WO2018157004A1

WO2018157004A1 - Methods for determining the incidence and/or intensity of effects of a chemical or biological agent on members of a target population using stem cells

Info

Publication number: WO2018157004A1
Application number: PCT/US2018/019590
Authority: WO
Inventors: Kevin P. Coyne; Shawn T. Coyne; Bernard Fermini
Original assignee: COYNE SCIENTIFIC LLC
Current assignee: COYNE SCIENTIFIC LLC
Priority date: 2017-02-24
Filing date: 2018-02-24
Publication date: 2018-08-30
Anticipated expiration: 2019-08-24

Abstract

Methods for determining the beneficial or detrimental effects, or incidence or severity thereof, of exposure of a target population to a biological or chemical agent in vivo, based on in vitro information from a test population. A test population exposed to the agent of interest in vivo and for which PK/PD data may be, or will become available is identified. An in vitro culture of hiPSDCs mimicking the genetic diversity of the test population are exposed to the chemical or biological agent of interest and results measured. Similar hiPSDCs from the target population for which one is seeking to predict the outcome of treatment are exposed to the same chemical or biological agent of interest and results measured, then results from both cultures are recorded as one or more endpoints. These results are analyzed to determine the impact of the chemical or biological agent of interest on the target population."

Description

METHODS OF DETERMINING THE EFFECTS OF A CHEMICAL OR BIOLOGICAL

AGENT ON MEMBERS OF A TARGET POPULATION

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/463,343, filed on February 24, 2017, which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] In a variety of scientific fields including environmental health, medicine, and the study of infectious diseases, there is a pressing need to develop in vitro tools that enable a better prediction of potential adverse effects of chemical or biological agents such as small and large molecules, biological products, and environmental chemicals, in a given target population prior to its exposure to such agents and products. The effects following exposure may be measured in terms of incidence, magnitude and/or distribution of the

pharmacokinetics (PK) and pharmacodynamic (PD) responses to a given agent or product within the population studied.

[0003] In some instances, safety and toxicology data for a given agent or product may be available in one population and, while one may be tempted to "extrapolate" directly the outcome observed in that population to another (e.g. the application of an adult dosing regimen to children), there is evidence suggesting that this may not always be applicable. For example, neonates have significant differences in the physiology affecting drug absorption, distribution, metabolism, and elimination making extrapolation of dosage from adults or older children inappropriate. Moreover, an important role has been observed for ethnicity in some cases of serious or severe adverse reactions to various agents (e.g. the incidence of drug-induced Stevens-Johnson syndrome in Han Chinese vs. Caucasian).

[0004] Consequently, simply extrapolating PK and PD results across different populations can be inaccurate and greatly reduce the ability of researchers, scientists or health professionals to correctly predict the risk for individuals within each population, and/or for the population as whole. [0005] Thus, the development of alternative in vitro methods that enable a better prediction of the potential impact of chemical or biological agents on a target population using data obtained for the same agent, but studied in a separate population, could contribute significantly to our understanding of the safety of these chemicals and biological products. This approach could also support reducing the time for development and approval of new or repurposed drugs, while improving safety and availability to patients in need.

SUMMARY

[0006] Methods are provided herein for estimating, or determining, the beneficial or detrimental effects of exposure of members of a target population to one or more chemical or biological agents. In accordance with the method, a population that has been, or can be, exposed to the agent of interest in vivo (the "test" population) and for which PK/PD data may be, or will become, available is identified and in vivo results are obtained. An in vitro culture of human induced pluripotent stem cell-derived differentiated cells (hiPSDCs) is derived from this test population (mimicking its genetic diversity). The hiPSDCs from the test population are exposed to the chemical or biological agent of interest and results measured and recorded as one or more endpoint. Similar hiPSDCs from a population for which one is seeking to determine, predict or estimate the outcome of treatment (the "target" population) are exposed to the same chemical or biological agent of interest and results measured and recorded as one or more endpoints. These results, or endpoints, are then analyzed in various combinations to determine, predict or estimate the impact or potential impact, or effect, of the chemical or biological agent of interest on the target population.

