IN HUMAN AND MINIPIG SKIN, LIVER AND KIDNEY

TECHNICAL FIELD

[0001] The present invention relates to the use of a minipig to model the activity of transporters such as Solute Carrier ("SLC") transporter, ATP Binding Cassette ("ABC") transporter and/or other transporters, substrate of the transporters, and substance that interacts with the transporters in humans, for example, in a pharmacokinetic setting.

BACKGROUND

[0002] A large animal model is needed to study human transporter activities, substrate of the transporters, and substance that interact with the transporters in human. This need has been unfulfilled until now.

BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Figure 1.Erreur ! Source du renvoi introuvable. shows a flow chart of the experiment design.

[0004] Figure 2. shows that the total RNA from human skin in organ culture, hepatocytes in primary culture, and kidney were analyzed by TaqMan Real-time RT-PCR.

[0005] Figure 3. shows that the total RNA from male and female Gottingen ^® minipig skin and liver, and total RNA from female Gottingen ^® minipig kidney were analyzed by TaqMan Real-time RT-PCR.

[0006] Figure 4. Gene expression of ABC transporters. Figure 4 compares the RNA expression profile of ABC transporters in Gottingen ^® minipig and human. The ABC

transporters shown are: ABCB1 (MDR1 ), ABCC1 (MRP1 ), ABCC2 (MRP2), and ABCG2 (BCRP). [0007] Figure 5. Gene expression of SLC transporters. Figure 5 compares the RNA expression profile of SLC transporters in Gottingen ^® minipig and human; the SLC transporters are: SLC22A6 (OAT1 ), SLC22A8 (OAT3), SLC22A1 (OCT1 ), SLC22A2 (OCT2), and

SLC47A1 (MATE1 ) (MATE1 was not cloned in Gottingen ^® minipig).

[0008] Figure 6. shows the constitutive RNA expression of ABC transporters in the skin of Gottingen ^® minipig. ABCC1 (MRP1 ) is the main transporter expressed in the skin of minipig. Constitutive expression of ABCC2 in the skin is very low. Constitutive expression of ABCC1 (MRP1 ) is higher in male than in female.

[0009] Figure 7. shows the constitutive RNA expression of ABC transporters in the liver of Gottingen ^® minipig. All the four ABC transporters are expressed in the liver of minipig, with ABCC2 being the main transporter expressed. Constitutive expression of ABCC2 (MRP2) is higher in female than in male.

[0010] Figure 8. shows the constitutive RNA expression of ABC transporters in the kidney of Gottingen ^® minipig. All the four ABC transporters are expressed in the kidney of minipig, with ABCC2 (MRP2) being the main transporter expressed. Constitutive expression of ABCB1 in the kidney is very low.

[0011] Figure 9. shows the constitutive RNA expression of SLC transporters in the liver of Gottingen ^® minipig. Only SLC22A1 (OCT1 ) is expressed in the liver of minipig. Constitutive expression of SLC22A1 is higher in the female than in the male.

[0012] Figure 10. shows the constitutive RNA expression of SLC transporters in the kidney of Gottingen ^® minipig. All the four SLC transporters are expressed in the kidney of minipig with SLC22A8 (OAT3) being the main transporter expressed.

[0013] Figure 1 1. shows the constitutive RNA expression of CYP in the skin of Gottingen ^® minipig. In the skin, constitutive expression of CYP1 A is higher than the other CYPs.

Constitutive expression of CYP1 A2 is very low in the skin. In the skin, constitutive expression of CYP1A, CYP2B22 and CYP3A29 are lower in female than in male. [0014] Figure 12. shows the constitutive RNA expression of CYP in the liver of Gottingen minipig. In the liver, constitutive expression of CYP1 A2 and CYP3A29 are almost similar in male and female. Constitutive expression of CYP1 A2 and CYP2B22 are higher in female than in male.

[0015] Figure 13. shows the constitutive RNA expression of CYP in the kidney of

Gottingen ^® minipig. In the kidney, constitutive expression of CYP2B22 is higher than the other CYPs. Constitutive expression of CYP1 A2 is very low in the kidney.

[0016] Figure 14. compares the constitutive mRNA expression of SLC04A (OATPE) in human skin, hepatocytes and kidney.

[0017] Figure 15. shows the effect of rifampicin on constitutive mRNA expression of SLC47A 1 (MATE1) and SLC47A2 (MATE2) in human skin.

[0018] Figure 16. shows the effect of rifampicin on constitutive mRNA expression of SLC47A 1 (MATE1) in human hepatocytes.

[0019] Figure 17. shows the effect of UV radiation through a solar simulator on

constitutive mRNA expression of SLC47A 1 (MATE1) and SLC47A2 (MATE2) ex vivo in human skin.

BRIEF DESCRIPTION

[0020] Herein described is the use of a minipig, preferably a Gottingen ^® minipig, as an animal model to evaluate or predict one or more biomedical property(ies), typically

pharmacokinetic property(ies), for example drug-drug interaction(s) or drug penetration (in the subject's body), of one or more drug candidate(s), for example topical drug(s), in a subject (typically in a human being) or in/on a subject's sample (for example human skin).

