Rapid assay to test anti-cancer drugs under physiological conditions

Medicilon offers fully integrated pharmaceutical services for the global scientific community. We focus on providing an exceptional client-centered experience and advancing the drug discovery process. Since the founding of our company in 2004, our integrated services across biology, chemistry and preclinical services are uniquely designed to help clients developing their research and discovery programs from the initial idea stage to the IND filing phase. Email:[email protected] Web:www.medicilon.com
This invention relates to an assay that allows for the rapid determination of the activity of a given drug against leukemic cells either taken from a patient or derived from a cell line. The assay is performed in the presence of whole blood or serum.
The research leading to the present invention was supported, at least in part, by a grant from the National Institutes of Health (NIH R01DE16133). Accordingly, the Government may have certain rights in the invention.
This invention relates to an assay that allows for the rapid determination of the activity of a given drug against leukemic cells either taken from a patient or derived from a cell line.
Each year, more than 60,500 people die of hematologic malignancies (leukemia, lymphoma, myeloma) with more than 110,000 new annual diagnoses in the US alone. Current treatment for these cancers includes the use of synthetic compounds that target the cell division process of nearly all cells of the body, not just the cancerous ones. Furthermore, a significant percentage of patients eventually show resistance to many of the drugs, thus rendering treatment largely ineffective. Indeed, there is an effort to identify agents that induce cancer cell death by methods other than damage to DNA or cell division (20).
The initial identification and testing of novel anti-cancer agents relies on in vitro killing assays using relevant cancer cell lines. While in vitro assays performed under cell culture conditions prove useful and necessary for preclinical testing of new therapeutics, extrapolation to the physiological conditions of a living organism is often difficult or impossible (27). Because of the high cost of drug development ($800 million), new drug screens are constantly being sought to more efficiently eliminate or identify candidate therapeutic agents (27). Indeed, increasing the clinical success rate from ⅕ to ⅓ because of more effective preclinical drug screens would reduce drug development costs by more than $200 million (27).
The activity, specificity, or toxicity of a compound in the physiological environment can vary significantly from cell culture conditions. While no in vitro assay or screen can represent the complexity of the human body, several assays have been developed to more closely mimic in vivo conditions. Several of these assays include the colony forming cell assay using bone marrow cells (27,29), hepatic drug biotransformation assays (3), and assays in whole blood (4,45). Because most chemotherapeutic agents are administered intravenously and are therefore immediately affected by blood cell components, screening for potential drugs in the presence of whole blood would be expected to yield more meaningful results. Blood contains biological components, such as proteases, antibody, and blood cells, which can affect the nature of a compound. For example, red blood cells and plasma proteins are known to affect the pharmacokinetics of drugs such as the anti-cancer agents docetaxel and gemcitabine (8,9). Vaidyanathan et al. (43) also reported that the cardioprotective drug, dexrazoxane, inhibits binding of the anti-cancer agent, doxorubicin, to red blood cells and that this interaction alters the pharmacokinetics of doxorubicin, and Clarke et al. (4) used an in vitro whole blood assay to study the binding affinity of a surrogate anti-CD11a monoclonal antibody to blood components. In addition, leukocytes produce a cytochrome P450 isoform (CYP2E1) that is involved in drug biotransformation (3). Thus, identifying and studying drugs in the presence of whole blood or blood components can offer a unique advantage over assays using cells in monoculture.
For studies on leukemia therapeutics, the cell line HL-60 is used as a standard target cell line. HL-60 cells were originally isolated from a 36-year-old female patient with acute promyelocytic leukemia (13). Testing the efficacy of anti-leukemia therapeutics against HL-60 cells in whole blood or other biological material is currently a challenge due to the inefficiency in differentiating the viability of HL-60 cells from other cells. Thus there remains a need to develop an efficient screen for anti-leukemia therapeutics and facilitate preclinical studies on a highly specific bacterial leukotoxin as a novel anti-leukemia therapeutic agent.
Accordingly, a stable bioluminescent HL-60 cell line whose viability can be rapidly and effectively determined in the presence of whole blood and live animals has now been developed along with an assay that allows for the rapid determination of the activity of a given drug against a leukemic cells either taken from a patient or derived from a cell line. The assay is carried out in the presence of whole blood or serum. This quantitative assay can screen thousands of drugs at a time or multiple concentrations of a drug in a 96- or 384-well format.
Screens for compounds and proteins with anti-cancer activity employ viability assays using relevant cancer cell lines. For leukemia studies, the human leukemia cell line, HL-60, is often used as a model system. To facilitate the discovery and investigation of anti-leukemia therapeutics under physiological conditions, HL-60 cells have been engineered that stably express firefly luciferase and produce light. Bioluminescent HL-60luc cells could be rapidly detected in whole blood with a sensitivity of approximately 1000 viable cells. Treatment of HL-60luc cells with a bacterial leukocyte-specific toxin or the drug chlorambucil revealed that the bioluminescent viability assay is more sensitive than the trypan blue dye exclusion assay. HL-60luc cells administered intraperitoneally (i.p) or intravenously (i.v.) were visualized in living mice using an in vivo imaging system (IVIS). The rapidity and ease of detecting HL-60luc cells in biological fluid indicates that this cell line can be used in high throughput screens for the identification of drugs with anti-leukemia activity under physiological conditions.
In vivo bioluminescence imaging (BLI) is a technology that allows visualization of live bioluminescent cells (mammalian, bacterial, viruses) in complex biological material and living animals (24,31). Firefly luciferase has been used extensively in reporter systems and its expression can be measured quantitatively using a luminometer or highly sensitive charge coupled device (CCD) camera. Rocchetta et al. (32) found that the CCD camera was approximately 25 times more sensitive than a luminometer, and so the IVIS 50 imaging system (Xenogen, Alameda, Calif.) was used for the work presented here. Luciferase reacts with its substrate, luciferin, to produce oxyluciferin and light (11). Because ATP and oxygen are required for the reaction, photon production has been used as a quantitative measurement of cellular viability (14). Animal studies have demonstrated a strong correlation between the abundance of emitted photons and number of cells present in a given tissue or animal (5,11).
In general, the field of oncology has utilized BLI extensively to study the effects of anti-cancer therapy in vivo (15,23). However, application of BLI to study hematologic malignancies has been limited (6,22,44), and to date, there are no bioluminescent hematologic cell lines commercially available (Xenogen Corp., Alameda, Calif.). Validation of BLI in preclinical study models has been carried out using currently available methods and evidence indicates that BLI has excellent sensitivity and offers unique advantages (5,25,31,33). For example, non-invasive BLI allows visualization of cells temporally and spatially, thus permitting small changes in cell number and localization to be detected over time (24,31). In addition, animals need not be sacrificed at each sampling time point, decreasing the number of animals that are required for an experiment and minimizing inconsistency from animal-to-animal variations. A bioluminescent HL-60 cell line has been engineered that can be visualized in whole human blood and living mice and whose viability can be rapidly determined. A WBC-specific bacterial toxin has been shown to be active in blood. The engineered HL-60luc cells of the invention behave similar to the parental HL60 cell line. The BLI signal peaked approximately one hour following the addition of luciferin but remained relatively high for several hours. This type of in vitro kinetics where an early peak in luminescence is followed by a slow decline is consistent with other BLI cell lines. The detection limit of 1000 viable cells is also consistent with other reports (35,36). Because human blood contains plasma proteins, such as antibody and proteases, and other cells, that may affect the activity, availability, or stability of a drug, the anti-leukemia assays with HL-60luc cells in the presence of blood can yield more physiological results than with buffer or growth media alone.
There is a significant difference between the sensitivity of BLI and the trypan blue dye. exclusion assay. For a cell to be detected as nonviable with the trypan blue assay, the dye must enter the cytoplasm of the cell. Trypan blue is a relatively large molecule (mw 960.8) and while many cells may be metabolically dead, their membranes could be sufficiently intact to exclude the dye to appear viable. In contrast, BLI detects killing sooner because ATP is no longer available in a metabolically dead cell. The results are in strong agreement with Kuzmits et al. (17) who found that an ATP/bioluminescent assay with HL-60 cells indicated nearly complete killing after a 24 hour incubation with 5.7 μmol/l doxorubicin, while the trypan blue assay indicated almost no killing after 48 hours with the same drug concentration. Furthermore, Petty et al. (30) reported that a bioluminescent ATP assay could detect as few as 1500 viable cells/well while the MTT assay could not detect less than 25,000 cells/well.