[0007] More specifically, the results for in vitro exposure of both the test and target populations to the chemical or biological agents of interest are determined along with the in vivo results observed when the test population is or was exposed to the chemical or biological agent in order to determine the effect of exposure of the target population to the chemical or biological agent in vivo.

[0008] Similarly, the results for in vivo and in vitro exposure of the test population to the chemical or biological agents of interest are determined along with the in vitro results observed when the target population is exposed to the chemical or biological agent in order to determine the effect of exposure of the target population to the chemical or biological agent in vivo. [0009] Exemplary populations to be compared include adult test populations and pediatric target populations, US test populations and non-US target populations, non-US test populations and US target populations, and an infected test population and two or more ethnically distinct non-infected target populations.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a schematic matrix showing values for in vivo exposure, in vitro exposure, a test population and a target population. A comparison of the results obtained from B to D are applied to A to determine or estimate C.

[0011] Figure 2 is a schematic matrix showing values for in vivo exposure, in vitro exposure, a test population and a target population. A comparison of results obtained from B to A are be applied to D to determine or estimate C.

[0012] Figure 3 is a bar graph with prophetic data illustrating the variability in effects in two distinct populations (Alpha and Beta) to the same compound when tested in vitro.

DETAILED DESCRIPTION

[0013] Titles or subtitles may be used in the specification for the convenience of a reader, which are not intended to influence the scope of the present invention.

[0014] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Thus, the endpoint of one range is combinable with the endpoint of another range. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 samples refers to groups having 1, 2, or 3 samples. Similarly, a group having 1-5 samples refers to groups having 1, 2, 3, 4, or 5 samples, and so forth.

Definitions

[0015] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0016] As used herein, the terms "a", "an", and "the" can refer to one or more unless specifically noted otherwise.

[0017] The use of the term "or" is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" can mean at least a second or more.

[0018] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a test result, the method being employed to determine the value, or the variation that exists among samples. It is to be understood, although not always explicitly stated, that all numerical designations may be preceded by the term "about."

[0019] The term "comprising" or "comprises" is intended to mean that the methods include the recited elements, but not excluding others. "Consisting essentially of when used to define methods, shall mean excluding other elements that materially affect the method. For example, a method consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed method. "Consisting of shall mean excluding more than trace amount of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0020] Pharmacokinetics, sometimes described as what the body does to a drug, refers to the movement of drug into, through, and out of the body— the time course of

its absorption, bioavailability, distribution, metabolism, and excretion. Drug

pharmacokinetics determines the onset, duration, and intensity of a drug's effect. [0021] Pharmacodynamics, described as what a drug does to the body, involves receptor binding, post-receptor effects, and chemical interactions.

[0022] The term "human induced pluripotent stem cell-derived differentiated cells" and its abbreviation "hiPSDCs" are defined as either iPSCs that have been obtained and replicated from a member of either the test population or the target population, or any functionally differentiated cells (such as, but not limited to: cardiomyocytes, hepatocytes, neurons, respiratory epithelial cells, etc.) derived from such cells, as well as any tissues or organs that consist entirely or partially of such cells.

[0023] The term "exposed" is defined herein to include all instances of one or more exposures, or combinations with, to include the chemical and biological agents described above, whether or not each of the incidences of exposure consists of the same concentration or dose, and/or of the same duration.

[0024] "Test population" and "target population" may be a human population or sub- population of any definition (e.g. the US population, victims of Huntington's disease, Caucasian females between 20 and 50 years old, etc.), or any defined mammalian population. "Optional" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0025] "Profile" refers to any quantitative measurement of the collective responses of a population, or a sample of a population. The term includes, but is not limited to such measures as: mean, median, range, binned distribution of responses, other measures of the distribution of responses, maximums, minimums, and any measures of values at chosen percentiles.

