used to determine various endogenous and transfected kinase activities within intact cells.
Post-translational modifications of proteins are carried out by enzymes within living cells.
Known post-translational modifications include protein phosphorylation and dephosphorylation as well as methylation, prenelation, sulfation, and ubiquitination. The presence or absence of the phosphate group on proteins, especially enzymes, is known to play a regulatory role in many biochemical pathways and signal transduction pathways. Hence together, specialized kinases and phosphatases regulate enzymatic activity.
A kinase function is to transfer phosphate groups (phosphorylation) from high-energy donor molecules, such as ATP, to specific target molecules (substrates). An enzyme that removes phosphate groups from targets is known as a phosphatase. The largest group of kinases are protein kinases, which act on and modify the activity of specific proteins. Various other kinases act on small molecules (lipids, carbohydrates, aminio acids, nucleotides and more) often named after their substrates and include:
Adenylate kinase, Creatine kinase, Pyruvate kinase, Hexokinase, Nucleotide diphosphate kinase, Thymidine kinase.
Protein kinases catalyze the transfer of phosphate from adenosine triphosphate (ATP) to the targeted peptide or protein substrate at a serine, threonine, or tyrosine residue. Protein kinases are distinguished by their ability to phosphorylate substrates on discrete sequences. Commercially available kinases can be in the active form (phosphorylated by supplier) or in the inactive form and require phosphorylation by another kinase.
A protein phosphatase hydrolyses phosphoric acid monoesters at phosphoserine, phosphothreonine, or phosphotyrosine residue into a phosphate ion and a protein or peptide molecule with a free hydroxy group. This action is directly opposite to that of the protein kinase. Examples include: the protein tyrosine phosphatases, which hydrolyse phospho-tyrosine residues, alkaline phosphatase, the serine/threonine phosphatases and inositol monophosphatase.