[0021] Also herein described is the use of one or more minipig transporter(s), preferably a Gottingen ^® minipig transporter(s), to evaluate one or more biomedical property(ies), typically pharmacokinetic property(ies), for example drug-drug interaction(s) or drug penetration (in the subject's body), of one or more drug candidate(s), for example topical drug(s), in a subject (typically in a human being) or in/on a subject's sample (for example pig skin or human skin). The one or more transporter(s), preferably minipig transporter(s), is preferably one or more ABC transporter(s) and/or SLC transporter(s) and/or a combination of ABC transporter(s) and SLC transporter(s). The one or more transporters is preferably selected from the group consisting of MDR1 , MRP1 , MRP2, BCRP, MATE1 , OAT1 , OAT3, OCT1 , OCT2 and a combination thereof. One or more of the transporters can be a biomarker for a skin disease. The skin disease is preferably selected from the group consisting of inflammatory skin disease, viral skin disease, fungal skin disease, bacterial skin disease, cancerous skin disease, and a combination thereof. The cancerous skin disease is preferably selected from the group consisting of actinic keratosis, basal cell carcinoma, melanoma, Kaposi's carcinoma, squamous cell carcinoma, and a combination thereof.

[0022] Further herein described is a method of evaluating one or more drug candidate(s) for the regulation of one or more mammalian transporter(s) as herein described, the method comprising exposing/contacting the one or more drug candidate(s) with mammalian cells, preferably with mammalian cells from a minipig, and obtaining an expression profile of the transporter(s) expressed by the skin cells to determine whether the drug candidate(s) regulate(s) the expression of the mammalian transporter(s).

[0023] Also herein described is a method of evaluating one or more drug candidate(s) as a treatment for cancer, the method comprising exposing/contacting the one or more drug candidate(s) with cancerous cells of a minipig, obtaining an expression profile of one or more transporter(s) as herein described expressed by the cancerous cells, comparing the expression profile with a control expression profile, wherein a difference between the expression profile and control expression profile indicates that the drug(s) is(are) potentially useful for treatment of the cancer. The cancerous cells may be for example cells obtained from the skin, kidney and/or liver of a minipig. The cancer can be a skin cancer and can be selected from the group consisting of actinic keratosis, basal cell carcinoma, melanoma, Kaposi's carcinoma, squamous cell carcinoma, and a combination thereof. The control expression profile is typically obtained from reference cells, preferably from cancerous cells of a minipig that was not contacted by the drug candidate(s).

[0024] Also herein described is a method of using an animal model as herein described, preferably a minipig animal model, even more preferably a Gottingen ^® minipig, to evaluate one or more biomedical property(ies) of one or more drug candidate(s), the method

comprising:

(a) administering or exposing/contacting one or more drug candidate(s) to a first location of the animal model;

(b) administering or exposing/contacting one or more transporter inhibitor(s), preferably inhibitor(s) of the herein described transporter(s), to the first location of the animal model;

(c) administering or exposing/contacting the same drug candidate(s) to a second location of the animal model; and

(d) comparing the amount of drug candidate(s) (collected in) from the first location of the animal model to that (collected in) from the second location of the animal model.

[0025] Also herein described is a method of using an animal model as herein described, preferably a minipig animal model as herein described, even more preferably a Gottingen ^® minipig, to evaluate one or more biomedical property(ies) of one or more drug candidate(s), the method comprising:

(a) administering or exposing/contacting one or more drug candidate(s) to a first animal model, for example to the skin, liver or kidney, or to a sample thereof, of the first animal model;

(b) administering or exposing/contacting one or more transporter inhibitor(s) to the first animal model, for example to the skin, liver or kidney, or to a sample thereof, of the first animal model;

(c) administering or exposing/contacting the same drug candidate(s) to a second animal model; for example to the skin, liver or kidney, or to a sample thereof, of the second animal model, and (d) comparing the amount of drug candidate(s) (collected in) from, for example at the skin, liver or kidney of, the first animal model to that of the second animal model.

[0026] Further herein described is a method of determining an effective dosage of one or more drug candidate(s), the method comprising:

(a) administering or exposing/contacting one or more drug candidate(s) to an animal model as herein described, preferably a minipig animal model as herein described, even more preferably a Gottingen ^® minipig;

(b) measuring an amount of drug candidate(s) (collected in) from the animal model; and

(c) determining the effective dosage of the one or more drug candidate(s) based on predetermined efficacy and toxicity measurements of the one or more drug candidate(s) in the animal model. Also herein described is a method as previously described further comprising an additional step (d) of determining the effective dosage of the one or more drug

candidate(s) in a subject, preferably in a human being, using a coefficient to correlate the effective dosage in the animal model, preferably in the minipig, to that of a human being.

DETAILED DESCRIPTION

Interpretations and Definitions

[0027] Unless otherwise indicated, this description employs conventional chemical, biochemical, molecular biology, genetics, pharmacology, pharmacokinetics, and biomedical methods and terms that have their ordinary meaning to persons of skill in this field. All publications, references, patents and patent applications cited herein are hereby incorporated herein by reference in their entireties.