[0026] The term "intensity" is defined herein to relate to a general degree of strength or effect, such as the intensity, or strength of a beneficial or adverse effect when a sample is exposed to a chemical or biological agent. The term "severity" is defined herein to mean the degree of something undesirable, bad, negative or serious and generally refers to a negative effect such as a side effect that is detrimental to the survival, health or well-being of a sample or individual. The term "intensity/severity" includes the altematives, intensity or severity, or includes both intensity and severity, depending on the context.

Methods to identify populations having different responses to agents

[0027] Methods are provided herein to determine the beneficial or detrimental effects, or incidence or severity thereof, of exposure of a target population to a biological or chemical agent based on information available on a test population. In accordance with the methods, a population that has been, or can be, exposed to the agent of interest in vivo (the "test" population) and for which PK/PD data may be, or will become available is identified. An in vitro culture, or cultures, of hiPSDCs is derived from a representative sample of donors, or members, from this test population (mimicking its genetic diversity). The hiPSDCs from the test population are exposed to the chemical or biological agent of interest and results measured and recorded for one or more endpoints for each member of the sample. Similar hiPSDCs from a representative sample of donors or members from the population for which one is seeking to predict or estimate the outcome of treatment (the "target" population) are exposed to the same chemical or biological agent of interest and results measured and recorded for one or more endpoints for each member of the samples. These results are then analyzed in various combinations to determine or estimate the impact of the chemical or biological agent of interest on the target population.

[0028] The present method provides information concerning the likely distribution of responses by the member of the target population to a chemical or biological agent {in vivo) by making comparisons among the three sets of endpoints to the agent of interest— i.e. 1) the test population's PK/PD profile based on in vivo results, effects, measurements or observations; 2) the outcome of in vitro testing using hiPSDCs derived from a sample of donors representing the "test" population; and 3) the outcome of in vitro testing using hiPSDCs derived from a sample of donors representing the "target" population.

[0029] In one embodiment, the results for in vitro exposure of both the test and target populations to the chemical or biological agents are determined along with the in vivo results observed when the test population is exposed to the chemical or biological agent in order to determine, estimate or predict the effect of exposure of the target population to the chemical or biological agent in vivo. [0030] In another embodiment, the results for in vivo and in vitro exposure of the test population to the chemical or biological agents are determined along with the in vitro results observed when the target population is exposed to the chemical or biological agent in order to determine the effect of exposure of the target population to the chemical or biological agent in vivo.

[0031] Exemplary populations to be compared include adult test populations and pediatric target populations, US test populations and non-US target populations, non-US test populations and US target populations, and an infected test population and two or more ethnically distinct non-infected target populations.

[0032] The present method addresses the above mentioned need, by: (1) selecting a population that has been, or can be, exposed to the agent of interest in vivo (the "test" population) and for which PK/PD data may be, or will become available, and establishing an in vitro culture of hiPSDCs from this test population (mimicking its genetic diversity); (2) exposing to the chemical or biological agent of interest, the hiPSDCs from a sample of donors representing the test population, and; (3) exposing to the chemical or biological agent of interest, similar hiPSDCs from a sample of donors representing the population for which one is seeking to predict the outcome of treatment (the "target" population). These results are then analyzed in various combinations to produce estimates of the impact of the chemical or biological agent of interest on the target population.

[0033] The present method enables inferences to be drawn about the likely distribution of the member of the target population to an agent by making comparisons among the three sets of endpoints to the agent of interest— i.e. the test population's PK/PD profile based on in vivo results, effects, measurements or observations; the outcome of in vitro testing using hiPSDCs derived from the "test" population; and the outcome of in vitro testing using hiPSDCs derived from the "target" population.

[0034] Figures 1 and 2 illustrate the potential logical patterns that can be used for such inference. In both figures, quantitative profiles associated with an effect of interest can be empirically established for A, B and D, while the quantity being estimated is C. The quantities in the Figures are:

[0035] A: the PK/PD profile of the test population [0036] B: the empirical results obtained from in vitro testing hiPSDCs from the test population.