Method for breeding experimental macaca mulatta

Medicilon offers fully integrated pharmaceutical services for the global scientific community. We focus on providing an exceptional client-centered experience and advancing the drug discovery process.Since the founding of our company in 2004, our integrated services across biology, chemistry and preclinical services are uniquely designed to help clients developing their research and discovery programs from the initial idea stage to the IND filing phase. Email:[email protected] Web:www.medicilon.com
The invention provides a method for breeding experimental macaca mulatta. The mode of female money single-cage raising and regular mating is adopted in the mating period, the mode that infant monkeys and a female monkey are raised in a concentrated mode in the lactation period and an adult male monkey is isolated is adopted, the monkey group fighting problem is effectively solved, the infant monkeys are raised in a single-breeding monkey cage in the ablactation period, the breeding quality is further improved, and the health condition of the macaca mulatta can be discovered and managed in time. The breeding rate and the pregnancy rate of the macaca mulatta are further improved, the phenomenon that the female monkey of the right age is infertile in the breeding period is greatly reduced, the oestrus, mating and farrowing activities of personal macaca mulatta can be observed conveniently, and the health condition of the macaca mulatta can be discovered and managed in time. According to the method, overall comprehensive breeding management is combined with rich and reasonable breeding equipment, and thus the yield and the quality of the experimental macaca mulatta are greatly improved.
said the monkey (Macaca mulatta) belong Primates (Primates) Cercopithecidae (Eereopithecidae) called Monkey (Macaca) animals, as «Endangered Species of Wild Fauna and Flora International Trade Convention (CITES)» Appendix II species.
macaque as human kinship with the closest species of non-human primates (Nonhuman Primates, NHP) and human genetic material more than 95% homology, and the organizational structure, immunity, physiology and metabolism respect human height approximation, which has a clear comparative advantage in biomedical research traitor, a traitor and basic research has become an ideal animal model to solve problems of human health and disease in pre-clinical research traitor. China SFDA and FDA clearly requires more than two types of new drugs in clinical trials before, must obtain reliable data on NHP animal experiments. China also states: «The neurological, psychiatric, anesthesia type, biological products category, appliances and other family planning medicines must be approved by NHP animal experiments approved before entering clinical trials.» At present, with AIDS, Parkinson's, cancer establish lung damage, liver damage and other long-standing human ills NHP disease models, as well as related drugs especially preclinical study based evaluation, NHP application is not around the past Hom. NHP proteins in particular the importance of new drugs biological products for drug development is evident.
monkeys, as the Chinese national animal protection, population traitor applied research in experimental animals has been increasing year by year in recent years, but mainly due to the current international breeding cynomolgus monkeys mainly just for monkeys reared in infancy, mainly from the wild, wild monkey experiment with one hand because there is no clear genetic background, a clear and stable microbial biological characteristics of the state, so the reliability of the experimental results is doubtful; the other hand, wild monkeys to capture inevitably result in excessive depletion of wildlife resources, and the development of human civilization very inconsistent.
The experimental primates China attaches great importance to the same resource, macaque experiments included in the «national» five «animals platform construction» project to provide support conditions for the development of national strategic emerging industries. Ministry formulated the «primate laboratory animal husbandry management practices and quality standards», set up a «primate laboratory animal industry strategic alliance.» It is because of the great value of the experimental monkeys, Western countries (including the country) will be listed as a strategic resource research. Some experts even predict that the future of competition in the pharmaceutical industry may be competitive experiments in primates resources.
Thus genetic differences to understand the genetic background of experimental monkeys and populations through existing genetic records management system to adjust the macaque experiments, genetic information management, the establishment of experimental animals macaque detailed individual files and pedigree systems, establish an effective, accurate and reliable, quick and easy monitoring of genetic background, while establishing a sound feeding, breeding standard operating procedures, epidemic prevention system and disease prevention and control program for the cultivation of macaque experimental populations to provide technical support is particularly important.
According to 2009 statistics, the breeding stock of 21 million Chinese monkeys, of which nearly 30,000 macaques. According to 2011 statistics, nearly a decade of international market demand for the experimental monkeys annual growth rate of over 20%. In 2010, global demand macaque experiment nearly 200,000, while the amount of global experimental macaque breeding year only 13 million only, the market demand gap is very obvious. According to the relevant forecast shows the next five years (2012-2016), the global market for medical experiments macaque demand will maintain strong growth momentum in 2016 demand will reach 25-30 million.
For the purposes of China's domestic demand, along with increasing levels of Chinese science and technology, and the comparative advantages of various costs, large international pharmaceutical companies and research institutions have the R & D center traitor into China, or the relevant R & D tasks Outsourcing. This is certainly experimental animals for China, but also certainly to the global development of animal experiments. Will effectively promote domestic monkeys and usage patterns of animals, in the foreseeable time frame will be a rising trend.
It has been reported worldwide each year for research Macaca animals only up to 10 million, China needs thousands every year. Although some countries have invested heavily in the establishment of a number of monkeys games, but still need to produce importers from China and Vietnam, a lot of rhesus macaques.
keeping the size and needs of Chinese monkeys gradually expanded, but because of long-term lack of standardized management, leading to disorderly competition between industries, violations of law, underdevelopment and low quality animal series of problems, not only cause for conservation a huge risk, but also damaged China's international reputation.

