FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
"IN VITRO ASSAY METHODS FOR
CLASSIFYING
EMBRYOTOXICITY OF COMPOUNDS"
RELIANCE LIFE SCIENCES PVT.LTD
an Indian Company having its Registered Office at
Chitrakoot, 2nd Floor,
Shree Ram Mills Compound,
Ganpath Rao Kadam Marg,
Worli, Mumbai - 400 013,
Maharashtra, India.
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-

FIELD OF THE INVENTION:
The present invention provides in vitro assay techniques for determining the teratogenic or embryotoxic potential of compounds as well as compositions using human embryonic stem cells ReliCell®hES of Indian origin. The method of this invention is in particularly useful for high throughput screening of potential drug candidates and is able to predict the effects on the molecular or cellular level of human body.
BACKGROUND OF THE INVENTION
A leading cause of drug candidate attrition is reproductive toxicity. Mutagenic, embryotoxic or teratogenic substances may exert direct cytotoxic effects and /or induce alterations of embryonic development as a result of mutations at the DNA level. Additionally, developmental defects may be generated by interference of mutagenic or embryotoxic substances with regulatory processes of proliferation and differentiation at the levels of gene and protein expression, respectively. Medical drugs and xenobiotics when administered during pregnancy may interfere with the embryonic development and as a consequence induce embryo lethal or teratogenic effects.
Most of the current understanding about the toxicity of various chemicals comes from animal data. The compounds that are toxic at the pre-clinical stage are ruled out of clinical trials. Toxic effects of the potential drugs and chemicals are done using animals to determine the safe and effective dose of drugs in humans. Further, the study on birth defects and the other reproductive effects are determined using pregnant animals and embryotoxicity test in the early stages of pregnancy by administering single or multiple doses of the test chemicals.
Although animal model studies are helpful in studying the reproductive effects of the drugs and chemicals, the negative animal studies do not guarantee that these agents
are free from reproductive effects. However there were many molecules in the past that did not demonstrate toxicity in animals but were proved to be toxic in humans. This is due to the probable difference in the genetic make up of the animals and humans, which fails in some cases to correlate the results. Fetal malformations caused
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by the use of the drug thalidomide constitute one of the most tragic chapters in modern pharmacology. Over the past decade, many individuals and organizations have used this episode to illustrate the inadequacy of animal testing, pointing out that extensive testing in animals did not reveal the teratogenic potential of the drug in human beings. The drug diflunisal was shown to be teratogenic in animal studies, but it is not so in human. Salicylates e.g. aspirin is a well-known therapeutic drug, when taken by pregnant women for years has shown no sign of being responsible for birth defects. However, aspirin causes birth defects in rats, mice, monkeys, guinea-pigs, cats and dogs.
Till date, all the drugs reaching the clinical trail stage pass through the testing of compounds on animal models for assessing the possible effects of chemicals on reproduction. Routinely, test chemicals are analyzed by segment studies, which cover pre-conceptional exposure and postnatal development including the lactation period (Spielmann, 1988). These in vivo tests are time consuming, expensive and have to be carried out on high numbers of laboratory animals (Schmidt et al., 2001). Therefore there is a need for alternatives to living animals to test the potential reproductive toxicity of chemical substances by in vitro systems.
New legislation enacted in many countries and regions of the world during the 1980s requires that laboratory animal use be reduced, refined and replaced wherever possible, for ethical and scientific reasons, in line with the Three Rs concept put forward by W.M.S. Russell and R.L. Bnurch in 1958, in The Principles of Humane Experimental Technique. Current uses and future prospects for the use of laboratory animal procedures and non-animal methods in the biomedical sciences are considered in five themes: the development of replacement alternative methods; the validation and regulatory acceptance of alternative test methods; reduction alternatives and the testing of biologicals (vaccines and hormones); refinement of animal procedures; and education, ethics and databases.
In the past 2 decades, cell culture systems are well established as cellular screening models in toxicology has been well established. Different cellular systems have been proposed, developed and established for in vitro tests for development toxicity which follow the OECD guidelines (Browm et al., 1995), including establishment of cell
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lines such as 3T3 fibroblasts (Spielmann et al., 1997), mouse ovarian tumor cells (Braun et al., 1982), primary culture of human embryonic palate mesenchymal cells (Pratt et al., 1982) and limbal bud cells in the micromass culture test (Flint and Orton, 1984). Studies utilize protein content (Hulme et al, 1990), colony size (Newall and Beedles 1994), enzyme activity (Spielmann et al, 1997; Laschinski et al, 1991) as methods of detection to have been employed. However in many cases, the in vitro models, using primary cultures or established cell lines do not represent the functional properties of specialized somatic cells. In vitro culture often results in a loss of
proliferation capacity, viability and tissue specific properties during long term cultivation (Rolletschek 2004; Wobus et al., 1994; Gottlieb, 2002) and tissue-specific characteristics may be impaired in established lines of cardiac, neuronal or pancreatic cells (see Greene and Tischler, 1976; Wobus et al., 1994a; Brismar, 1995; Murayama etal.,2001).
In vitro tests are performed with mammal embryos (Neubert and Merker, Cell culture techniques—applicability for studies on prenatal differentiation and toxicity, de Gruyter, Berlin-New York (1981)) like of rat (whole embryo culture, WEC) (Steele et al 1983), Xenopus (FETAX test) or chicken (CHEST) (Browm et al., 1995, Flint 1983) and with embryonic organs for teratogenicity tests. However, these tests procedures have the major disadvantage that they require the use of a large number of live mammals, in particular rats and mice. The use of these systems for embryotoxicity evaluations was rare because the predictive values using these systems were about 70% (Genschow et al., 2002). Further the teratogenic animal studies are time consuming, more laborious, required high level of technical skill and fall under various animal welfare governing bodies for which the approval needs to be sought.
Most of these tests used for in vitro embryotoxicity evaluation for drug discovery testing research were typically obtained from primary tissue, immortalized tumor cells or genetically normal (that is, diploid), yet have very limited survival times in culture, which affects the applicability of primary explants in screening technology. Also, the inconsistent availability, and the inherent donor variation, of human primary cells types restricted the opportunities for the use of primary cells in drug discovery testing. Immortalized cells derived from tumors or oncogenic transformation offer more consistent sources of cellular reagents, which make them suitable for use in high
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throughput screening (HTS) and secondary assays. Immortalized cells can be
maintained indefinitely and transfected with DNA constructs that express target proteins or reporters. However, these cells are typically genetically abnormal (aneuploid), and conclusions based on gene function could be limiting.
One of the most recent developments in the recent past has been the use of stem cells.