[0037] C: the projected PK/PD profile of the target population for which we are seeking to predict the outcome of treatment.

[0038] D: the empirical results obtained from in vitro testing hiPSDCs derived from the target population.

[0039] In Figure 1, a comparison of the results obtained from B to D allow the researcher or scientist to develop a relative relationship (through any statistical or graphic method of the researcher's choosing), that can then be applied to A to determine or estimate C.

[0040] In Figure 2, a comparison of the results obtained from B to A allow the researcher to develop a relative relationship (through any statistical or graphic method of the researcher's choosing), that can then be applied to D to determine or estimate C.

[0041] The present invention also includes any combination of the methods illustrated in Figures 1 and 2.

[0042] Figure 3 provides a representative example illustrating the variability in effects in two distinct populations (Alpha and Beta) to the same compound when tested in vitro. Thus, it is illustrative of the B to D relationship described in Figure 1.

[0043] Investigators may choose to base his/her conclusions as to the relationship between the populations on any statistical, mathematical or graphical comparison of the results of the experiments such as presented in Figure 3, where any or all of the following could be used: comparisons of each population's mean mode or medians; comparisons any percentile of results (e.g. the 80th percentile) of the two populations; a notation that 44 percent of the observations for levels of effects on Alpha fall substantially below the lowest observed level of effect for any member of Beta. This list is illustrative, and is not intended to be comprehensive.

[0044] Various samples can be collected from subjects and are not particularly limited. Samples can include, but are not limited to, cells (e.g. skin cells or oral swabs), tissues (e.g., biopsied tissue such as tumors), blood (e.g. cord blood), hair or nails, sputum, mucosal and other bodily secretions, etc. Samples may be obtained by technicians (e.g. phlebotomist) by any method known in the art, and can be collected via, for instance, donation banks, hospitals/clinics, or post-mortem. Samples are collected and stored under conditions to maintain the integrity of the sample and avoid contamination (e.g. in sterile cryo-tubes).

[0045] Generally, certain attributes of population studies depends in part on the number of objects (n) used in the study. Generally speaking, as the number (n) increases, so does the accuracy, statistical significance, and repeatability of the study. In some embodiments, the number of samples to be collected and analyzed in the herein described methods is preferably more than about 10, 20, 25, 100 or 300. The maximum total number of samples included in any disclosed method is not limited. The number of samples selected for use in the disclosed methods may be less than the total number of samples collected from subjects. The total number of samples included in any disclosed method depends in part on factors such as sample availability and integrity, and the selection criteria for populations and

subpopulations. In the method provided herein, the preferred number of objects (or subjects) from whom samples are taken are at least six.

[0046] In some embodiments, samples can include cells which can be reprogrammed in vitro to induced pluripotent stem cells (iPSCs). In some embodiments, a portion of the donated sample from a subject is reprogrammed to iPSCs, and the iPSCs are subjected to the methods disclosed herein. Where desirable, reprogrammed iPSCs may be differentiated into functional cells. In some embodiments a portion of the functional cells derived from iPSCs are subjected to the methods disclosed herein.

[0047] The agent can be any biological or chemical agent which can be exposed to a biological sample collected from a subject. An agent may be a combination of multiple component agents, such as two or more pharmaceutical compounds. Numerous agents may be of interest and hence, are not particularly limited. Non-limiting examples of agents include biological agents such as antibodies, proteins, lipids and glycolipids, steroids, hormones, neurotransmitters, viruses, viral vectors, bacteria, liposomes, biological extracts such as plant extracts, and chemical agents such as small molecules, carbon-based molecules, synthetic and derivative molecules, drugs such as therapeutic drugs, and a wide range of other agents. For example, certain therapeutic and/or pharmaceutical compounds may be of interest to a researcher for a particular situation, but a different set of therapeutic and/or pharmaceutical compounds may be of interest in a different situation. As an example, in situations in which it is desirable to investigate which agents are effective to treat radiation exposure, the agent can be an FDA-approved or experimental drug administrable to treat radiation exposure. In situations in which it is desirable to investigate which agents are effective to treat a condition but avoid cardiotoxicity, the agent can be an FDA-approved or experimental drug administrable to treat that condition that is evaluated for cardiotoxic effects in the disclosed methods.