[0028] As used in this specification and the appended claims, the following general rules apply. Singular forms "a," "an" and "the" include plural references unless the content clearly indicates otherwise. General nomenclature rules for genes and proteins also apply. That is, genes are italicized or underlined (e.g.: MATE1 or MATED, but gene products, such as proteins and peptides, are in standard font, not italicized or underlined (e.g.: MATE1 ).

General nomenclature rules for organism classification also apply. That is kingdom, phylum, class, order, suborder, and family are capitalized and not italicized. Binomial nomenclature are italicized with capitalized genus and lower case species.

[0029] As used herein, the following terms shall have the specified meaning. The term "about" takes on its plain and ordinary meaning of "approximately" as a person of skill in the art would understand. The term "comprise," "comprising," "contain," "containing,"

"include," "including," "include but not limited to," or "characterized by" is inclusive or open-ended and does not exclude additional, unrecited elements.

[0030] As used herein, the following terms shall have the specified meaning:

[0031] "ABC transporters" means ATP binding cassette transporters, are a type of integral membrane protein present in cells that is responsible for the transport of substrates across a membrane. In general, ABC transporters require ATP hydrolysis to actively transport substrate across the membrane.

[0032] "ABCB1 " means ATP-binding cassette sub-family B member 1. See MDR1.

[0033] "ABCC1 " means ATP-binding cassette sub-family C member 1. See MRP1 .

[0034] "ABCC2" means ATP-binding cassette sub-family C member 2. See MRP2.

[0035] "ABCG2" means ATP-binding cassette sub-family G member 2. See BCRP.

[0036] "administering" means introducing a target to a subject by any means, including administering by means of auricular (otic), buccal, conjunctival, cutaneous, dental, electro- osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, inhalational, interstitial, intra-abdominal, intra- amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronal, dental, intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary,

intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, unassigned, unknown, ureteral, urethral, vaginal, and combinations thereof. Contrary to the term "administering", the terms "exposing" and "contacting" do not include or refer to an invasive step, in particular an invasive step carried out on an animal, typically a mammal, in particular a human being.

[0037] "BCRP" means breast cancer resistance protein. It is found in the gastrointestinal tract, liver, kidney, brain, mammary tissues, testes, and the placenta. BCRP is also known as ABCG2 or MXR.

[0038] "biomedical" or "biomedical property" means all the properties of a substance in biomedical science. The substance can be constitutively expressed in the subject. One such example is an protein that is important in a targeted pathway. The substance can also be introduced to the subject. On such example is a therapeutic candidate administered to the subject. Biomedical properties broadly encompasses properties such as pharmacokinetic, pharmacodynamics, biochemical, physiological, efficacy, potency, toxicity, degradation, activation, activity, interaction, and localization, of the substance, whether in physiological or non-physiological conditions.

[0039] "cDNA" means complementary deoxyribonucleic acid.

[0040] "Ct" means cycle threshold or comparative threshold cycle.

[0041] "CYP" means cytochromes or the cytochrome P450 protein. [0042] A "disorder" means an abnormal condition characterized by an identifiable group of signs, symptoms or both, of a part of, a organ of, or a system of an organism that is a result from one or more causes, including an infection, inflammation, environmental factors, and a genetic defect. An example of a disorder is a skin disease.

[0043] "ex vivo" means outside an organism or body. It generally involves working with intact organs or tissues with minimal alterations to mimic in vivo conditions as much as possible.

[0044] "FAM" means 6-carboxyfluorescein. FAM is a fluorescent dye with an absorption wavelength of 495 nm and an emission wavelength of 517 nm.

[0045] "GAPDH" means glyceraldehyde-3-phosphate dehydrogenase.

[0046] "in situ" means outside an organism or body. It generally involves working with intact and living organs or tissues and considered to best mimic in vivo conditions. An example of an in situ experiment is studying cell functions within a whole, intact and living organ under perfusion.

[0047] "in vitro" means outside a living organism or body. It generally involves working with a test tube, culture dish, and nonliving samples.

[0048] "in vivo" means in a living organism or body.

[0049] "KS" means Kaposi's sarcoma.

[0050] "LTC4" means leukotriene-C4.

[0051] A "marker" or "biological marker" means a biological marker associated with the possible presence or with the possible absence of a particular disease, such as cancer. The biological markers can be a particular macromolecule in the subject, including a piece of DNA, such a gene; a piece of RNA, such as a DNA transcript; and a polypeptide, such as a gene product. [0052] "MATE1" means multi-antimicrobial extrusion protein. It is also known as multidrug and toxin extrusion or multidrug and toxic compound extrusion. It is encoded by the

SLC47A 1 gene.

[0053] "MDR" means multidrug resistance.

[0054] "MDR1" means multidrug resistance protein 1. It is found in the intestine, kidney, liver and the brain. MDR1 is also known as P-gp or ABCB1.