Embryonic stem (ES) cells are derived from the inner cell mass (ICM) of the
mammalian blastocyst (Evans & Kaufman 1981; Martin 1981). These cells are
pluripotent, thus capable of developing into any organ or tissue type. ES cells are
capable of indefinite proliferation in vitro in an undifferentiated state; maintaining a
normal karyotype through prolonged culture. They also have the capability to
differentiate into derivatives of all three embryonic germ layers (i.e., mesoderm,
ectoderm and endoderm) (Itskovitz -Eldor et al., 2000). Embryonic stem cells
represent a powerful model system for the investigation of mechanisms underlying
pluripotent cell biology and differentiation within the early embryo as well as
providing opportunities for genetic manipulation. Appropriate proliferation and
differentiation of ES cells can be used to generate an unlimited source of cells.
However, differentiated cells from stem cells after considerable advantages, compared
with primary or immortalized cells, in that these cells are genetically normal,
demonstrate uniform physiological responses are maintained in culture in long periods
of time and are grown at scale, all of which enhances their usefulness in screening
processes. Furthermore, unique advantage of ES cells is their ability to undergo
homologous recombination at a relatively high frequency, which enables the selection
of reproducible and precise genetic modifications of the endogenous genome. Under
certain culture conditions e.g. absence of leukemia inhibitory factor, ES cells can
differentiate in vitro into an embryo-like aggregates, so-called embryoid bodies,
derivatives of all three germ layers (ectoderm, mesoderm and endoderm), i.e. in
cardiogenic cells (Doetschmann et al., J. Embryol. Exp. Morphol. 87, 27-45, 1985), in
myogenic cells (Rohwedel et al., Dev. Biol. 164, 87-101, 1994), neuronal and
haematopoietic cells (Wiles and Keller, Development 111, 259-267, 1991). Thus ES
cells offer several important advantages over primary or immortalized cells such as
they are genetically normal, demonstrate uniform physiological response, can be
maintained in culture for long periods of time and are grown at scale, all of which
enhance their usefulness in drug screening (McNeish, 2004).
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Studies have shown that retinoic acid causes changes in tissue specific genes at specific times of embryoid body differentiation and to activation, repression, or modulation of the expression of the myocardial-specific genes or somatic-specific genes thus proving to be teratogenic which is specifically incorporated herein by reference (Wobus et al., Roux's Arch.Dev.Biol.204, 1994(36-45). Hence it is demonstrated that Embryoid bodies can be used as a toll for checking the embryotoxicity of known compounds.
Embryonic stem cells have been isolated from the inner cell mass (ICM) of blastocyst stage embryos in multiple species (Bhattacharya et al., 2005) like mice (Solter and Knowles, 1975), porcine (Chen et al., 1999), non-human primates (Thomson et al., 1995) and humans (Reubinoff et al., 2000, Arundhati et al., 2006).
The use of blastocyst-derived pluripotent ES cells has been used to develop in vitro methods for testing various medical drugs and xenobiotics. Most of these studies include parameters as protein content (Hulme et al., 1990), colony size (Newall and Beedles 1994), enzyme activity (Laschinski et al., 1991, Newall and Beedles, 1994, Spielmann et al., 1997) and surface receptor expression (Hooghe and Ooms, 1995). However, the so called "embryonic stem cell test" (EST) for the first time included the EB model of ES cell differentiation (Spielmann et al., 1997, Scholz rt al., 1999). By using EST the effects of test compounds on developing processes of early ESC differentiation are determined. The EST has been validated in a study coordinated by European Centre for the Validation of Alternative Methods (ECVAM) (Genschow et al., 2000 and Spielmann et al., 2001a). The validation study included in vitro cultivation of post implantation of rat whole embryo culture, the micromass test and the differentiation analysis of a pluripotent mouse embryonic stem cell line into the EST (Genschow et al., 2000). The EST involves 1) cytotoxic effects of test substances on differentiated 3T3 fibroblasts, 2) cytotoxic effects on the undifferentiated ES cells and 3) the influence of test compounds on ES cell derived cardiac differentiation. For statistical evaluation, a prediction model for the embryotoxic potential of a given substance was established (Scholz et al., 1999). During the EST pre-validation study, 10 compounds have been tested for their embryotoxic potential: 100, 88.9 and 91.7%
of the prediction for non embryo toxic (Class 1), weekly embryo toxic (Class 2) and strongly embryotoxic (Class 3) substances, respectively, were in accordance with
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classification derived from in vivo data (Scholz et al., 1999, Genschow et al 2000). During the pre-validation of EST, it was observed that there is a high percentage of resemblance between data generated by EST and in vivo studies (Rohwedel et al., 2001). The validation study using pluripotent ES cells under differentiation conditions supports the idea that ES cells are valuable tool to investigate the embryotoxic potential of environmental factors in vitro. In fact, the companies that were involved in the ECVAM validation study have already established the EST as an in vitro test procedure for embryotoxicity.
The mouse EST procedure is already used not only to test chemical compounds but also physical factors, such as electromagnetic fields emitted by digital mobile communication system (Rohwedel et al., 2001, Schonborn et al., 2000). However, despite the recent improvements of embryotoxicity testing using the EST protocol, there are some drawbacks of the mouse EST regarding the differentiation analysis and the parameters used. One of the main concerns of the protocol is that it utilizes manual counting of beating cardiomyocytes, which is cumbersome, time consuming and requires technical expertise.
While the mouse model provides the foundation for studying stem cell biology, distinct differences between mouse embryonic stem cells (mES) and human stem cells (hES) have been observed (Abeyta et al 2004). The morphology, cell surface markers and growth requirement of embryonic cells from other species are significantly different from mouse ES cells. Further, mouse and human embryos differ significantly in temporal expression of embryonic genes, such as in the formation of the egg cylinder versus the embryonic disc (Kaufman, The Atlas of Mouse Development; London; Academic Press, 1992) in the proposed derivation of some early lineages (O'Rahilly and Muller; Develomental stages in Human Embryos, Washington; Carriage Institution of Washington, 1987) in the structure and function of the extra-embryonic membranes and placenta (Mossman, Vertebrate Fetal membranes; New Brunswick; Rutgers, 1987) in growth factor requirement for
development (e.g., the hematopoietic system- Lapidot Lab. Animal Sciences 1994) and in adult structure and function (e.g., central nervous system). Apart from the development differences there are certain molecular differences that make mES cells different from hES cells. In particular, leukemia inhibitory factor
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(LIF) activity which is regulated by gpl30 and JAK/STAT pathways is sufficient to maintain mES in undifferentiated state, whereas addition of exogenous LIF too is not sufficient to maintain hES in undifferentiated state (Thomson et al., 1998). Apart from this role of gpl30 in JAK/STAT pathway is not fully understood (Rose-John, 2002). Only hES cells and not mES cells express specific cell surface antigens like SSEA 3 and SSEA 4 however SSEA 1 is expressed only by mES cells (Henderson et al., 2002).