[0048] Samples can be exposed to one or more agents under a wide array of conditions. Some conditions which can, but not necessarily need to, influence observable responses of a sample to exposure to an agent include duration of exposure, incubation temperature, agent concentration, amount of sample, agent activation state, presence of additional factors (e.g. co-factors, substrates, enzymes, etc.), condition of samples (e.g. clumped vs. dispersed cells), and other variables. Preferably, the agent is exposed to samples for a time sufficient for a response to be observed and recorded.

[0049] Samples exposed to an agent can be assayed in numerous ways known to those of skill in the art. Assays can be designed to control for a particular condition, e.g. agent concentration. In some embodiments, the agent is exposed to replicates of samples in an array. For instance, various concentrations of the agent may be exposed to numerous sample replicates in a population study. More than one agent can be included in an array. As an example, samples can be exposed to a constant concentration of a first agent and increasing (or decreasing) concentrations of a second agent. Additional variables can be simultaneously tested in the same array. For instance, samples exposed to a constant concentration of a first agent and increasing (or decreasing) concentrations of a second agent can additionally be incubated at varying temperatures. Further, the methods can include samples from numerous populations and subpopulations. Thus, two or more subpopulations can be exposed to the agent (or agents) and assayed (under one or more conditions).

[0050] Methods disclosed herein can identify populations having different responses to agents. As used herein, a "response" or "reaction" refers to any observable change which occurs after exposing the agent to the population samples. The change can be in the type of response, magnitude of response, time of response or combination thereof. Examples of responses, which are changes in type, include cells that normally produce one protein but produce another upon exposure to the agent, and living cells that die upon exposure to the agent. Examples of responses, which are changes in magnitude, include cells that produce a protein but produce less upon exposure to the agent, as well as a percentage of cells that continue to live upon exposure to the agent. In some embodiments, sub-populations, which are compared in the disclosed methods, can demonstrate a substantially similar response, e.g. not different to a statistically significant degree. In some embodiments, sub-populations, which are compared in the disclosed methods, can demonstrate a statistically significant different response.

[0051] Responses by an individual member of a population observed in the methods should be evaluated, quantitatively or qualitatively, to develop meaningful conclusions based on the results of the methods. Likewise, responses across members of a population or across the representative samples of the population should be combined in ways that elucidate their similarities and differences. Quantitation of responses typically depends on the components involved and the nature of the exposure, as understood by one of skill in the art. For instance, agent toxicity to cell samples can be measured in various live/dead stains, metabolic assays, visualization and scoring (e.g. microscopy), and/or other experimental procedures. Endpoints of a response represent a means to compare the results of one sample/agent exposure to the results of another sample/agent exposure. Like selection of quantitation methods, selection of endpoints of a response typically depend on the nature of the study and the selected quantitation method. For instance, an endpoint of a response, as it relates to e.g. cellular toxicity, may be the percentage of metabolically active cells remaining after exposure to the agent for a period of one minute. As another example, an endpoint of a response, as it relates to e.g. cellular toxicity, may be the duration of exposure after which no observable change in metabolic activity occurs. While the former example can be evaluated using a single data point obtained at one minute post-exposure, the latter example can be evaluated using numerous data points over time, or alternatively, selecting a single data point from the numerous data points as representative of the observed response. Other means of quantitation such as averages or other derivative data are also anticipated.

[0052] Data derived from samples can be used in statistical analysis of population studies. For instance, endpoints obtained from samples exposed to an agent in an array can be analyzed as a distribution of endpoints. Distributions, inclusive of averages, means, trend lines, and other methods to compare bulk data, permit the identification of population trends, tendencies, and other characteristics. As such, the distribution of endpoints of a first population of samples can be compared to the distribution of endpoints of a second population of samples. Based on said comparison, a method user can determine whether the response of the first population samples to exposure to the agent is statistically different from the response of the second population samples to exposure to the agent.