Systems and methods of fluidic sample processing

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
One advantage of the current invention is that assay results can be substantially immediately communicated to any third party that may benefit from obtaining the results. For example, once the analyte concentration is determined at the external device, it can be transmitted to a patient or medical personnel who may need to take further action. The communication step to a third party can be performed wirelessly as described herein, and by transmitting the data to a third party's hand held device, the third party can be notified of the assay results virtually anytime and anywhere. Thus, in a time-sensitive scenario, a patient may be contacted immediately anywhere if urgent medical action may be required.
In some embodiments a method of automatically selecting a protocol to be run on a fluidic device comprises providing a fluidic device comprising an identifier detector and an identifier; detecting said identifier with said identifier detector; transferring said identifier to an external device; and selecting a protocol to be run on said fluidic device from a plurality of protocols on said external device associated with said identifier.
By detecting each fluidic device based on an identifier associated with the fluidic device after it is inserted in the reader assembly, the system of the present invention allows for fluidic device-specific protocols to be downloaded from an external device and run on the fluidic device. In some embodiments the external device can store a plurality of protocols associated with the fluidic device or associated with a particular patient or group of patients. For example, when the identifier is transmitted to the external device, software on the external device can obtain the identifier. Once obtained, software on the external device, such as a database, can use the identifier to identify protocols stored in the database associated with the identifier. If only one protocol is associated with the identifier, for example, the database can select the protocol and software on the external device can then transmit the protocol to the communication assembly on the reader assembly. The ability to use protocols specifically associated with a fluidic device allows for any appropriate fluidic device to be used with a single reader assembly, and thus virtually any analyte of interest can be detected with a single reader assembly.
In some embodiments multiple protocols may be associated with a single identifier. For example, if it is beneficial to detect from the same patient an analyte once a week, and another analyte twice a week, protocols on the external device associated with the identifier can also each be associated with a different day of the week, so that when the identifier is detected, the software on the external device can select a specific protocol that is associated with the day of the week.
In some embodiments a patient may be provided with a plurality of fluidic devices to use to detect a variety of analytes. A subject may, for example, use different fluidic devices on different days of the week. In some embodiments the software on the external device associating the identifier with a protocol may include a process to compare the current day with the day the fluidic device is to be used based on a clinical trial for example. If for example, the two days of the week are not identical, the external device can wirelessly send notification to the subject using any of the methods described herein or known in the art to notify them that an incorrect fluidic device is in the reader assembly and also of the correct fluidic device to use that day. This example is only illustrative and can easily be extended to, for example, notifying a subject that a fluidic device is not being used at the correct time of day.
In some embodiments, the present invention provides a method of obtaining pharmacological data useful for assessing efficacy and/or toxicity of a pharmaceutical agent from a test animal utilizing the subject fluidic devices or systems.
When using laboratory animals in preclinical testing of a pharmaceutical agent, it is often necessary to kill the test subject to extract enough blood to perform an assay to detect an analyte of interest. This has both financial and ethical implications, and as such it may be advantageous to be able to draw an amount of blood from a test animal such that the animal does not need to be killed. In addition, this can also allow the same test animal to be tested with multiple pharmaceutical agents at different times, thus allowing for a more effective preclinical trial. On average, the total blood volume in a mouse, for example, is 6-8 ml of blood per 100 gram of body weight. A benefit of the current invention is that only a very small volume of blood is required to perform preclinical trials on mice or other small laboratory animals. In some embodiment between about 1 microliter and about 50 microliters are drawn. In an embodiment between about 1 microliter and 10 microliters are drawn. In preferred embodiments about 5 microliters of blood are drawn.
A further advantage of keeping the test animal alive is evident in a preclinical time course study. When multiple mice, for example, are used to monitor the levels of an analyte in a test subject's bodily fluid over time, the added variable of using multiple subjects is introduced into the trial. When, however, a single test animal can be used as its own control over a course of time, a more accurate and beneficial preclinical trial can be performed.
In some embodiments a method of automatically monitoring patient compliance with a medical treatment using the subject fluidic devices or systems is provided. The method comprises the steps of allowing a sample of bodily fluid to react with assay reagents in a fluidic device to yield a detectable signal indicative of the presence of an analyte in said sample; detecting said signal with said fluidic device; comparing said signal with a known profile associated with said medical treatment to determine if said patient is compliant or noncompliant with said medical treatment; and notifying a patient of said compliance or noncompliance.
Noncompliance with a medical treatment, including a clinical trial, can seriously undermine the efficacy of the treatment or trial. As such, in some embodiments the system of the present invention can be used to monitor patient compliance and notify the patient or other medical personnel of such noncompliance. For example, a patient taking a pharmaceutical agent as part of medical treatment plan can take a bodily fluid sample which is assayed as described herein, but a metabolite concentration, for example, detected by the reader assembly may be at an elevated level compared to a known profile that will indicate multiple doses of the pharmaceutical agent have been taken. The patient or medical personnel may be notified of such noncompliance via any or the wireless methods discussed herein, including without limitation notification via a handheld device such a PDA or cell phone. Such a known profile may be located or stored on an external device described herein.

Facilitating a Transaction between a Client

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
Successful life science research service providers and their clients must devote significant time, effort and money to sample management, data management and analysis, accumulating knowledge of publicly accessible standard control samples, web site development, and sales and marketing. Building these essential business functions uses resources that otherwise could be used to develop core research technologies and expand service offerings. Moreover, the absence of these functions often prevents the association of, and subsequent transactions between, life science research providers and their clients.
Therefore, there is a need for companies that can provide these services in an efficient, accurate, and cost effective manner to clients and/or life science research service providers in order to facilitate transactions between clients and life science research service providers. The present invention provides solutions to these and other needs in the art of life sciences.
A reference in this specification to any prior publication (or information derived there from), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that the prior publication (or information derived there from) or known matter forms part of the common general knowledge in the field to which this specification relates.
A functional life science objective refers to a desired assessment goal of a client for a particular test sample requiring the performance of a series of life science research services (e.g., assays) in order to reach the desired assessment goal. The desired assessment goal is related to the life sciences field and answers a functional question regarding the test sample. For example, the sample may be a potential drug. The client may have the assessment goal of determining whether the potential drug is sufficiently non-toxic to file an investigational new drug application (IND) with the Federal Drug Administration (FDA). In this embodiment, the functional life science objective is a preclinical safety study requiring a plurality of assays to assess the safety of the potential drug. Such assays may include, for example, drug-drug interaction assays, cardiotoxicity assays, genotoxicity assays, cell toxicity assays, and target selectivity profiling assays. These safety assays may be performed by one or a plurality of life science research providers, some or all of which may be contacted by the facilitator company in order to fill the client's order.
Because many clients and research providers do not have knowledge of the battery of life science research services required to achieve a functional life science objective, the facilitator company provides valuable knowledge to the client and/or research provider for achieving a given assessment goal of the client. Thus, by identifying particular life science research services required to achieve a requested functional life science objective, and in some embodiments identifying one or more life science research service providers capable of performing one or more of the required life science research services, the facilitator company associates clients with service providers that may otherwise not be associated thereby facilitating transactions between a given client and service provider.
A wide array of life science research objectives may be provided by the facilitator company using the selectable internet-based listing. Appropriate objective include, for example, such as preclinical safety studies, target identification studies, target validation studies, target selectivity profiling studies, drug metabolism studies, biomarker identification studies, chemical optimization studies, drug formulation studies and the like.
Preclinical safety assays include tests designed to evaluate the safety of a potential drug (e.g., a prospective investigational new drug) prior to clinical testing. Preclinical safety studies typically include assays performed with cells and animal models to determine the possible effects of the prospective investigational new drug on the human body in a clinical setting. Target identification studies include tests used to identify one or more molecular or cellular entities that are modulated by a particular chemical or biological test sample (e.g., a prospective drug). Target validation studies include tests designed to confirm or rebut a theory concerning the ability of a chemical or biological sample to modulate one or more molecular or cellular entities. Target selectivity profiling studies include tests to determine whether a particular chemical or biological test sample (e.g., a prospective drug) modulates one or more of a family of related targets (e.g., 10, 100, 500 kinases or all currently known nuclear receptors). Drug metabolism studies include tests to measure the metabolism of a test sample in in vitro assays or in a test animal. Biomarker identification studies include tests to identify surrogate markers for a disease state. Chemical optimization studies include tests to determine optimal chemical synthetic routes for the synthesis of new chemical leads with improved drug properties. Drug formulation studies include tests to identify the optimal salt and crystalline forms of a chemical test sample.