Studies of genetics of gene expression in Saccharomyces cerevisiae, for example, demonstrated that the expression of more than 1500 genes differed between two closely related strains and that the expression of differences were modulated by complex genetic differences the strains (Cavalieri et al., 2000, Bremer et al., 2002). It further affirms the notion that there would be a difference in the expression of genes as well as effects of teratogenic compounds when compared between mES and hES cells. Since hES cells are believed to be the closest in vitro system to humans (McNeish, 2004), the present invention has taken the objective that hES cells could be used to evaluate embryotoxicity based on human fibroblast IC5o, undifferentiated hES cells IC50 and differentiated hES cells ID5o and classify compounds into non-, weak and strong teratogens.
While the mouse model provides the foundation for studying stem cell biology, distinct differences between mouse embryonic stem cells (mES) and human stem cells (hES) have been observed (Abeyta et al, 2004). There are hundred of genes that are not found in both the species and there are significant differences in the gene expression patterns of many genes (McNeish 2004). hES cells differ from mES cells in their growth expression of cell surface markers (Henderson et al, 2002) and the rate of growth of hES cells is slower (Amit et al, 2000). Unlike mES cells, hES cells do not maintain undifferentiated state and proliferate in the presence of LIF (Burdon et al., 2002).
US patent 5,811,231 presents a method of screening the toxic compounds by determining the level of transcription of genes linked to selected stress promoters in cells of various cell lines.
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WO 97/01644 describes a method of creating a molecular profile of a chemical composition namely toxicity screening using transgenic embryoid bodies containing tissue specific promoters and reporter genes. The method involved is embryonic stem cells and embryonic germ cells are stably transfected with a reporter gene/ promoter construct and allowed to differentiate into Embryoid bodies in the presence of test substances. The expression of the reporter gene is then detected.
WO 97/13877 provides a method for assessing the toxicity of a compound in a test organism by measuring the gene expression profile of selected tissues which are measured by massively parallel signature sequencing of cDNA libraries constructed
from mRNA extracted from selected tissues.
WO 00/34525 provides methods and systems for identifying and typing toxicity of chemical compositions as well as for screening new compositions for toxicity. The PCT publication involves detecting the alteration in genes or protein expression and hence establishing molecular profiles in isolated mammalian embryoid bodies contacted with various chemical compositions of known and unknown toxicities, and correlating the molecular profiles with toxicities of the chemical compositions. The alterations in levels of gene or protein expression can be detected by use of a label selected from any of the following: fluorescent, colorimetric, radioactive, enzyme, enzyme substrate, nucleoside analog, magnetic, glass, or latex bead, colloidal gold, and electronic transponder.
As the traditional method for testing embryotoxic potential of industrial and pharmaceutical chemicals is laboratory animal testing performed on pregnant animals. Many mammalian and non-mammalian in vitro models using permanent cell lines have been developed (Huuskonen, 2005). The new methods are not aimed to replace animal testing but to reduce the number of animals used. Apart from consumption of animals, during early discovery phase of drugs or pesticides, time and amount of compound critically limit the applicability of the in vivo testing (Zur Nieden et al,
2004). Looking into the need for a more sensitive, reliable and robust method for drug toxicity evaluation especially in embryotoxicity, which will overcome the species differences, the present invention has focused in providing an assay method for determining the embryotoxicity in terms of qualitative and quantitative techniques.
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The present invention provides an in vitro embryotoxicity testing method, which is a simple and effective method for assessing the toxicity of chemicals and /or compositions and provides a high throughput screening of the chemicals ands/or compositions. The present invention provides the use of multiple markers, which focuses on improving the efficiency of the test and aims to provide an objective measurement instead of subjective assessment. The techniques provided results, which are reliable, reproducible and highly sensitive than the conventional mouse embryonic screening models. The present invention also focuses on the use of hES of INDIAN origin for the detection of embryotoxicity of chemicals and/or compositions.
OBJECT OF THE INVENTION
The object of the present invention is to provide a routinely employable in vitro embryotoxicity test method for the detection of chemically induced embryotoxic / teratogenic effects.
It is the object of the present invention to provide a parallel method for pre-clinical in vitro embryotoxicity testing of chemicals and their compositions using human embryoid bodies.
It is the object of the present invention to provide an in vitro embryotoxicity testing method, which can be used for high throughput screening of chemicals.
It is the object of the present invention to classify compounds, drugs and xenobiotics as non-embryo, weakly and strongly embryotoxic.
It is the object of the present invention to provide IC50 (inhibitory concentration) and ID50 (inhibition of differentiation concentration) for the known and unknown compounds or xenobiotic or medicinal drugs or pesticides or metals or any other chemical that has human usage
It is the object of the present invention to provide new method to calculate ID50 values based on gene expression profiles of genes that are predominantly involved in early developmental stages of pregnancy or fetal developmental stage.
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It is the object of the present invention to provide an in vitro embryotoxicity testing method, which is quick and less expensive than animal pre-clinical toxicity studies as well as more closer to human clinical trials.
It is the object of the present invention to provide an in vitro embryotoxicity-testing model, which yields results that is same or similar as compared with the animal preclinical toxicity studies and also show the differences that are not shown in animal studies due to species differences between animal and humans.
It is the object of the present invention to provide an in vitro embryotoxicity testing method for detection of embryotoxicity/ teratogenic properties of the chemical by giving suitable indications of possible developmental disturbances and differentiation disturbances during early development of adults.
It is the object of the present invention to provide an in vitro embryotoxicity testing method, which can predict the effects of the chemicals on the organs of ectoderm, mesoderm and endoderm origin which gives rise to different cell types that form a complete individual.
It is the object of the present invention to provide an in vitro embryotoxicity testing method, which has highly controlled experimental conditions that gives results, which are easily quantified.
It is the object of the present invention to provide an in vitro embryotoxicity testing method, which predicts the cellular and molecular effects of a chemical or drug.
It is the object of the present invention to provide an in vitro embryotoxicity testing method, which provides consistent results in a sensitive manner.
It is the object of the present invention to provide an in vitro embryotoxicity testing method, which correctly converts the results into useful prediction of toxicity so that appropriate safety assessment can be made.
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It is the object of the present invention to provide an in vitro embryotoxicity testing method, which is reliable wherein the results are reproducible following application of a clearly stated prediction model.
It is the object of the present invention to provide an in vitro embryotoxicity testing method, which is relevant in establishing the scientific meaningfulness and usefulness of results for a particular purpose in terms of hazard prediction.
It is the object of the present invention to provide an in vitro embryotoxicity testing method to predict the mechanism of toxic chemical at molecular and cellular level thus providing the manufacturers the data so as to chemically modify the appropriate substituents in the chemical structures of the toxin.
SUMMARY OF THE INVENTION
The present invention provides an in vitro embryotoxicity testing method, which is a simple and effective method for assessing the toxicity of chemicals and /or compositions.
In one embodiment the present invention has provided a technique to evaluate the cytotoxic potential of the test compounds on the 3 different cell types namely
a) the human foreskin fibroblasts which represents the mature adult cell types
b) embryonic stem cells resembling germ cells and
c) embryoid bodies which represents the early developmental stages of pregnancy or fetal developmental stage.