[0053] The number of populations used in a disclosed method is limited by employed statistical parameters and constraints such as the total available pool of samples from various subjects. Because subjects can include members of the same species, e.g. humans, from anywhere in the world, the pool of potential subjects is extensive. As the actual pool of subjects from which samples are collected increases in number, the array of selectable subpopulations increases in number and diversity. Thus, although the number of populations used in a disclosed method is generally not limited, such numbers can be limited by the number and diversity of subjects in the overall populations from which samples are collected. As such, the methods can comprise at least 2 (e.g. a first and a second population), at least 3 (e.g. a first, a second, and a third population), at least 5, at least 10, or at least 20 populations. The methods can comprise 2, 3, 4, 5, 10, 20, 50, or 100 populations. Further, a subset of populations in a study can be selected for comparison. For instance, numerous populations can be exposed to an agent (or agents) and assayed, yet a method user may opt to compare a first and a second population; a first and a third population; a first, second, and third, population; and so on.

[0054] A user may choose to base conclusions as to the relationship, or differences, between the populations on any statistical, mathematical or graphical comparison of the results of the experiments. Statistical analysis of the data also depends on the nature of the study performed and is inclusive of a wide array of statistical studies, programs, and techniques known to those of skill in the art. Whether a difference is significant, or statistically significant, depends on the parameters selected for the method. In some embodiments, the threshold for statistical significance is determined by a statistical p-value. As non-limiting examples, any or all of the following could be used: comparisons of each population's mean mode or medians; comparisons of any percentile of results (e.g. the 80th percentile) of the compared populations; a notation that 44 percent of the observations for levels of effects on a first population fall substantially below the lowest observed level of effect for any member of a second population, etc. This list is illustrative, and is not intended to be comprehensive. Any applicable technique to evaluate the difference in responses of sub-populations to exposure to an agent may be used, as would be understood by one of skill in the art. Guidance for evaluating the significance of any differences in data can be found, for example, in Navidi, W.C. et al , Elementary Statistics, McGraw-Hill Higher Education (Oct. 2014); and in Lane, D. et al , Online Statistics Education: A Multimedia Course of Study (available online at http://onlinestatbook.com/), each of which are fully incorporated by reference herein.

[0055] Disclosed herein are methods which can be used with an array of materials useful for carrying out any one or more disclosed method. Where a method is disclosed and a number of modifications to the method are discussed, each and every combination and permutation of the method, and the modifications that are possible, are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure. Thus, if there are a variety of additional steps that can be performed, it is understood that each additional step can be performed with any specific method step or combination of method steps of the disclosed methods, and in any order or permutation, unless otherwise indicated, and that each such combination or subset of combinations is specifically contemplated.

[0056] Publications cited herein are hereby specifically incorporated by reference in their entireties and at least for the material for which they are cited.

EXAMPLES

[0057] The examples below are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims.

[0058] Example 1: Pediatric Medicine. A pharmaceutical compound has been widely prescribed over several years for treating various cancers in adults - enough time and usage to obtain significant data on the incidence and severity of cardiotoxic adverse effects, including arrhythmias, which occur in 7 to 10 percent of those taking the compound at therapeutic doses. [0059] The compound has not been used in children, although the pharmaceutical company believes the compound may be beneficial to children suffering from those same cancers. A barrier to using the treatment on children is the potential for a high incidence and/or severity of cardiotoxic side effects. The company and the regulators are reluctant to evaluate the safety of the compound on children in clinical trials unless and until an a priori estimate can be made as to its cardiotoxic effects.

[0060] An in vitro assay is developed and shown to predict changes in heart rate suggesting increased potential for proarrhythmic effects. Cord blood samples are obtained from 24 newborns, and selected to represent the distribution of the US population in terms of race and gender (such as to mimic genetic diversity). Peripheral blood is then obtained from 24 adult donors, also chosen to represent the normal distribution of the US population in terms of race and gender.