Process for production of recombinant proteins as a soluble form

Medicilon is a leading provider of comprehensive, high quality recombinant protein and bioprocess services. We offer a variety of recombinant protein expression platforms along with a host of other protein services like chemical protein synthesis, protein refolding and structural biology services.Email:[email protected] Web:www.medicilon.com
A target protein is prepared as soluble protein using a recombinant protein expression system. An expression vector is used that includes (1) an expression-inducible promoter sequence; (2) a first coding sequence including a polynucleotide coding for a polypeptide that is represented by the formula (Z)n; and (3) a second coding sequence that includes a polynucleotide that codes for a target protein. A method of producing the target protein is also used that includes expressing protein using this expression vector.
1. Field of the Invention
The invention relates to a method of producing a target protein as soluble protein.
2. Related Art
A large number of recombinant protein expression systems have been developed to date, including, for example, cell-free translation systems and recombinant protein expression systems within hosts such as bacteria, yeast, insects, transgenic animals, and transgenic plants. Escherichia coli is widely used as an expression system for heterologous protein because it is easily grown to high densities and because of the progress in research on host vector systems.
However, when a target protein is expressed using these recombinant protein expression systems, incorrect folding by the expressed protein can prevent expression of the functionality of the original protein and can result in the not insignificant formation of insoluble aggregates, known as inclusion bodies. Even when, for example, refolding is carried out in such cases after solubilization of the inclusion body with a denaturant or surfactant, the correctly folded protein exhibiting its native functionality is not necessarily obtained. In addition, even when protein expressing its original functionality is obtained, in many instances a satisfactory recovery rate is not obtained.
Against this background, a method of suppressing the formation of inclusion bodies of an expressed recombinant target protein has not been established to date. As a stand in for such a method, expression as a soluble protein is attempted by fusing the insoluble target protein with the soluble high molecular weight (40,000) maltose-binding protein or glutathione S-transferase (GST) (Fox, J. D. and Waugh, D. S., “Maltose-binding protein as a solubility enhancer.” METHODS MOL. BIOL., 205: 99-117 (2003); Ausubel, F. M. et al., editors, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol. 2, 16.0.1 (1996)). However, there have been problems such as, for example, the soluble protein may not exhibit its original activity or functionality and the target protein may become insoluble when the maltose-binding protein or GST is removed.
The ZZ domain is a synthetic IgG binding region developed on the basis of the IgG binding region of protein A (refer, for example, to Nilsson B. et al., Protein Eng., 1: 107-113 (1987)).
However, in those cases where the ZZ domain of the IgG binding region has been expressed fused with a target protein, there have been no reports of an effect whereby the solubility of the fusion protein is increased, nor have there been reports of an activity that contributes to an efficient refolding of the target protein to the active form of the protein. Up to the present time, the use of the ZZ domain originating from protein A has not gone beyond use, after expression of the fusion protein with a target protein, as a ligand in IgG antibody affinity chromatography in target protein purification. In addition, IgG antibody columns are expensive and there are only limited instances where they can be used even when a genetic recombinant fusion protein utilizing the ZZ domain is employed in mass production.
The Target Protein
There are no particular limitations on the target protein in the fusion protein of the invention. For example, even proteins that are prone to form inclusion bodies when expressed in a recombinant protein expression system can be advantageously used.
The target protein in the invention can be exemplified by protein (viral antigen), e.g., coat protein, core protein, protease, reverse transcriptase, integrase, and so forth, encoded in the genome of a pathogenic virus, e.g., hepatitis B virus, hepatitis C virus, HIV, influenza, and so forth; the Fab and (Fab)2 of antibodies; growth factors such as platelet-derived growth factor (PDGF), stem cell growth factor (SCF), hepatocyte growth factor (HGF), transforming growth factor (TGF), nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), and so forth; cytokines such as tumor necrosis factor, interferon, interleukin, and so forth; hematopoietic factors such as erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, thrombopoietin, and so forth; peptide hormones such as luteinizing hormone-releasing hormone (LH-RH), thyrotropin-releasing hormone (TRH), insulin, somatostatin, growth hormone, prolactin, adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), vasopressin, oxytoxin, calcitonin, parathyroid hormone (PTH), glucagon, gastrin, secretin, pancreozymin, cholecystokinin, angiotensin, human placenta lactogen, human chorionic gonadotropin (HCG), cerulein, motilin, and so forth; analgesic peptides such as enkephalin, endorphin, dynorphin, kyotorphin, and so forth; enzymes such as superoxide dismutase (SOD), urokinase, tissue plasminogen activator (TPA), asparaginase, kallikrein, and so forth; peptide neurotransmitters such as bombesin, neutrotensin, bradykinin, substance P, and so forth; as well as albumin, collagen, proinsulin, renin, al antitrypsin, and so forth. However, the target protein is not limited to the foregoing.