[0055] A "minipig" means miniature pig, which is several breeds of pigs developed and used for medicinal research. It is also known as micro pig or teacup pig. One example of a breed of minipig is Gottingen ^® minipig.

[0056] A "modulator" means a substance that causes changes to the target marker. Examples of preferred modulators here include macromolecules such as a small organic molecule; a polynucleotide, DNA, RNA, mRNA, miRNA, siRNA, any other antisense RNA, PNA, polypeptide, protein, antibodies (such as a monoclonal antibody), lectin and peptide; and combinations thereof and the like.

[0057] "mRNA" means messenger ribonucleic acid.

[0058] "MRP" means multidrug resistance associated proteins

[0059] "MRP1" means multidrug resistance associated p_rotein 1. MRP1 is also known as ABCC1.

[0060] "MRP2" means multidrug resistance associated protein 2. MRP2 is also known as ABCC2 or cMOAT.

[0061] "NHEK" means normal human epidermal keratinocytes.

[0062] "OAT" means organic anion transporter. It includes OAT1 (also known as

SLC22A6) and OAT3 (also known as SLC22A8).

[0063] "OCT" means organic cation transporter. It includes OCT1 (also known as

SLC22A1 ) and OCT2 (also known as SLC22A2).

[0064] "PBPK" means physiologically-based pharmacokinetic. [0065] "PBS" means phosphate buffered saline.

[0066] "PCR" means polymerase chain reaction.

[0067] "P-gp" means p-glycoproteins. See MDR1.

[0068] "pharmacodynamics" or "pharmacodynamics property" means the relationship between the concentration of a substance at the site of action and the intensity of the effect of the substance. While the substance is typically a therapeutic, such as a drug, it can also be nutrients, hormones, toxins, pollutants, pesticides, and a combination thereof.

[0069] "pharmacokinetic" or "pharmacokinetic property" means the properties of absorption, distributions, metabolism, and excretion of a substance and the process that governs the concentration of the substance in the body, the different organs and tissues of the body, and/or the fluids of the body. While the substance is typically a therapeutic, such as a drug, it can also be nutrients, hormones, toxins, pollutants, pesticides, and a combination thereof. Pharmacokinetic is crucial to the discovery, development and clinical and preclinical evaluation of a therapeutic as well a clinical use of a therapeutic.

[0070] "rpm" means rotation per minute.

[0071] "RQ" means fold change.

[0072] "RT" means reverse transcription.

[0073] "SEM" means standard error of means.

[0074] A "skin disease" means a disorder of the skin. Some common skin diseases include, inflammatory skin disease, viral skin disease, fungal skin disease, bacterial skin disease, and cancerous skin disease.

[0075] "SLC transporters" means Solute Carrier transporters, are a type of integral membrane protein present in cells responsible for the transport of substrates across a membrane. In general, SLC transporters do not require ATP hydrolysis to transport substrate across the membrane, and use either electrochemical potential or ion gradient to transport substrate across the membrane. [0076] "SLC22A6" means the solute carrier family 22 member 6. See OAT.

[0077] "SLC22A8" means the solute carrier family 22 member 8. See OAT.

[0078] "SLC22A1" means the solute carrier family 22 member 1. See OCT.

[0079] "SLC22A2" means the solute carrier family 22 member 2. See OCT.

[0080] A "subject" can be any organism or a portion of any organism. As used herein, the preferred embodiment of the subject is a mammal, further preferably a minipig, further preferably a human, and further preferably a skin, liver, kidney or combination thereof of a human and/or a minipig.

[0081] A "substrate" means a substance that travels through a transporter. Examples of preferred substrates include a molecule, such as a small organic molecule, an inorganic molecule, and ion; a polynucleotide, DNA, RNA, mRNA, miRNA, siRNA, any other antisense RNA, PNA, polypeptide, protein, antibodies (such as a monoclonal antibody), lectin, peptide, and amino acid; a lipid; a carbohydrate; and combinations thereof and the like.

[0082] A "target" means a composition under inquiry. Examples of preferred targets include a molecule, such as a small organic molecule, an inorganic molecule, and ion; a polynucleotide, DNA, RNA, mRNA, miRNA, siRNA, any other antisense RNA, PNA, polypeptide, protein, antibodies (such as a monoclonal antibody), lectin, peptide, and amino acid; a lipid; a carbohydrate; and combinations thereof and the like.

[0083] "treatment" means the administration and/or application of therapeutic product or method to a patient with a certain disease, and includes, among others, monitoring the efficacy of a type of treatment for the disease.

ABC Transporters

[0084] ABC transporters are a type of integral membrane proteins present in cells. They are responsible for the transport of substrates across a membrane and are presented in all species of Archaea, Eubacteria, and Eukaryota. In general, ABC transporters require ATP hydrolysis to actively transport substrate across the membrane. The human genome encodes 48 ABC transporter genes.