[0061] The researcher reprograms each of the samples into iPSCs using a kit

commercially available from Stemgent-Reprocell of Lexington Massachusetts. The resulting iPSCs are then differentiated into cardiomyocytes using technology licensed from the Wisconsin Alumni Research Fund (WARF). These cells are the hiPSDCs to be used in the analysis below.

[0062] The researcher then conducts the assays on the resulting cardiomyocytes (using identical concentrations of the compound on samples derived from both the adults and from the newborns), and separately calculates the distributions of effects (i.e. histograms showing the number of individuals experiencing an effect within each binned concentration range (as shown in Figure 3), on the hiPSDCs that originated from the two populations (adults and newborns).

[0063] Using the calculated results from the hiPSDCs in vitro assay for the sample of adults, and the distribution of effects when the compound was previously administered to adult humans, the researcher develops a mathematical model that describes the relationship between the two distributions: this is an example of the "B to A" type relationship illustrated in Figure 2. The researcher then applies this model to the distribution of results from the hiPSDCs in vitro assays conducted on the samples of cells drawn from cord blood: this step is an example of the "D to C" application in Figure 2 of the B to A transform just developed above. [0064] This analysis predicts that the likelihood of proarrhythmic effects in the newborn population is significantly less than that for adults, at the same dose levels. Based on this analysis, the researcher concludes that the development process for the potential use of the compound should be continued.

[0065] Example 2: Bridging studies. A compound to treat a serious disease has been approved by the FDA for usage in the United States, and has been shown to be efficacious but carries a high incidence of adverse effects. The pharmaceutical manufacturer of the compound desires to market the compound in Japan.

[0066] The Pharmaceuticals and Medical Devices Agency, which regulates the use of pharmaceuticals in Japan, requires that clinical trials and/or so-called "bridging studies" be conducted on persons of Japanese genealogy before the compound of interest can be marketed in Japan. Either process is costly and time consuming. Therefore, the

pharmaceutical company would like to estimate the incidence and severity of adverse effects in the Japanese population prior to incurring such time and expense.

[0067] A researcher obtains information from public and company records as to any cardiac, hepatic and neuronal adverse drug reactions (ADR) associated with the use of that drug. Based on these findings, the researcher identifies relevant in vitro assays that can be conducted on human induced pluripotent stem cell (hiPSC) derived cardiomyocytes, hepatocytes and neurons, and identifies the various endpoints that would result from the conduct of these assays.

[0068] The researcher then collects peripheral blood from representative samples of the US population and the Japanese population, and develops the required hiPSDCs from each of the members of the two samples. The researcher then conducts the various assays on the resulting hiPSDCs, and separately calculates the distributions of effects on the cells that originated from the two populations (US and Japanese).

[0069] Using the calculated results from the two sets of hiPSDCs in vitro assays, the researcher develops a separate mathematical formulation for each endpoint that describes the relationship between the two distributions: this is an example of the "B to D" type relationship illustrated in Figure 1. [0070] The researcher observes that for every endpoint, the two distributions are remarkably similar, in terms of median, mode and 90th percentile observation.

[0071] Given that the drug has been approved for use in the US, and that the B to D relationships are similar, the researcher concludes that the likelihood of success of a clinical trial on Japanese patients for the purpose of assessing safety is relatively high, and recommends that the company proceed.

[0072] Example 3: First in Human Trials. A common practice of the pharmaceutical industry is to conduct so-called "first in human" trials in countries outside of the United States. However, later stage trials must be conducted on samples of the US population to obtain FDA approval to market the drug in the United States.

[0073] Therefore, a pharmaceutical company that has conducted first in human (FIH) trials on a particular compound in China desires to understand whether similar adverse effects as those observed in the China-based trial can be expected in participants in a US-based FIH trial.