Process for producing recombinant protein and fused protein

Medicilon is a leading provider of comprehensive, high quality recombinant protein and bioprocess services. We offer a variety of recombinant protein expression platforms along with a host of other protein services like chemical protein synthesis, protein refolding and structural biology services.Email:[email protected] Web:www.medicilon.com
This invention provides a recombinant protein expression system using a host and a cell-free translation system capable of universally expressing a large amount of any proteins as soluble proteins while preventing expression of the toxicity of a desired protein in hosts, formation of inclusion bodies and decomposition with proteases by expressing the desired protein as a fusion protein with chaperonins, that are, about 60 kDa molecular chaperones, 60 kDa heat shock proteins, or thermosomes and accommodating it certainly in the inside of a stereostructure of a chaperonin.
A process for producing a protein, which comprises transcribing and translating a gene containing a gene encoding the linked chaperonin subunits and a gene encoding a desired protein thereby synthesizing a fusion protein having the desired protein linked via a peptide linkage to the linked chaperonin subunits.
Up to now, recombinant protein expression systems in many hosts such as bacteria, yeasts, insects, animal and plant cells, and transgenic animals and plants and cell-free translation systems have been established. Particularly in production of recombinant proteins by mammalian cultured cells, the proteins are subjected to suitable posttranslational modification, and thus this production system is becoming a standard system for production of therapeutic agents. However, the protein synthesis level in this system is lower than in the system with microorganisms as the host, thus necessitating a larger culture chamber, which would cause shortage of production facilities in biotechnology industry pursuing new medicines (Garber, K., Nat. Biotech. 19, 184-185, 2001). Protein production techniques using transgenic animals and plants attempted to improve production efficiency in recent years still do not attain full confidence (Garber, K., Nat. Biotech. 19, 184-185, 2001).
In the recombinant protein expression systems developed so far, it is often difficult to obtain a large amount of active protein. If a desired protein is toxic to the host to a certain degree, synthesis of the protein is inhibited to decrease the expression level. Further, even if the desired protein is expressed as soluble protein, the protein may be decomposed by proteases in the host so that the amount of the protein produced is reduced to a very low level. In addition, even if the desired protein is expressed, the protein may fail to achieve suitable folding, resulting in formation of an inclusion body. In this case, even if the protein is solubilized and folded again, the amount of the finally obtained active protein is very low. Particularly when a cell-free translation system is used, the inclusion body is easily formed.
When the inclusion body is formed, it is attempted to solve this problem by using a method of expressing the protein in the form of a fusion protein with e.g. glutathione-S-transferase (GST) (Smith, D. B., et al., Gene 67, 31-40, 1988), with thioredoxin (LaVallie, E. R. et al., Bio/Technology 11, 187-193, 1993), or with a maltose-binding protein (Guan, C., et al., Gene 67, 21-30), but there are few cases where formation of the inclusion body is suppressed at high efficiency. Alternatively, there is a method wherein a desired protein is co-expressed with a chaperonin i.e. a protein group supporting protein-folding reaction to increase the amount of the desired protein expressed in the soluble fraction (Nishihara et al., Apply. Environ. Microbiol., 64, 1694-1699, 1998), but at present, this method cannot achieve a remarkable increase in the amount of the active protein.
As a method of solving the problem of decomposition of the desired protein by proteases in the host, a method of using a host deficient in a part of protease structural genes, for example in lon, ompT etc. in the case of E. coli, has been devised (Phillips et al., J. Bacteriol. 159, 283-287, 1984), there are few cases where the influence of decomposition with proteases can be avoided, while if the host is made deficient in all proteases, other problems can occur, thus failing to essentially solve the problem of decomposition with proteases.
As described above, the conventional protein expression techniques have serious problems such as toxicity to hosts, decomposition with host proteases, and formation of inclusion bodies, and thus the expression level is significantly varied depending on the type of protein to be expressed, and expression conditions for each protein should be examined in trial and error. Accordingly, there is demand for development of techniques for essentially solving the problems described above.
In view of the foregoing, the object of this invention is to provide a recombinant protein expression system using a host and a cell-free translation system capable of universally expressing a large amount of any proteins as soluble proteins while preventing expression of the toxicity of a desired protein in hosts, formation of inclusion bodies and decomposition with proteases by expressing the desired protein as a fusion protein with a chaperonin subunit, that is, about 60 kDa molecular chaperones, 60 kDa heat shock proteins, or thermosomes and accommodating it certainly in the inside of a stereostructure of chaperonin.
This invention relates to a process for producing a protein, which comprises transcribing and translating a gene containing a gene encoding a chaperonin subunit and a gene encoding a desired protein thereby synthesizing a fusion protein having the desired protein linked via a peptide linkage to the chaperonin subunit.
Preferably, the fusion protein comprises 1 to 20 chaperonin subunits linked to one another and a desired protein linked via a peptide linkage to the N-terminus of the linked chaperonin subunits, the C-terminus of the linked chaperonin subunits, or a linking region of the chaperonin subunits.
In this invention, a gene containing a gene encoding the linked chaperonin subunits and a gene encoding a desired protein may be introduced respectively into 2 different plasmids each capable of coexistence and replication in the same host, and then co-expressed in the same host, or a gene containing a gene encoding the linked chaperonin subunits and a gene encoding a desired protein, and a gene encoding the linked chaperonin subunits only, may be introduced respectively into 2 different plasmids each capable of coexistence and replication in the same host, and then co-expressed in the same host.
Preferably, the desired protein, while being in a state linked via a peptide linkage to the chaperonin subunits, is accommodated in the inside of a chaperonin ring.