[0085] Substrates of ABC transporters can range from small ions to drugs, lipids, proteins, amino acids, peptides, inorganic ions, among others. In eukaryotes and prokaryotes, ABC transporters generally function as exporters or effluxers that pump toxins and drugs out of the cell. Accordingly, ABC transporters are important in regulating drug pharmacokinetics, can play crucial roles in drug absorption, distribution, metabolism, and excretion, and can affect the pharmacological effect and toxicity of the target drug. To illustrate, it is known that ABC transporter expressed in intestinal epithelial cells and cerebrovascular endothelial cells can have a great influence on the bioavailability and migration of orally administered drugs to the central nervous system. As another example, an overexpression of ABC transporters, such as P-gp and MRP1 , in cancerous cells during chemotherapy increase drug excretion, reduce drug efficacy, and lead to drug tolerance. Some have found that the function of ABC transporters on drug efflux can be independent of the route of administration, whether oral or enteral. It is thought that ABC transporters would function in a viable skin layer (viable epidermis and dermis), but not in stratum corneum that consists of dead cells. However, currently, little is known about the functional role of transport proteins constitutively expressed in the skin, including human skin, as applied to dermatological products, such as topical drugs.

[0086] Nine (9) human transporters are associated to a multi-drug resistance phenotype, due to their ability to extrude out of the cells a large variety of xenobiotics. These transporters include the multidrug resistance protein 1 ("MDR1", also known as P-gp or ABCB1 ), the multidrug resistance associated proteins ("MRPs", namely MRP1 -MRP7, also known as ABCC1 -6 and ABCC10), and breast cancer resistance protein ("BCRP", also known as ABCG2).

[0087] The specific function and distribution of ABC transporters varies. For example, real time PCR showed that MRP1 was the most highly expressed of the drug transporter genes in human skin. (Smith et al., J. Invest. Dermatol. 121 , 390 (2003)). Reports also show MRPs are expressed in normal human epidermal keratinocytes ("NHEKs") (Baron et ai, J. Invest. Dermatol. 116, 541 (2001 )). However, the exact function of MRPs is unclear.

[0088] As another example, the transporters MDR1 and P-gp are in particular widely distributed in the organism and play an important role in absorption regulation, tissue distribution and substrate elimination (Thiebaut et ai, Proc. Natl. Acad. Sci. 84, 7735 (1987)). Immunohistochemistry studies show a strong expression of P-gp (MDR1 ) in various compartments of human skin such as sweat ducts, vessels, nerve-sheaths and muscles, but only moderate expression of P-gp (MDR1 ) in the basal epidermis layer (Shazik et ai, J. Exp. Derm. 20, 450 (201 1 )). More recently, using the same real-time PCR analysis, MDR1 mRNA expression in whole human skin samples was found to be 360-fold higher than that of NHEKs. Strong expression of MDR1 mRNA was especially detected in human dermis (approximately 300-fold higher than in NHEKs), whereas epidermal mRNA expression was only slightly higher (12-fold) than expression in NHEK (Shazik et ai, J. Exp. Derm. 20, 450 (201 1 )).

SLC Transporters

[0089] SLC transporters are another type of integral membrane protein present in cells, that like ABC transporters, are also responsible for the transport of substrates across a membrane. SLC transports can transport an extraordinarily diverse number of solutes, including charged and uncharged organic molecules, as well as inorganic ions. In general, SLC transporters do not require ATP hydrolysis to transport substrate across the membrane, and use either electrochemical potential or ion gradient to transport substrate across the membrane. Accordingly, SLC transporters include facilitative transporters that allow solutes to flow passively with their electrochemical gradients, or be coupled to secondary active transporters to allow solutes to flow against its electrochemical gradient.

[0090] Examples of SLC transporters include, but are not limited to, SLCO1 B1

(OATP1 B1 ), SLCO1 B3 (OATP1 B3), SLC22A1 (OCT1 ), SLC22A2 (OCT2), SLC22A6 (OAT1 ), SLC22A8 (OAT3), SLC02B1 (OATPB), SLC03A1 (OATPD), SLC04A1 (OATPE), SLC47A1 (MATE1 ), and SLC47A2 (MATE2). Among these, MATE has been identified as efflux transporter in liver and kidney for xenobiotics, such as oxaliplatin. Thus, expression levels and pattern of MATE can be relevant for skin cancer treatment, such as actinic keratosis, basal cell carcinoma, melanoma, Kaposi's sarcoma ("KS") and squamous cell carcinoma.

[0091] Most identified drug transporters are either ABC transporters or SLC transporters. Further, recent research indicates that these transporters play an important role in the absorption, distribution and excretion of drugs, and are involved in clinically relevant drug- drug interactions. Some drug transporter-based interactions have been documented in liver and kidney for systemic drugs. However very little is known about the role of drug

transporters in human skin in the disposition of topically applied drugs, especially SLC transporters. For example, the expression profile of SLC transporters is not known in humans, particularly in human skin. This is particularly true for MATE. Thus, there is a need for tools and/or methods to evaluate how drugs, such as topical drugs are absorbed and transported in mammals, particularly in humans, and especially in human skin. The ideal tool and/or method should allow for in vivo, in situ, ex vivo and/or in vitro investigations. The tool and/or method is also expected to greatly advance the discovery of, treatment of, and/or diagnosis of diseases, such as skin diseases.