In another embodiment the present invention has provides the effects of the compounds on the differentiation potential of human embryoid bodies (hEBs) into different cell types based on gene expression levels.
In one embodiment, the method involves the use of human embryoid bodies. The embryoid bodies are formed from human embryonic stem cells. In the preferred embodiments the embryoid bodies are obtained from human embryonic stem cells ReliCell®hES of Indian origin
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The present invention provides assays techniques which comprises of following:
1. Analysis for qualitative assessment of embryotoxicity by MTT assay or Fluroscent activated cell sorter (FACS) or luminescent methods
2. Analysis for quantitative toxicity by molecular end points for detection of gene expression levels.
In one embodiment the present invention provides a human embryonic stem cells as a model to test the effects of three reference compounds which are well know in vitro and in vivo studies as strongly embryotoxic, weakly embryotoxic and non embryotoxic. In one preferred embodiment the present invention has provided a test method and checked on three reference compounds such as strongly embryotoxic (5-Fulurouracil 5FU), weakly embryotoxic (caffeine) and non- embryotoxic (penicillin G). The aim in selecting the three compounds mainly relies on the fact that teratogenic effects of penicillin G are neither observed in mouse or human (Boucher and Delost 1964), where as 5FU is well known as a cytostatic drug with strong teratogenic potential in vivo (Shuey et at, 1994). The present invention provides data that suggests caffeine down regulates NFH expression suggesting neurotoxicity, which is in agreement with the published report in neonatal rats and adult rats (Kang et ai, 2002) where it was observed that intraperitoneal administration of caffeine caused neuronal death in various brain areas of neonatal rats within 24 h. Enns et al. (1996) too reported potential deleterious effects of caffeine in hippocampal neurons.
In another embodiment the present invention aims to further validate the test by screening known / predetermined compounds as well as unknown compounds. In one preferred embodiment the present invention has selected from a list of drugs with predetermined toxicity such as
a) Non embryotoxic eg: Penicillin G, Saccharin, Ascorbic acid, Isoniazid
b) Weakly embryotoxic eg: Caffeine, Lithium chloride, Diphenhydramine, Indomethacin, Aspirin, Dexamethasone, Methotextrate, Diphenylhydantoin
c) Strongly embryotoxic eg: 5-Flurouracil, Hydroxyurea, Busulfan,
Cytosinearabinoside, retinoic acid
In another embodiment the present invention has compared the cytotoxic effects of the above stated 3 compounds on 3 different cell types. The HFF represents the adult
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or mature cell types, the hES cells mimic the early growth stages of development post fertilization or the germ lineages and the hEBs which represent the stages of development during early pregnancy.
In one embodiment the present invention has used molecular end points to increase the sensitivity and reproducibility of the assay. Previously beating cardiomyocytes were used as an indictor of developmental toxicity. More recently, literature too supports our hypothesis to use hES cells and utilizing PCR and FACS as a tool for developing better end points for testing to developmental toxicity (Huuskonen, 2005). Researchers have also suggested the use of automated in vitro screening methods for teratogens which are based on cytotoxicity and cell morphology (Walmod et al, 2004). Further, Pellizer et al. (2004) reported use of PCR as a detection method for mouse embryonic stem cells. They used myosin heavy chain, Oct-4, Brachyury and Nkx2.5 as an indicator of teratogenic effects based on the decrease in expression at different days of development. It is now known that a single marker could not be conclusive in determining developmental toxicity of a compound and hence the use of representative lineage specific markers have been utilized in the present invention
In one preferred embodiments the present invention has utilized and tested a panel of gene representative of some of the major organs in the process of development. Selection of genes was done based on earlier published reports from our group (Mandal et al., 2006) and other groups (Bhattacharya et al., 2005; Noaksson et ai, 2005; Mitalipova et al, 2005).
In one preferred embodiment the protocol of in vitro embryotoxicity testing method involves culturing of human embryonic stem cells by "hanging drop" method wherein the cells are seeded onto the lid of the culture dish and grown for 3 days in presence of the concentration range of test chemical. The embryoid bodies thus formed i.e. the aggregates of cells are then transferred to the bacteriological petri dishes containing the appropriate concentration of test chemical for another 2 days. On the 5th day the
embryoid bodies are then seeded into a 96 well plate and incubated for another 5 days under controlled conditions. After 15 days the test of MTT and RT -PCR are
performed on these
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In another embodiment, the method involves the use of 3-(4,5,-di-methylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) for qualitative assessment of embryotoxicity of chemicals and/or compositions by determining the viability of the cells The test procedure is based on the capacity of mitochondrial dehydrogenase enzymes in living cells to convert the yellow substrate MTT into a dark blue formazan product, which is then detected quantitatively using a micro plate ELISA reader.
In one embodiment the method involves qualitative method for assessment of effect of compounds on various lineages by detection of gene expression after isolating the total RNA using the TRIzol method (Invitrogen, USA) or RNAeasy Spin Columns (Qualigens, USA). The isolated RNA is used to study the expression of genes specific for ectoderm, mesoderm and endoderm, by an appropriate method, like RT-PCR, qPCR, Micro-array, TaqMan Low Density Array (TLDA) etc. The changes in the gene expression in presence or absence of the drug are then compared.
In one aspect, the test chemical composition can be the same as the chemical composition having predetermined toxicities. For example, the test chemical is identified through this testing as exhibiting the identical molecular profile as the known chemical composition.
In one aspect of the present invention, the toxicity of a test chemical composition can be ranked according to a comparison of its molecular profile in EB cells to those of chemical compositions with predetermined toxicities.
In one aspect, the present invention provides a embryotoxicity prediction model by which correctly converts the results in to prediction of toxicity. This method aims to provide results that appropriately classifies as not embryotoxic, weak embryotoxic and strong embryotoxic.
In one aspect, the present invention provides a method, which can predict the molecular or cellular mechanism of chemicals exhibiting toxicity. This method aims to provide data on the molecular and cellular mechanism, which will be, enable the respective manufactures to change the substitutions accordingly in the chemical structures.
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In another preferred aspect, the present invention provides a method for determining the IC 50 values of the test compounds. In one aspect, the present invention aims to provide results of embryotoxicity, which are reliable, reproducible and relevant, and proving to be quicker and less expensive.
Further, the chemical compositions can be therapeutic agents (or potential therapeutic
agents), of agents of known toxicities, such as neurotoxins, hepatic toxins, toxins of hematopoietic cells, myotoxins, carcinogens, teratogens, or toxins to one or more reproductive organs. The chemical compositions can further be agricultural chemicals, such as pesticides, fungicides, nematicides, and fertilizers, cosmetics, including so-called "cosmeceuticals," industrial wastes or by-products, or environmental contaminants. They can also be animal therapeutics or potential animal therapeutics. They can be biopharmaceutical products wherein the testing in human is mandatory rather than on animals.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form the part of the present invention and are included to substantiate and demonstrate the important aspects of the disclosure. The present invention may be better understood by the following drawings in combination with the detailed description of the specific embodiments presented herein.