Methods and compositions for therapeutic drug monitoring

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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
In certain embodiments, the present invention provides a method for therapeutic drug monitoring of an individual treated with a drug. The method involves constructing a pharmacokinetic profile of the drug for the individual using concentrations of drug in at least two samples obtained from the individual at time points suitable to construct a pharmacokinetic profile. The samples are collected at point-of-care or point of use by sampling or self-sampling on point-of-care devices or point of use devices, each capable of quantitating the drug, or on matrices suitable for storage of the at least two samples prior to quantitation of the drug by a laboratory. The pharmacokinetic profile includes pharmacokinetic parameters suitable for guiding dosing of the drug for the individual.
The samples are collected by at point-of-care or point of service, e.g., by self- sampling. Samples may be applied to a lateral flow device for quantitation of the drug, and the results transmitted to the physician or physician's agent for pharmacokinetic analysis. In other embodiments, the samples are collected at point- of-care or point of service, e.g., by self-sampling, on a suitable storage matrix, e.g., filter paper, prior to delivery of the samples to a laboratory for quantitation and analysis.
In certain embodiments, samples collected at various times from the individual through point-of-care or point-of-use by self-sampling are obtained by a laboratory. The laboratory then tests the samples to quantitate the drug of interest and, based on the results, constructs a pharmacokinetic profile. The results of the pharmacokinetic profile may be presented, optionally along with recommendations to increase the individual's dosage of the drug in order to enhance efficacy or to reduce the dosage in order to reduce risk of toxicity.
In another aspect it provides a kit for therapeutic drug monitoring of an individual treated with a drug using pharmacokinetic profiling. Advantageously, the kit may be used to perform the method of claim 1. The kit comprises a plurality of point-of-care device or a point of use device capable of quantitating the drug in the at least two samples, or matrices suitable for storage of the samples prior to quantitation by a laboratory.
The present invention relates to methods by which pharmacokinetic profiles of drugs may be constructed for individuals receiving a drug using samples obtained at various time points following dosing. Data for use in the construction of the pharmacokinetic profiles is obtained from samples collected at point-of-care or point of use. Advantageously, the samples may be obtained by self-sampling. In certain embodiments, the samples may be delivered to a point-of-care device to quantitate the drug, and the results thus obtained are reported to the physician or his agent. Alternatively, the samples are collected using a matrix or vessel suitable for collection and storage of the samples until receipt and analysis by a laboratory. Examples of matrices suitable for collection and storage of the samples include, but are not limited to commercially available biological sampling filter paper systems such as Whatman 3 MM, GF/CM30, GF/QA30, S&S 903, GB002, GB003, or GB004. Several categories of blotting materials for blood specimen collection are available, e.g., S&S 903 cellulose (wood or cotton derived) filter paper and Whatman glass fiber filter paper. The blood spot is placed in one or more designated areas of the filter paper, allowed to dry, and then mailed along with a test request form to the laboratory. This method of collection has the advantage of obviating the need for collection of samples at a doctor's office or clinic. Thus, multiple samples may be conveniently collected by the patient over a period of from 0 to 72 hours at considerable savings of cost and time. This has the advantages of increased efficiency and reduced delays in transmitting results of the analysis to the treating physician, who may use the information to adjust treatment as necessary, and contact the patient to convey the new treatment regimen.
Prior to the instant invention, the potential value of pharmacokinetic-guided dosing had not been exploited in part because collecting the samples needed for individual pharmacokinetic profiles was inconvenient and prohibitively expensive, in that collection of samples typically requires extended hospital stays of up to 72 hours. For a typical drug PK study, pharmacokinetic studies have been limited to phase I studies in which PK/PD on a few patients forms the basis for the use of the drug throughout the population. To compensate, a population PK study is usually performed during phase III of the drug development; however, due to the same limitations, these studies are performed under the sparse sampling procedure. Due to the imprecision associated with sparse sampling, only an approximation of population PK can be obtained. For full pharmacokinetic testing, at least 12 data points collected over a period of 48-72 hours are needed to adequately characterize the PK parameters for each particular patient. It is not possible to perform this for every patient enrolled in a phase III (or even for a phase II trial for that matter). Obtaining blood samples to assess drug levels immediately after drug administration for Cmax are not typically problematic. However, obtaining blood samples at subsequent time points to define «Clearance» and «Concentration at Steady State» are difficult as the inflection points are 4-24 hours after treatment. It is often inconvenient or burdensome for patients to return to the hospital for more blood work, and keeping the patient overnight would be expensive both in terms of cost and services.
A phase 3, multicenter, randomized study (N=208) in which 5-FU dosing was optimized at steady state levels by LC/MS demonstrates the benefits of pharmacokinetic-guided dosing for both efficacy and toxicity (Gamelin, E, Delva, R, Jacob, J, et al: «Individual fluorouracil dose adjustment based on pharmacokinetic follow-up compared with conventional dosage: Results of a multicenter randomized trial of patients with metastatic colorectal cancer.» J Clin Oncol. 13:2099-2105, 2008.). Half of the patients were dosed with 5-FU based on body surface area (BSA). The other half were initially dosed based on BSA, with subsequent cycle doses adjusted based on blood tests that measured the actual concentration of chemotherapy in the patients' blood plasma. The primary endpoint was tumor response; the secondary endpoint was treatment tolerance. The study concluded that: 1 ) Response rates were nearly doubled in the dose-adjusted group versus the BSA group (33.6 percent versus 18.3 percent) with statistical significance; 2) Overall survival at two years among patients with personalized 5-FU dose management improved by 48 percent with an improved median survival of 22 months versus 16 months in the BSA arm. The survival data was leaning towards significance; 3) Grade lll/IV 5-FU related toxicities were found to be significantly lower in patients with personalized dose adjustment; 4) Fifty eight percent of patients were found to be under-dosed (sub-therapeutic and less effective drug levels) and had their doses adjusted upwards; and 5) Seventeen percent were found to be over-dosed (increasing the risk of severe side effects) and had their doses adjusted downward. However, most intravenously administered drugs are not dosed to achieve steady state with prolonged infusion of 24 hours Instead, they are dosed as single short infusions (30 min- 3 hours) not allowing for long sustained steady state drug concentrations in the blood.