Minipigs and biomedical research

[0092] Developing animal models for biomedical research is crucial and necessary for the advance of scientific research. A good animal model, for example, possesses inherited, naturally acquired or induced biological processes, which, in one way or another, closely resemble that of a human. Accordingly, important scientific inquiries, including inquiries on pathology, toxicity and pharmacokinetics, can be addressed, in vivo, using the animal model, before they are being explored in humans.

[0093] In the field of pharmacokinetics, for example, physiologically-based

pharmacokinetic ("PBPK") modeling and simulation in animals is critical to understanding the pharmacokinetic processes and allows for better prediction of human pharmacokinetic and/or physiological effects. Through an animal model, researchers can study the pharmacokinetics of a substance. These pharmacokinetic studies can include (1 ) evaluating the properties of absorption, distributions, metabolism, and excretion of a substance; (2) evaluating the process that governs the concentration of the substance in the body, the different organs and tissues of the body, and/or the fluids of the body; (3) designing and evaluating dosage forms; (4) evaluating drug formulations; (5) conducting pharmacological testing; (6) conduct toxicological testing; (7) evaluating organ functions; and (8) designing dosing regimens. (For pharmacokinetics generally, see Hedaya, Basic Pharmacokinetics (2012)). The substance under study can be composition of matter, including nutrients, hormones, toxins, pollutants, pesticides, but is typically a therapeutic, such as a drug. Accordingly, pharmacokinetic along with pharmacodynamics is crucial to the discovery, development, and clinical and preclinical evaluation of a therapeutic, as well as clinical use of a therapeutic.

[0094] Traditionally, mouse, rat, dog and non-human primates have served as animal models for PBPK studies to understand, for example, the design, evaluation and

interpretation of pharmacokinetic, toxicokinetic and formulation of a drug candidate. Recently, there is a growing interest in using minipigs, such as the Gottingen ^® minipigs, for biomedical research. However, no PBPK model for minipig has yet been published. Here, the inventors have provided evidence that minipigs, such as the Gottingen ^® minipigs, have expression profiles similar to that of humans. Moreover, the inventors have described here a novel large animal model and a novel method of using the model for biomedical studies, including pharmacokinetic, drug-drug interaction, and drug-transporter interaction studies.

[0095] Minipigs, such as Gottingen ^® minipig, have been found to share numerous anatomical, physical, genetic, and biochemical similarities with humans. (Suenderhauf et al., Pharm. Res. 30, 1 (2013)). The Gottingen ^® minipig, which was the first European minipig breed, was developed in the 1960s by Dr. Fritz Haring at the Institute of Animal Breeding and Genetics at the University of Gottingen, in Germany. They have been the predominant laboratory pig breed, due to favorable characteristics such as, small size (they weigh about 450 g at birth and 35 kg at adulthood), well defined health status, and strictly managed but not inbred genetics. So far, they have been mainly used for regulatory toxicity testing and some specialized studies, such as cardiovascular, diabetes, orthopaedic, dental and surgical studies. However, due to certain drawbacks, recent studies have not yet found a way for the model to be appropriate for preclinical and pharmaceutical studies. (Suenderhauf et al., Pharm. Res. 30, 1 (2013)). In order to integrate the minipig fully into preclinical research as a large animal model for biomedical research, it is essential to establish reliable physical data for minipigs (Suenderhauf et al., Pharm. Res. 30, 1 (2013)). This includes, for example, understanding the expression profiles of drug transporter in pigs and minipigs, which are not generally known (Suenderhauf et al., Pharm. Res. 30, 1 (2013))

[0096] Here, the inventors have, for the first time, described the constitutive RNA expression of transporters in minipigs, such as the Gottingen ^® minipig (See for example, Example 1 and Figure 4-Figure 10). The inventors have further shown that the minipig constitutive transporters have unique RNA expression profiles, for example in the skin, liver and kidney (See for example, Example 1 and Figure 4-Figure 10). As seen in, for example, Example 1 and Figure 4-Figure 10, the SLC22A1 (OCT1 ) is the predominant SLC transporter expressed in minipig liver, whereas SLC22A8 (OAT3) is the predominant SLC transporter expressed in minipig kidney. Similarly, ABCC1 (MRP1 ) is the predominant ABC transporter expressed in minipig skin, whereas ABCC2 (MRP2) is the predominant ABC transporter expressed in minipig liver and kidney (See for example, Example 1 and Figure 4-Figure 10).

[0097] In addition, the inventors have, for the first time, compared the expression profiles of the minipig to that of humans. Surprisingly, the inventors have found that the constitutive RNA expression profiles of minipig is highly comparable to that of human. (See for example, Example 2 and Figure 4-Figure 5). For example the transporter predominantly expressed in an organ of a minipig is also predominantly expressed in the same organ of a human. This is generally true for all of the transporters and organs tested. (See for example, Example 2 and Figure 4-Figure 5).