FIG. 1. Photomicrographs show the dose dependent effects of 5-fluorouracil (A), caffeine (B) and penicillin G (C) on day 5 human embryoid bodies (hEBs). Al-6, represent different doses of 5FU, 1- Control (no treatment), 2- 0.0001 μg/ml, 3- 0.001 μg/ml, 4- 0.01 μg/ml, 5- 0.1 μg/ml, 6- 1 μg/ml. Bl-6, represent different doses of caffeine, 1- Control (no treatment), 2- 0.05 μg/ml, 3- 0.5 μg/ml, 4- 5 μg/ml, 5- 50 ,g/ml, 6- 500 μg/ml. Cl-6, represent different doses of penicillin G, 1- Control (no treatment), 2- 1 μg/ml, 3- 10 μg/ml, 4- 100 μg/ml, 5- 1000 μg/ml, 6- 5000 μg/ml.
Photographs are representative of 3 experiments.
FIG. 2. Graphs represent survival percentage in a dose dependent effect of 5-fluorouracil (A), caffeine (B) and penicillin G (C) on day 15 on human foreskin
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fibroblast (HFF), human embryonic stem cells (hES) and human embryoid bodies
(hEBs). Survival percentage for HFF and hEBs was performed by MTT whereas for hES FACS was utilized. Data represent mean ± SE (n=3). * Significantly different from control, p < 0.05.
FIG. 3. Gene expression profile following treatment with different doses of 5FU on hEBs on day 15. A, shows the gene expression pattern at different doses lane 1-Control (no treatment), 2- 0.0001 ug/ml, 3- 0.001 ng/ml, 4- 0.01 u-g/ml, 5- 0.1 ug/ml. B, graphs shows percentage relative gene expression after normalizing with internal control (GAPDH). Data represent mean ± SE (n=3). * Significantly different from control, p < 0.05
FIG. 4. Gene expression profile following treatment with different doses of caffeine on hEBs on day 15. A, shows the gene expression pattern at different doses lane 1-Control (no treatment), 2- 0.05 μg/ml, 3- 0.5 μg/ml, 4- 5 μg/ml, 5- 50 μg/ml, 6- 500 μg/ml. B, graphs shows percentage relative gene expression after normalizing with internal control (GAPDH). Data represent mean ± SE (n=3). *Significantly different from control, p < 0.05
FIG. 5. Gene expression profile following treatment with different doses of penicillin G on hEBs on day 15. A, shows the gene expression pattern at different doses lane 1-Control (no treatment), 2- 1 μg/ml, 3- 10 μg/ml, 4- 100 μg/ml, 5- 1000 μg/ml, 6- 5000 μg/ml B, graphs shows percentage relative gene expression after normalizing with internal control (GAPDH). Data represent mean ± SE (n=3). * Significantly different from control, p < 0.05
DETAILED DESCRIPTION
As used herein ,"embryoid body ,"EB"or"EB cells" typically refers to a morphological structure comprised of a population of cells, the majority of which are derived from embryonic stem ("ES") cells that have undergone differentiation
Toxicity," as used herein, means any adverse effect of a chemical on a living organism or portion thereof. The toxicity can be to individual cells, to a tissue, to an
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organ, or to an organ system. A measurement of toxicity is therefore integral to determining the potential effects of the chemical on human or animal health, including the significance of chemical exposures in the environment. Every chemical, and every drug, has an adverse effect at some concentration; accordingly, the question is in part whether a drug or chemical poses a sufficiently low risk to be marketed for a stated purpose, or, with respect to an environmental contaminant, whether the risk posed by its presence in the environment requires special precautions to prevent its release, or quarantining or remediation once it is released. See, e. g., Klaassen, et al., eds., Casarett and Doull's Toxicology: The Basic Science of Poisons, McGraw-Hill (New York, NY, 5th Ed. 1996).
"Chemical composition,""chemical,""composition,"and"agent,"as used herein, are generally synonymous and refer to a compound of interest. The chemical can be, for example, one being considered as a potential therapeutic, an agricultural chemical, an environmental contaminant, or an unknown substance or biochemical entity or biopharmaceuticals.
The term IC50 as used herein refers to the inhibitory concentration at which 50% of the cells show cytotoxicity.
The term ID50 as used herein refers to the inhibitory concentration at which 50% of the cells show inhibition of differentiation to a cell type
The present invention herein provides details of the study protocol wherein the chemicals and/or compositions can be evaluated for their embryotoxicity potential. The techniques provided herein involves the qualitative assessment of the toxicity by MTT assay and its effect on various lineages by using specific markers and the quantitative detection of IC50 values. The test provided herein is compared against standard known toxic or safe compounds.
The present invention has provided the techniques or test procedure which would be able to present to the researcher or the inventors, the correlation of the toxicity effects on various lineages and thus enables them to observe the toxicity and the efficacy potential of various groups (for example in NCE's) present in one main structure.
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The present invention has provided test procedures, which would be able to identify more than just embryotoxicity potential in that it gives the toxicity data on the various lineages. This data would be helpful to those researchers and inventors to enable them to alter the drug delivery techniques in order to avoid the toxic effects on that particular lineage.
Thus the present invention has provided embryotoxicity assay techniques which depicts the toxicity of the chemicals and /or compositions in a more elaborate manner. The present invention also focuses on providing results which can be compared with mouse ES cells to highlight the sensitivity of the test by using human ES cells.
The present invention also focuses on providing testing on certain gene expression markers specific for the Asian or Indian population.
The present invention has provided the use of human embryonic stem cells for embryotoxicity test because as seen from the literature the hES cells are believed to be the closest in vitro system that mimic humans and the toxicity in hES cell could resemble post fertilization stages. Further the present invention provides a test system that provides a comparative effects of the compounds on different lineages during development and the test system being based on gene expression, is more sensitive, reproducible, robust and requires lesser dependency on visual expertise which is a must in the conventional techniques where the test is based on visual observation of beating cardiomyocytes.
Apart from the in vitro data available, similar results have been shown using mouse embryonic stem cells (Zur Nieden et al, 2004). Our results too are in accordance with the published data about the non-toxic and toxic effects of penicillin G and 5FU respectively. The data also indicated that the method is sensitive and specific as it does not cause gene down regulation in a non-specific manner, as only endodermal markers are down regulated in case of penicillin G where as all the three lineage markers are effected by caffeine and 5FU.
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In summary, this present invention provides evidence that the use of human embryonic stem cells for drug toxicity evaluations is a far better choice that the use of any other alternative method for toxicity testing because (1) hES cells are believed to be the closest in vitro system to mimic humans and the toxicity in hES cell could resemble post fertilization stages; (2) the test system provides a comparative effects of the compounds on different lineages during development, and (3) the test system being gene expression based is more sensitive, reproducible, robust and requires lesser expertise than visual observation of beating cardiomyocytes. However, there is scope to increase the number of markers used in order to get a clearer picture of the developmental toxicity in humans.