[0074] The company is able to procure peripheral blood samples from all of the participants in the Chinese trial. The researcher then collects peripheral blood from representative samples of the US population, and develops the required hiPSDCs (i.e.

cardiomyocytes, hepatocytes and neurons) from each of the members of the two samples.

[0075] The researcher then proceeds through steps that are similar to those in the "Bridging Studies" example.

[0076] Example 4: The study of infectious diseases. An infectious respiratory disease has begun to spread in one ethnic population in Africa (referred to herein as Population 1). However, thus far, the disease has not spread beyond this one population. Public health officials are seeking information that can help them target their efforts. Specifically, these officials are wondering if they should concentrate all of their limited resources on the affected population (Population 1), or divide their resources by investing some resources in disease prevention for the surrounding, ethnically distinct populations (Populations 2 and 3).

[0077] Central to this issue is whether the surrounding populations are equally, more, or less susceptible to the disease that the population that is already affected. [0078] A researcher obtains peripheral blood from representative samples of all three populations (1, 2 and 3). The researcher develops hiPSC derived pulmonary epithelial cells using the method developed by Huang et al, Nature Biotechnology 32, 84-91 (2014) doi:10.1038/nbt.2754 01 December 2013.

[0079] The researcher then exposes each of the hiPSDCs samples from each member of each of the three sets of donors using techniques well known to those familiar with the field, and measures whether each member becomes infected. The researcher then determines the percentage of the entire sample from each population that become infected, and compares these percentages across the three populations. (This is an example of the "B to D" relationship shown in Figure 1).

[0080] The researcher then combines the "B to D" relationship of Populations 1 and 2, with the actual rate of infection in Population 1 to develop an estimate of the likely infection rate in Population 2 if that population is exposed to the same degree that Population 1 has been. The researcher then repeats this step for Populations 1 and 3.

[0081] The results show that Population 2 is significantly more vulnerable to the disease than Population 3. The health officials use this information to allocate more resources to preventing exposure within Population 2 than Population 3.

Claims

1. A method of determining the effect on members of a target population when exposed to a chemical or biological agent in vivo, comprising: a. obtaining collective information on the incidence and/or intensity/severity of a beneficial or adverse effect on individual members of a test population when exposed to the chemical or biological agent in vivo, b. identifying an in vitro assay comprised of hiPSDCs derived from members of the target population, and/or cells or tissues derived therefrom, wherein the assay measures one or more endpoints of the effect identified in (a), c. obtaining source tissue from a sample of each of at least six members of the test population, and deriving hiPSDCs from those source tissues, d. obtaining source tissue from a sample of each of at least six members of the target population, and deriving hiPSDCs from those source tissues, f. conducting in vitro assays on cells of the samples of (c) by exposing the hiPSDCs to the chemical or biological agent, measuring results and quantifying one or more endpoints for each member of the test population, g. conducting in vitro assays on cells of the samples of (d) by exposing the hiPSDCs to the chemical or biological agent measuring results and quantifying one or more endpoints for each member of the target population, h. comparing the distributions of incidence and/or intensity/severity of results for the in vitro endpoints for the test population in (f) with the distributions of incidence and/or intensity/severity of results for the in vitro endpoints for the target population from (g), and, based on the in vivo information obtained in (a), determining the effect of exposure to the chemical or biological agent on members of the target population in vivo, or i. comparing the in vitro endpoints for the test population in (f) with the in vivo information obtained in (a), and, based on the in vitro endpoints for the target population from (g), determining the effect of exposure to the chemical or biological agent on members of the target population in vivo.

2. The method of Claim 1, wherein the effect is an adverse effect.

3. The method of Claim 1 wherein the effect is a beneficial effect.

4. The method of Claim 1 wherein the incidence of effect is determined.

5. The method of Claim 1 wherein the intensity/severity of effect is determined.

6. The method of Claim 1, further comprising differentiating the hiPSDCs into functional cells prior to conducting the assays.

7. The method of Claim 1, wherein more than one chemical or biological agent is exposed to the samples in the assay.