[0098] The inventors further have described, here, a novel large animal model, the minipig, and methods of using the same for predications in humans. Importantly, because transporters are crucial for the transport and absorption of drugs and other therapeutics and have significant impact on drug efficacy, toxicity and metabolism, the novel large animal model and methods described here have tremendous applications in biomedical studies, including pharmacokinetic, drug-drug interaction, and drug-transporter interaction studies. Furthermore, because studies can now be conducted in live systems, the model and methods advantageously allows for the evaluation of target behaviors, transporter behaviors, and/or interaction behaviors, in vivo, and/or in situ, in addition to ex vivo, and/or in vitro. The flexibility of this novel minipig model, then allows for more accurate predictions of target behaviors, transporter behaviors, and/or interaction behaviors in human and has significant advanced biomedical and pharmaceutical field.

A novel animal model to evaluate biomedical properties

[0099] The inventors have described a novel animal model to evaluate in vivo, in situ, ex vivo, and/or in vitro one or more biomedical property(ies), preferably one or more

pharmacokinetic property(ies), of one or more target(s), preferably of one or more drug candidate(s), further preferably of one or more target drug(s), and further preferably of one or more topical target drug(s), using a minipig, preferably a Gottingen ^® minipig. The novel animal can advantageously predict one or more biomedical property(ies) of target(s) in a subject, preferably a mammal, further preferably a human, further preferably a human skin, liver, kidney or combination thereof. The preferred biomedical property to be evaluated and/or predicted in a subject can include the absorption, toxicity, metabolism, efficacy of target, or a combination thereof. In a preferred embodiment, the target is a substrate, preferably a substrate for an ABC transporter and/or a SLC transporter, further preferably a substrate for an ABC transporter constitutively expressed on: (a) the skin of a minipig, further preferably an ABC transporter selected from the list of MDR1 , MRP1 , MRP2, and BCRP, and further preferably the MRP1 ; and/or

(b) the liver of a minipig, further preferably an ABC transporter selected from the list of MDR1 , MRP1 , MRP2, and BCRP, and further preferably the MRP2; and/or

(c) the kidney of a minipig, further preferably an ABC transporter selected from the list of MDR1 , MRP1 , MRP2, and BCRP, and further preferably the MRP2.

[00100] In another preferred embodiment, the target is a substrate for a SLC transporter constitutively expressed on:

(a) the skin of a minipig, further preferably a SLC transporter selected from the list of MATE1 , OAT1 , OAT3, OCT1 , and OCT2, and further preferably MATE1 ;

(b) the liver of a minipig, further preferably a SLC transporter selected from the list of MATE1 , OAT1 , OAT3, OCT1 , and OCT2, and further preferably OCT1 ; and/or

(c) the kidney of a minipig, further preferably a SLC transporter selected from the list of MATE1 , OAT1 , OAT3, OCT1 , and OCT2, and further preferably OAT3.

[00101] The novel animal model can also be used to predict optimum dosage of a target in vivo, in situ, ex vivo, and/or in vitro for a subject, preferably a mammal, further preferably a human.

A novel method to evaluate biomedical properties of a target using the minipig model.

[00102] The inventors have described a novel method to evaluate one or more biomedical property(ies), preferably one or more pharmacokinetic property(ies) of one or more target(s), preferably of one or more drug candidate(s), further preferably of one or more target drug(s), and further preferably of one or more topical target drug(s), using the minipig model, preferably the Gottingen ^® minipig. Advantageously, the method can be used to evaluate one or more biomedical property(ies) of target(s) in vivo, in situ, ex vivo, and/or in vitro.

[00103] In a preferred embodiment, the novel method can be used to predict one or more biomedical property(ies) of one or more target(s) in a subject, preferably a mammal, further preferably a human, further preferably a human skin, liver, kidney or a combination thereof. The preferred biomedical property to be evaluated and/or predicted in a subject can include the absorption, toxicity, metabolism, efficacy of target, or a combination thereof. In a preferred embodiment, the target is a substrate, preferably a substrate for an ABC transporter and/or a SLC transporter, further preferably a substrate for an ABC transporter constitutively expressed on:

(a) the skin of a minipig, further preferably an ABC transporter selected from the list of MDR1 , MRP1 , MRP2, BCRP, a combination thereof and the like, and further preferably the MRP1 ; and/or

(b) the liver of a minipig, further preferably an ABC transporter selected from the list of MDR1 , MRP1 , MRP2, BCRP, a combination thereof and the like, and further preferably the MRP2; and/or

(c) the kidney of a minipig, further preferably an ABC transporter selected from the list of MDR1 , MRP1 , MRP2, BCRP, a combination thereof and the like, and further preferably the MRP2.

[00104] In another preferred embodiment, the target is a substrate for a SLC transporter constitutively expressed on:

(a) the skin of a minipig, further preferably a SLC transporter selected from the list of MATE1 , OAT1 , OAT3, OCT1 , OCT2, a combination thereof and the like, and further preferably

MATE1 ;

(b) the liver of a minipig, further preferably a SLC transporter selected from the list of MATE1 , OAT1 , OAT3, OCT1 , OCT2, a combination thereof and the like, and further preferably OCT1 ; and/or

(c) the kidney of a minipig, further preferably a SLC transporter selected from the list of MATE1 , OAT1 , OAT3, OCT1 , OCT2, a combination thereof and the like, and further preferably OAT3.