Treatment of disorders relating to the serotonergic system

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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 possible antidepressant activity of deramciclane has also been evaluated in various conventional animal models. In learned helplessness tests in rats (Giral et al. Reversal of helpless behavior in rats by putative 5-HT1A agonists. Biol. psychiatry 23: 237-242), deramciclane dose dependently attenuated helpless behaviour induced by inescapable electric foot shocks, when given intraperitoneally 1 or 10 mg/kg, repeatedly 8 times, twice a day, before the test. The effect of deramciclane was found to be negligible, even at relatively high oral doses, 48-160 mg/kg, when evaluated for tetrabenazine-induced ptosis in mice according to the method of Howard et al. (Howard, J. L. et al., (1981) Empirical behavioral models of depression with emphasis on tetrabenazine antagonism. In Enna S. J., Malick J. B., Richelson E. (eds.): Antidepressants: Neurochemical, Behavioral, and Clinical Perspectives. New York: Raven Press, p 107). In the forced swimming test in rats deramciclane was clearly ineffective at oral doses of 25 and 100 mg/kg.
Thus, deramciclane has been effective in some animal models of anxiety after oral doses in a range from 1 mg/kg to 30 mg/kg in mice and rats. Further, deramciclane has shown negligible effects in animal models of depression even after high peroral doses in mice and rats, which is in line with the results reporting that 5-HT2C-receptor agonists are effective in animal models of depression.
In a whole body autoradiography distribution study with tritium labeled deramciclane in rats at a dose of 3 mg/kg, it was found that after intravenous administration there was high radioactivity (reflecting amount of deramciclane) in several organs including blood and the brain, but after oral administration the amount was substantially lower, especially in the brain.
In a comparative pharmacokinetic study of orally administered deramciclane in rats, dogs, rabbits and humans, it was shown that the plasma concentration curves obtained after the administration of a single 3 mg/kg oral dose of deramciclane to rats (dogs, rabbits) and human show considerable species specific differences. In the peak plasma concentration (Cmax) values there were significant differences: Cmax was 5.4 ng/ml in rat and 217.5 ng/ml in human after the same 3 mg/kg oral dose. Thus a 40-times lower oral dose of deramciclane could be used in man to result in the same maximal plasma concentration as in rat. Furthermore, the total amount of deramciclane absorbed into blood, calculated as Area Under Curve values (AUC 0-∞) from plasma concentrations as a function of time, showed more considerable species difference. The mean AUC 0-∞ values after single oral administration of deramciclane were 11.9 ng h/ml and 3737.8 ng h/ml in rat and human, respectively. Thus, over 300 times lower oral doses should result in equal exposure in humans than in rats. Basing only the Cmax difference between rat and man, it can be predicted that considerably lower doses should be centrally active in humans than in rat. The minimum oral effective anti-anxiety dose in rats was 1 mg/kg (1-30 mg/kg the full range; see above), i.e. in a 70 kg-man this would mean 70 mg dose. To reach the same pharmacologically active plasma concentration in humans as was shown to be efficacious in rat, one should divide the rat dose by 40. This would result in 70 mg/40=1.75 mg (i.e. 0.025 mg/kg) as an effective dose in man.
The binding of deramciclane to serotonin 5-HT2A-receptors in frontal cortex of healthy male volunteers after a single oral dose of 20, 50 and 150 mg of deramciclane is discussed in Kanerva, H. et al., Psychopharmacology (1999) 145:76-81. The determination of the brain 5HT2A-receptor occupancy of deramciclane in humans has shown that 90% and 50% receptor occupancies were reached at a deramciclane plasma concentration of about 70 ng/ml and 21 ng/ml, respectively. The pharmacokinetics of a single dose of deramciclane and during oral dosing of 10 mg, 30 mg and 60 mg twice a day for seven days are discussed in Kanerva, H., Pharmacokinetic studies on deramciclane. Kuopio University Publications A. Pharmaceutical Sciences 39.1999. After a single oral administration of 20 mg and 30 mg doses of deramciclane, the Cmax-values were 24±9.4 ng/ml and 27±6.1 ng/ml, respectively. During repeated administration of deramciclane for one week the Cmin and Cmax for 60 mg and 20 mg daily doses were shown to range between 48-91 ng/ml and 16-33 ng/ml, respectively.
As the above experimental animal and human data does not disclose repeated administration of deramciclane rendering steady state plasma concentrations in treated patients, it was impossible to predict the oral dosages of deramciclane that would be effective in treating anxiety in humans. Furthermore, it was totally unexpected that deramciclane would be effective in treating depressive symptoms.
Anxiety is a normal emotional feeling related to different situations of threat or fear. External threat is experienced as a fear whereas obscure and unidentified feeling of threat may be experienced as anxiety. When anxiety persists it can develop into a pathological disorder. Anxiety disorders are divided more specifically in diagnostic disorders e.g., panic disorder, phobias, and GAD. GAD is a chronic illness associated with excessive anxiety and worry lasting for at least six months. In addition, the anxiety and worry are associated with restlessness, fatigue, difficulties in concentrating or mind going blank, irritability, muscle tension, and sleeping disturbances. The symptoms may be triggered by different events of life, and the control of anxiety is very difficult for the patient.
Anxiety is currently treated with benzodiazepines, SSRI's and buspirone, which are not optimal treatments due to adverse drug reactions and their efficacy profiles. Moreover, relapse of the disease, different kinds of withdrawal effects, development of tolerance, as well as relapse and recurrence, often happen when traditional anxiolytics are used. For example, to avoid withdrawal effects, doctors usually gradually taper the dosage of the medicine (i.e. gradually diminish its daily dosage) before the treatment may be stopped. Patients tend to develop tolerance to those traditional compounds as well. Development of tolerance occurs when, for example, a patient requires greater quantities of a compound over time to achieve the same therapeutic effect.
In the treatment of psychiatric disorders with a chronic course, such as anxiety, it is important to prevent the relapse and recurrence of the disease. After the acute treatment phase, the improved condition can be maintained, and relapses can thus be prevented by continuing the treatment in those who have responded to the treatment or who have reached remission during it. After the continuation treatment phase, when recovery has been reached, the disease can be prevented by continuing the treatment further by the so-called maintenance treatment, during which the daily dosage may be decreased, for example, to a half from the original.
There has thus been a long felt need in the art to obtain an anxiolytic medicament, which is void of withdrawal and discontinuation effects and does not cause development of tolerance in patients. Furthermore, sufficient efficacy in relapse and recurrence prevention are important qualities of a well functioning anxiolytic drug. It is believed that deramciclane satisfies this need in the art.
Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Pharmaceutical compositions

Medicilon is the first CRO to offer preclinical animal testing service for the global pharmaceutical companies in China. We provide the IND filing for the preclinical services. Medicilon is the CRO that fulfill both the Chinese and US GLP standards. Medicilon could submit the application for both US FDA and CFDA for your new drug. Since 2004, we have successfully helped our clients to submit their new drug application to US FDA and CFDA and met the requirements of the US FDA and CFDA. We have undergone several inspections and passed all of them. Medicilon will provide an efficient, cost-effective and professional service to help our clients to achieve their goals.Email:[email protected] Web:www.medicilon.com
The present invention is directed to pharmaceutical compositions comprising: (a) a cyclopolysaccharide and (b) a compound of Formula (I) or its pharmaceutical acceptable salt: wherein X1, X2, Q, Z, and m are defined herein. Also disclosed is a method for treating a neoplastic disease or an immune disease with these compositions.
Example 5: Single Dose IV Toxicity Study in Mice with the NL-101 first generation formulation:
A single dose of NL-101 1st generaton formulation (20, 40, 60, 80 or 100 mg/kg) was slowly administered (iv, injection time>30 seconds), to mice and change in body weight was measured over 14 day to assess toxicity of the various doses of NL-101. We found that up to 60mg/kg of NL-101 did not result in a significant change in body weight.
However, we found that this first generation formulation has many disadvantages such as low pH value, potential precipitation after i.v. injection, and series side effects such as damaged mice tail after iv injection. More seriously, sometimes we observed that quick iv injection (e.g injection time <5 seconds) of NL-101 may lead to mice sudden death. Example 6: Single Dose IV Toxicity Study in Mice with the HP CD-based NL-101 formulation:
A single dose of HP CD-based NL-101 formulation (20, 40, 60, 80, 100, or 150 mg/kg) in 10% HP CD was administered (iv) to mice and change in body weight was measured over 14 day to assess toxicity of the various doses of NL-101. We found that up to 60mg/kg of NL-101 did not result in a significant change in body weight.
We are surprised to found that the HP CD-based NL-101 formulation can significantly reduce the cardiotoxicity in vivo. The mice even can survive under quick injection (t<5 seconds) of as high as 150mg/kg NL-101. More importantly, we didn't observe cardiorespiratory stress in mice at therapeutically effective dose of 60mg/kg. In addition, this formulation also has many other advantages, such as neutral pH, clear and stable injection solution, no precipitate issue after iv injection, and no damaged mice tail after iv injection. Therefore, HP CD-based NL-101 formulation will be an ideal formulation to be used in NL-101 MTD, PK, in vivo efficacy study, and IND enabling study. We are actively developing the HP CD-based NL-101 formulation for future human clinical trial.
Example 7: Single Dose IV Toxicity Study in Mice with the Captisol™-based NL-101 formulation:
A single dose of Captisol™-based NL-101 formulation (20, 40, 60, 80, 100, or 150 mg/kg) in 10% Captisol™ was administered (iv) to mice and change in body weight was measured over 14 day to assess toxicity of the various doses of NL-101. We found that up to 60mg/kg of NL-101 did not result in a significant change in body weight.
We are glad to found that the Captisol™-based NL-101 formulation can also significantly reduce the cardiotoxicity in vivo. The mice even can survive under quick injection (t<5 seconds) of as high as 150mg/kg NL-101. More importantly, we didn't observe cardiorespiratory stress in mice at therapeutically effective dose of 60mg/kg. In addition, this formulation also has many other advantages, such as neutral pH, clear and stable injection solution, no precipitate issue after iv injection, and no damaged mice tail after iv injection. Therefore, Captisol™-based NL-101 formulation will be also an ideal formulation to be used in NL-101 MTD, PK, in vivo efficacy study, IND enabling study, as well as in future human clinical trial. Example 8: Multiple Doses IV Toxicity Study in Mice with the HPpCD-based NL-101 formulation:
Multiple doses of HPpCD-based NL-101 formulation (60 mg/kg) in 10%
HP CD was administered (iv) to mice and change in body weight was measured to assess toxicity of the various doses of NL-101. We found that mice can well tolerate multiple doses of 60mg/kg of NL-101 without significant change in body weight. For example, the mice can be dosed at 60mg/kg at day 1, 4, 8, 11, 18, 25. Another feasible dosing scheme is 60mg/kg at day 1, 2, 8, 15, 22, 29.