The following steps are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
The protocol of the embryotoxicity testing involves the following sequence of steps: PART A (Studies with Embryoid Bodies):
i) Preparation of the various concentrations of test solutions in culture medium or
desired solvent,
ii) Preparation of the cell suspension by enzymatic digestion of human embryonic stem
cell colonies for embryoid bodies,
iii) Culturing of the cell suspension for formation of embryoid bodies in presence and
absence of test substance and incubation for 3 days in hanging drops,
iv) Cultivation of the embryoid bodies in bacteriological petri dish for 2 days under
controlled conditions.
v) Transfer of the embryoid bodies into 96well plate and incubation for 10 days in presence of test solution.
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vi) On the 15 day, detection of the viability of embryoid bodies is done by MTT or fluroscent or luminescent method assay and RNA is isolated for studying the gene expression.
PART B (Studies with human foreskin fibroblast (HFF):
i) Preparation of the various concentrations of test solutions in culture medium or
desired solvent,
ii) Plating of HFF cells in 96 well plates in the presence or absence of test compounds
for 15 days with change of media and test chemicals every 2 days,
iii) On the 15th day, detection of the viability of HFF is done by MTT assay or fluroscent
or luminescent method.
PART C (Studies with human embryonic stem cells):
i) Preparation of the various concentrations of test solutions in culture medium or
desired solvent,
ii) Preparation of the hES colonies by manual passage in presence and absence of test
substance and incubation for 15 days on matrigel coated plates for 15 days with
change of media and test chemicals every 2 days,
iii) On the 15th day, disruption of the colonies with enzymes for single cells formation,
iv) Single cells are labeled with fluorescent labeled dye like PI and acquisition of the
percentage viability with FACS as a measure of cytotoxicity.
As outlined above, the details of sequence of steps is described further herein in the
specification.
The various concentration range of test chemicals are prepared in culture medium
which will be termed as "test solution" further in this invention. The culture medium
comprises 80% DMEM/F-12, 15% ES tested FBS, 5% Serum replacement, 1%
nonessential amino acid solution, ImM glutamine (Gibco), 0.1% beta
mercaptoethanol, 4ng/ml human bFGF and 20 ng/ml of LIF.
The preparation of the cell suspension of human embryonic stem cells involves the enzymatic digestion of 3-4 days old human embryonic stem cell colonies. The process involves the washing of the colonies with 2 ml DPBS and then 2 ml of collagenase (2 mg/ml) was added for 15-20 min. After the incubation, Collagenase is removed and 1
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ml of trypsin (0.05 %) is added for 1 min. The trypsin is then removed and colonies are collected in the medium and cell suspension is prepared by trituration. A cell
suspension of approx 10,000 cells is prepared with appropriate test solution.
The culturing of the cell suspension is done by dispensing about 20μl of the cell suspension along with test chemicals on the inner side of a 100mm bacteriological petri dish. Around 50-80 drops are dispensed for each concentration as well as untreated control and solvent control. The lid is turned carefully into its regular position and is put on the top of petri dish containing 5 ml of phosphate buffer saline (PBS). The "hanging drops" were incubated for 3 days in a humidified atmosphere with 5% C02 at 37°C.
On the day 3, freshly prepared 5ml of test solution is added to the lid of the "hanging drops" petri dish. The embryoid bodies are carefully transferred from the lid of the "hanging drops" petri dish to a 60mm bacteriological petri dish. And the suspension cultivated for 2 days in a humidified atmosphere with 5% CO2 at 37°C.
On the 5th day, the EBs is transferred into 96 well plates and by taking 10 EBs in each well. About 150 μl freshly prepared test solutions is added to each well and 3 wells were used for each concentration. The 96 well plates are incubated for 5 days in a humidified atmosphere with 5%C02 at 37°C. The remaining EBs were plated on a 24 well plate coated with 0.2% gelatin and were allowed to differentiate. Media was replaced on every second day till day 15th.
On 15th day, MTT assay is performed. MTT solution (5 mg/ml) was prepared in culture medium containing test chemical. This MTT solution is added to all wells and incubated at 37°C in a humidified atmosphere of 5% CO2 for 4 hours after which the MTT desorb solution (acidified isopropanol) was added to each well. The plate is shaken on a micrometer plate for 15 min to dissolve the formazan. The absorbance is measured at 550-570 nm in a microtiter plate reader using 630 nm as a reference wavelength. The absorbance is recorded.
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On 15 day, differentiated cells that are adherent were washed with 2 ml DPBS and trypsinized (0.05%) from 24 well plates and collected in a tube. The tube is centrifuged at 2000 rpm for 10 mins to pellet down the cells. The total RNA is isolated using TRIzol (Invitrogen, USA) as per the manufacturer's instructions. The RNA was quantified by its absorbance at 260 nm and 1-2 μg of RNA of control and drug treated cells was converted to cDNA using Superscript cDNA syntheis kit (Invitrogen, USA) as per the manufacture's instruction. CDNA was used to study the expression of genes specific for ectoderm, mesoderm and endoderm, by an appropriate method, like RT-PCR, qPCR, Micro-array etc. Marker indicative of each linage were used.
The changes in the gene expression in presence or absence of the drug are compared.
Changes in mesodermal marker representative of cardiogenesis was used to calculate ID50 values of the tested compounds and the densitometry of the band intensities performed in case of RT-PCRs or CT values were used in case of qPCR to calculate the ID50 values.
Thus the above sequence of steps were used to study the effects of known chemicals 5- Fluro uracil, Penicillin G and caffeine on the fibroblast, hES and hEBs as can be seen in the figures. The effects were compared with control embryoid bodies, which were not treated with the drug.
EXAMPLE 1: PREPARATION OF TEST MATERIALS
a) Preparation of test solutions
Cells were treated with log doses of the compounds. 5-flurouracil (5FU) was treated at a concentration of 0.0001-1 μg/ml, Caffeine at a concentration of 0.1-500 μg/ml where as penicillin G at a dose of 0.1- 5000 p-g/ml. These doses were selected based on the previously published reports (Genschow et al, 2000). Caffeine and penicillin G was dissolved in media where as 5FU was dissolved in DMSO and further diluted in media.
b) Preparation of cell suspension
Undifferentiated ES cells, ReliCell®hESl, were cultured on feeder layer of primary mouse embryonic fibroblast on 0.1% gelatin coated petri plates in high glucose
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(4.5g/l) DMEM supplemented with 10% FCS, 5% KnockOut Serum, 2mM glutamine, penicillin-streptomycin, NEAA, 0-ME, and hLIF as described earlier (Mandal et al., 2006). Human foreskin fibroblast (HFF, SCRC-1042) was obtained from ATCC and was maintained in DMEM with 10% serum. Cultures were maintained at 37°C under 5% CO2 and 95% humidity and were routinely passaged every 3 days.