Microrna dosing regimens

Medicilon is the first CRO to offer preclinical animal testing service for the global pharmaceutical companies in China. We provide the IND filing for the preclinical services. Medicilon is the CRO that fulfill both the Chinese and US GLP standards. Medicilon could submit the application for both US FDA and CFDA for your new drug. Since 2004, we have successfully helped our clients to submit their new drug application to US FDA and CFDA and met the requirements of the US FDA and CFDA. We have undergone several inspections and passed all of them. Medicilon will provide an efficient, cost-effective and professional service to help our clients to achieve their goals.Email:[email protected] Web:www.medicilon.com
A method of treating a subject, for example for a subject with a solid tumor or hematologic malignancy, can include administering a therapeutic treatment cycle to the subject, the cycle including daily microRNA mimic administrations on the first 3-7 consecutive days of the cycle followed by no microRNA administration on the next 7-21 consecutive days of the cycle.
Micro-ribonucleic acids (microRNAs) belong to a class of small non-coding RNAs. They regulate many biological processes, including the cell cycle, cell growth and differentiation, stress response and apoptosis. Alterations in microRNA synthesis occur in human cancers and these are often linked to tumor development, progression and metastasis. Epigenetic alterations and mutations of microRNA expression may promote tumor formation as well as increased tumor aggressiveness, invasion, metastasis and resistance to chemotherapy and radiotherapy. It has been postulated that deregulation of microRNA synthesis, which regulates protein synthesis, is one of the most important factors implicated in cancer development.
These findings suggest novel ways of blocking cancer-related cell proliferation, by re-expression of microRNAs inhibited or silenced by cancer development or by inhibiting oncogenic microRNAs. This might be achieved by introducing molecules that mimic the expression of protective microRNAs that are down-regulated in cancer, or by introducing synthetic antisense molecules complementary to the microRNA of interest and which inhibit oncogenic microRNAs overexpressed in cancer cells (i.e. antagomiRs, anti- miRs).
One of the best-characterized microRNAs to date is microRNA-34 (miR-34).
Human miR-34 comprises three family members: miR-34a, miR-34b and miR-34c. These miR-34 genes are frequently inactivated or expressed at reduced levels in numerous cancer types. miR-34a-c frequently functions downstream of p53 by regulating genes that induce cell cycle arrest, cellular senescence and apoptosis.
The re-introduction of miR-34a inhibits cancer cell growth both in vitro and in vivo. Therapeutic activity of miR-34a has been demonstrated in animal models of non-small cell lung cancer, prostate cancer, melanoma, pancreatic cancer and lymphoma, generally showing 50% to 83% tumor growth inhibition. In order to efficiently deliver miR-34a to tumors in vivo upon intravenous administration, Mirna Therapeutics has evaluated multiple existing delivery systems that are in pre-clinical development or have already entered clinical testing with other oligonucleotide therapeutics. Based on this systemic evaluation program, Mirna Therapeutics has selected a liposomal delivery formulation which is complexed with synthetically produced mimics of miR-34a, and which constitutes the therapeutic drug candidate, MRX34. Evaluations of efficacy in murine cancer models, microRNA bio- distribution and preliminary safety have been performed.
Nucleic acid delivery technologies are being developed in connection with various nucleic acids therapeutic candidates. One delivery technology is liposomes, for example amphoteric liposomes like Marina Biotech's SMARTICLES. Amphoteric liposomes are a class of liposomes, which are pH dependent charge-transitioning particles that can provide for the delivery of a nucleic acid payload (e.g., siRNA, microRNA, antisense, etc.) to cells either by local or systemic administration. Amphoteric liposomes can be designed to release their nucleic acid payload within the target cell where the nucleic acid can then engage a number of biological pathways, and thereby exert a therapeutic effect.
ProNAi Therapeutics has used the NOV340 SMARTICLES® liposomal formulation encapsulating a single-stranded DNA that targets BCL2. With ProNAi' s formulation 2 complete remission and 1 partial remission were observed out of 6 patients with either follicular lymphoma or diffuse large B-cell lymphoma. Out of 9 patients with evaluable safety information, the following drug-related adverse events were seen: nausea (8 pts); chills (6 pts); diarrhea (5 pts); fever, tumor pain, vomiting (5 pts each); and anorexia, back pain, fatigue (3 pts each). Most of these adverse events were of low grade and no grade 4 toxicity was observed.
ProNAi Therapeutics has completed a phase I study (ClinicalTrials.gov
Identifier: NCT01191775) in Patients With Advanced Solid Tumors, and has an ongoing phase II study (ClinicalTrials.gov Identifier: NCT01733238) for Treatment of Relapsed or Refractory Non-Hodgkin's Lymphoma, both using a liposome encapsulated oligonucleotide (DNA Interference, or DNAi) drug substance that was administered by intravenous infusion once daily for 5 consecutive days of a 21 -day cycle.
Tekmira Pharmaceuticals has used lipid nanoparticles which share some similarity with NOV340 SMARTICLES® to deliver oligonucleotides directed against PLK and found tumor responses in patients with adrenocortical carcinoma and neuroendocrine tumor. [0010] As of March 2013, Mirna Therapeutics (Austin, TX) has completed the preclinical development program to support the manufacture of cGMP-materials and the conduction of IND-enabling studies for a miR-34-based supplementation therapy (MRX34). Mirna Therapeutics evaluated the toxicity as well as the pharmacokinetic profile of the formulation containing miR-34 mimic in non-GLP pilot studies using mice, rats and non- human primates. These experiments did not show adverse events at the predicted therapeutic levels of MRX34, as measured by clinical observations, body weights, clinical chemistries (including LFT, RFT and others), hematology, gross pathology, histopathology of select organs and complement (CH50). In addition, miRNA mimics formulated in lipid nanoparticles do not induce the innate immune system as demonstrated in fully immunocompetent mice, rats, non-human primates, as well as human whole blood specimens. A more detailed review of the pre-clinical data is provided in Bader, Front Genet. 2012; 3: 120. Clinical trials are ongoing and, as of March 27, 2014, twenty-nine patients have been treated with MRX34, three at 10 mg/m 2, six at 20 mg/m 2, three at 33 mg/m 2, eight at 50 mg/m 2, seven at 70 mg/m 2, and two at 93 mg/m on a twice weekly dosing schedule.