EXAMPLE 2: Method for performing the embryotoxicity assay
a) For test on the human foreskin fibroblast (HFF):
HFF, fibroblast (Human Foreskin Fibroblast) were trypsinized and a cell suspension of lxl04cells/ml in routine culture medium was prepared. Using a multi-channel pipette, dispense 50 μl volumes of the cell suspension of 1x10" cells/ml (= 500 cells/well). Viability of the cells can be checked by staining an aliquot of the cell suspension with trypan blue. A viability of >90% is acceptable. Incubate the cells for 2hrs in a humidified atmosphere with 5% CO2 at 37°C. This incubation period allows adherence of cells.
After 15 days of culture with medium changes (containing appropriate test compound concentrations) on every 3rd day, the viability of the cells was determined using MTT test, which was then detected quantitatively using a microplate ELISA reader at 570nm with a 630nm reference filter. The percent viability at each test concentration was expressed based on the absorbance, where controls absorbance was considered as 100% viable cells and 50% inhibitory concentration were calculated from the concentration-response curve (IC50 HFF).
b) For test on the human embryonic stem cells:
hES cells are trypsinised and added last, after preparation of test chemicals in medium to avoid prolonged storage outside the incubator. Using a pipette, dispense 20u.l of cell suspension containing the appropriate test chemicals (5000 cells) on the inner side of a 100 mm tissue culture petri dish lid. 50-80 drops are pipetted per lid.
After 2 hrs incubation, add 150 μl assay medium containing the appropriate concentration of test chemical (note that the 150μl vol has to contain 1.333x the final chemical concentration). Appropriate blanks without the chemicals are kept along with the test samples Incubate cell cultures at 5% C02 and 37°C for 3 days. On Day
24

3, test solution was removed with the care that it does not to destroy the cell layer on the bottom of the wells. Add 200 μl freshly prepared test solution (final concentration/well as on day 0). Incubate cell cultures at 5% C02 / 37°C for 3 days. This process was repeated on day 6, 9, 12. Determination of cell growth inhibition was performed at day 15 of the assay using MTT reagent. Cytotoxicity for hES cells was calculated using flow cytometry. Briefly, cells were placed on matrigel coated plates along with different test compound concentrations. Media was replaced on every 3rd day along with the different test compound concentrations. At day 15th, hES cells were collected by trypsinization and after washing with PBS the cells were incubated with PI for 10 minutes in dark. Percent viability was for various test compound concentrations was done using flow cytometry.
d) For test on human embryoid bodies
Hanging drops are made in hES media with LIF and bFGF for calculating ICso-hES cells and in hES media without LIF and bFGF (hEB media) for the formation of EBs. On day 3 the hES colonies were collected and along with the respective drug dilution where as the hEBs were collected and transferred to 60mm bacteriological petriplates for another 2 days. Morphology of the EBs was seen under the microscope for the formation of EBs and recorded. On day 5 the hEBs were plated in a 96 well plate for MTT as well as on a 6 well plate for RNA extraction. The media along with the appropriate drugs concentration was replaced for the 96 well plate, 6 well plate on day 7, 9, 11, and 13. On day 15, MTT was performed on the hES plate as well as the differentiated EBs. RNA was extracted from all the doses of 5-Fluorouracil and lug RNA was converted to cDNA to perform RT-PCR or real time PCR or TLDA.
EXAMPLE 3: ANALYSIS/ RESULTS
1) Morphological evaluation of embryotoxicity
Photomicrographs show the dose dependent effects of 5FU, caffeine and penicillin G on the growth and formation in day 5 old embryoid bodies (fig. 1). Fig 1A shows the dose dependent effects of 5FU on day 5 EBs; there was a significant change in the
morphology of the EBs at a dose of 1 p.g/ml (fig. 1 A6) when compared to the controls (fig. 1 Al). There was a loss of compactness and decrease in the size of the EBs, suggesting that the effects at this dose were detrimental for growth. The hEBs at
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the dose of 1 μg/ml of 5FU did not survive till the day 15 of experiment. Fig IB represents the effects of caffeine in a dose dependent manner. The morphology did not show any major change as in case of 5FU but there was a significant darkening of the cells as the dose increased (fig 1 B5-6). The effects were more prominent at a dose of 500 ng/ml when compared to the lower doses. There was no significant change observed in the penicillin G EBs (fig. 1C) up to 1000 μg/ml dose (fig 1C5). However, there was some non-significant darkening of the EBs at the highest dose suggesting even penicillin G at a very high dose could cause some toxicity (5000 μg/ml, fig 1C6). The data also suggest that morphology evaluations at an early stage (day 5) of the treatment could give some indications of the effect of different compounds that are utilized enabling to understand the toxic effects of different compounds.
2) Cytotoxicity effects of various compounds on hES, HFF and hEBS
Figure 2A represents the dose dependent effects of 5FU on three cell types, HFF, hES cells and hEBs. The results suggest that there is a significant decrease in the cell viability of all the three cell types following treatment with 5FU. At a dose of 1 μg/ml of 5FU, the survival percentage decreased to 7 % in hEBs and HFF cells where as to 30% in case of hES cells. There was no major effect of 5FU in HFF cells and hES up to 0.01 ug/ml, however there was a significant effect on hEBs cells at this dose, suggesting that the compound was more toxic to the differentiating cell rather than the hES cells. The graph (fig 2B) shows the effects of caffeine in a log dose response. The highest dose of 500 (μg/ml of caffeine caused a significant decrease in the survival of all the three cell types to about 10%. hEBs seems to be more vulnerable to toxic effects of caffeine rather than hES cells and HFF cells. A dose of 50 μg/ml caused a significant decrease in cell survival percentage when compared to controls (fig 2B). Effects of penicillin G on the survival patterns in a doses dependent manner are presented in fig 2C. All the three cell lines showed a statistically significant decrease in cell survival at a dose of 5000 fig/ml. There was a 30% cell survival in hEBs where as there was 1-2 % survival in hES and HFF cells at the highest dose. hEBs showed more cytotoxic effects at a dose of 1000 μg/ml when compared to hES cells and HFF suggesting the fact that differentiating cells are more susceptible to toxic effects than hES and HFF cells.
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3) Effects of the various drugs on the gene expression profile of hEBs
Fig 3 A represents the effects of the 5FU on gene expression profiles of the EBs. The genes were selected in such a way that they could provide indication on the organogenesis of major organs from each lineage i.e., ectoderm, mesoderm and endoderm. In the highest dose (1 μg/ml) of 5FU cells did not survive till day 15 for the analysis of gene expression as the dose was highly toxic and hence could not be used for the study. There was a significant decrease in the NFH levels (60% at 0.01p.g/ml) suggesting toxicity of the compound to the neuronal lineage (Fig 3B). Down regulation of gene expression too was observed in AFP (80% at 0.1 μg/ml) and
albumin (40% at 0.001|ig/ml) levels following treatment with 5FU showing its effects on endodermal development and liver function. Changes too were observed in nanog and Msxl expression (25% and 15% down regulation at 0.lug/ml respectively). However, there was no change observed in the gene expression levels of c-actin, keratin, CD34, BMP 4 and BMP 5 gene expression levels (Fig 3). Fig 4A shows the gene expression profile of day 15 EBs following treatment with caffeine. There was a significant down regulation of various genes at a dose of 500 μg/ml of caffeine. NFH expression decreased by 80 % at a dose of 50 μg/ml, keratin by 85 % at a dose of 50 jig/ml, c-actin by 40% at 500 μg/ml and AFP 80 % at a dose of 50 μg/ml (fig 4B). However there was no significant change observed in expression levels of other markers like nanog, CD34, BMP4, Msxl and BMP5. Fig 5A depicts the toxic effects of penicillin G on various tissue specific markers. There was no significant change observed in any of the marker of ectoderm, mesoderm and endoderm except a significant decrease in AFP levels and albumin levels. The AFP levels decreased to 40 % at a dose as low as 100 μg/ml where as albumin levels decreased at a dose of 10ng/ml. There was a complete loss of expression of AFP and albumin at the highest dose of penicillin G, suggesting its toxic effects is more prominent in the endoderm lineage than the other two lineages (fig 5B).
RNA extraction and RT-PCR. RNA was extracted from different drug treated groups
using the trizol method. One microgram of RNA was converted to cDNA using superscript reverse transcriptase. PCR was performed with the initial denaturation cycle at 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, annealing
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temperature varying (table 1) for 30sec, 72°C for 1 min, followed final extension of 72°C for 5 min. Electrophoresis was performed on 1.5 % agarose gels.
Statistical analysis. Paired comparisons were conducted using a paired t test, and all data are presented as mean values ± S.E. Differences were considered significant at a 0.05 level of confidence.
TABLE 1: List of primers used in the study with their respective annealing temperature, product size and gene accession numbers.

NAME OF THE GENE PRIMER SEQUENCE Annealingtemperature(Deg C) Product size (bps) AccessionNo
GAPDH 5' -TGAAGGTCGG AGTC AACGG ATTTGGT-3' 5 '-C ATGTGGGCCATGAGGTCCACCAC-3' 60 890 J 04038
Nanog 5' -CCTCCTCC ATGGATCTGCTTATTC A-3' 5 '-C AGGTCTTC ACCTGTTTGTAGCTGAG-3' 52 262 AB 093576
NFH 5'-TGAACACAGACGCTATGCGCTCAG-3' 5'-CACCTTTATGTGAGTGGACACAGAG-3' 58 400 X 15307
Keratin 15 5'- GGAGGTGGAAGCCGAAGTAT-3' 5'- GAGAGGAGACCACCATCGCC-3' 58 194 X 07696
Cardiac Actin 5 '-TCTATGAGGGCTACGCTTTG-3' 5' -CCTGACTGGAAGGTAG ATGG-3' 50 630 NM 005159
Msx 1 5'- CCTTCCCTTTAACCCTCACAC-3' 5'- CCGATTTCTCTGCGCTTTTC-3' 62 285 BC067353
CD 34 5'- TGAAGCCTAGCCTGTCACCT-3' 5'- CGCACAGCTGGAGGTCTTAT-3' 60 200 BC 039146
AFP 5' - AG AACCTGTC AC AAGCTGTG-3' 5'-GACAGCAAGCTGAGGATGTC-3' 50 680 J 000077
Albumin 5' -CCTTTGGCAC AATGAAGTGGGTAACC-3' 5'-CAGCAGTCAGCCATTTCACCATAGG-3' 58 450 M12523
BMP 4 5'- ACCTGAGACGGGGAAGAAAA-3' 5'- TTAAAGAGGAAACGAAAAGCA-3' 55 348 NM 130850
BMP 5 5'- AAGAGGACAAGAAGGACTAAAAATAT-3' 5'- GTAGAGATCCAGCATAAAGAGAGGT-3' 55 303 M 60314
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EXAMPLE 4:Analysis for finding the dose range
For calculating ICso-hES and ID50-hES cells, Use one petri dish per concentration of the test chemical as well as for the untreated control (= assay medium) and the solvent control. Turn lid carefully into its regular position and put on top of the petri dish
filled with 5 ml PBS. Incubate the "hanging drops" for 3 days in a humidified atmosphere with 5% C02 at 37°C. The changes in the gene expression in presence or
absence of the drug are compared. Changes in mesodermal marker representative of cardiogenesis was used to calculate ID50 values of the tested compounds and the densitometry of the band intensities performed in case of RT-PCRs or CT values were used in case of qPCR to calculate the ID50 values.
Based on the log dose responses of all the three drugs, inhibitory concentrations (IC50) were calculated and are represented in table 2 The data suggest that 5FU shows the lowest IC50 values when compared to the other two compounds. The data also suggest that hEBs are more susceptible to toxic effects when compared to the HFF and hES cell toxicity. Based on the significant changes ID50 (inhibitory concentration at which 50% differentiation of cells is inhibited) were calculated and it was observed that ID50-NFH was 0.00289 μg/ml, ID50-AFP was 0.0524 ug/ml where as ID50-Albumin was 0.000814 ug/ml, suggesting that albumin levels are affected more prominently followed NFH levels and AFP levels. The ID50 values were calculated for the most significant changes in gene expression and were IDS0-NFH was 17.38 μg/ml, ID50-Keratin was 17.08 μg/ml, 1D50-AFP was 1.28 μg/ml and ID50-Albumin was 57.30 μg/ml, suggesting that caffeine showed maximum inhibition of AFP expression followed by NFH and keratin and albumin. AFP and albumin ID50 were calculated to be 84.11|a.g/ml and 28.11 μg/ml respectively, suggesting that both the endodermal markers showed toxicity to penicillin G.
TABLE 2: Inhibitory concentration for cytotoxicity (IC50) on the three cell types (HFF, hES and hEBs) following treatment with 5FU, caffeine and penicillin G.

SNo Compound IC50-HFF IC50-hES IC50-hEB
1 5-Flourouracil 0.1103 0.546 0.001136
2 Caffeine 79.259 115.4 81.629
3 Penicillin G 1660.74 2067.65 1300.22
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Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, are within the scope of the invention.
Dated this 6th day of March 2007
For Reliance Life Sciences Pvt. Ltd.
K. V. Subramaniam President

ABSTRACT
The present invention provides methods useful for high throughput screening of potential drug candidates and is able to predict the effects on the molecular or cellular level of human body using human embryonic stem cells ReliCell®hES of Indian origin.. The method disclosed in the present invention thus correlates well with the animal preclinical toxicity studies done in a clinical trial setup.