[00105] In one embodiment, the method includes the steps of:

(a) administering or exposing one or more transporter inhibitor(s) to a first location of a minipig, preferably a Gottingen ^® minipig;

(b) administering or exposing one or more target(s) to the first location of the minipig;

(c) administering or exposing a blank control to a second homologous location of the minipig;

(d) administering or exposing the same target(s) to the second homologous location of the minipig; and

(e) comparing the amount of target(s) (collected in) from the first location of the minipig to that of the second homologous location of the minipig,

wherein an increase of a target in the first location of the minipig as compared to the control is:

(i) indicative that the inhibited transporter is involved in the efflux of the target at the location in the minipig,

(ii) predicative that the same transporter is involved in the efflux of the target at the same location of a human, and/or

(iii) predicative that the target will have low absorption, low toxicity, low efficacy and high metabolic profiles at the same location in a human; and

wherein a decrease of a target in the first location of the minipig as compared to the control is:

(i) indicative that the inhibited transporter is involved in the uptake (or absorption) of the target at the location in the minipig,

(ii) predicative that the same transporter is involved in the uptake of the target at the location in a human, and/or

(iii) predicative that the target will have high absorption, high toxicity, high efficacy and low metabolic profiles at the location at the location in a human.

The first and second locations can be areas/locations on the skin, liver and/or kidney of the minipig.

[00106] In another embodiment, two minipigs are used, using the same general steps as above, wherein one minipig is treated with the inhibitor(s) and the other minipig is treated with the control. [00107] In a preferred embodiment, the transporter is MRP1 and the location is skin; the transporter is MRP2 and the location is liver and/or kidney; the transporter is MATE1 and the location is skin, the transporter is OCT1 and the location is liver; and/or the transporter is an OAT3 and the location is kidney.

[00108] In another embodiment, the method is used to predict an effective, preferably optimum, dosage of one or more target(s) in a subject, preferably a mammal, further preferably a human, wherein the method comprises an additional step (f) of reducing the dosage of the target for use in the subject when the inhibited transporter is involved in the uptake of the one or more target(s) in the minipig, and increasing the dosage of the one or more target(s) for use in the subject when the inhibited transporter is involved in the uptake of the target in the minipig. In another embodiment, the dosage method further comprises an additional step (g) of determining the dosage of one or more target(s) in a subject using a predetermined correlation coefficient.

A novel method to modulate a transporter using the minipig model.

[00109] The inventors have described a novel method to modulate a transporter using the minipig model, preferably the Gottingen ^® minipig. Advantageously, the method can be used to modulate one or more transporter(s) in vivo, in situ, ex vivo, and/or in vitro. In one embodiment, the method includes the steps of:

(a) administering or exposing one or more modulator(s) at a first location of a minipig, preferably a Gottingen ^® minipig;

(b) administering or exposing one or more substrate(s) of one or more transporter(s) to the first location of the minipig;

(c) administering or exposing a blank control to a second homologous location of the minipig;

(d) administering or exposing the same substrate(s) to the second homologous location of the minipig; and

(e) comparing the amount of substrate(s) (collected in) from the first location of the minipig to that of the second homologous location of the minipig, wherein if the transporter is an uptake transporter and

(i) the modulator increases the substrate in the first location of the minipig as compared to the control is:

(A) indicative that the modulator is an activator of the transporter;

(B) predicative that the modulator will activate the same transporter in humans, and/or

(C) predicative that the modulator will increase absorption, increase toxicity, increase efficacy, reduced drug tolerance, and reduce metabolic of a substrate of the transporter in a human; or

(ii) the modulator decreases the substrate in the first location of the minipig as compared to the control is:

(A) indicative that the modulator is an inhibitor of the transporter;

(B) predicative that the modulator will inhibitor the same transporter in humans, and/or

(C) predicative that the modulator will decrease absorption, decrease toxicity, decrease efficacy, increased drug tolerance, and increase metabolic of a substrate of the transporter in a human; and

wherein if the transporter is an efflux transporter and

(i) the modulator decreases the substrate in the first location of the minipig as compared to the control is:

(A) indicative that the modulator is an activator of the transporter;

(B) predicative that the modulator will activate the same transporter in humans, and/or

(C) predicative that the modulator will decrease absorption, decrease toxicity, decrease efficacy, increase drug tolerance and increase metabolic of a substrate of the transporter in a human;

(ii) the modulator increases the substrate in the first location of the minipig as compared to the control is: (A) indicative that the modulator is an inhibitor of the transporter;

(B) predicative that the modulator will inhibite the same transporter in humans, and/or

(C) predicative that the modulator will increase absorption, increase toxicity, increase efficacy, reduced drug tolerance and reduce metabolic of a substrate of the transporter in a human.

[00110] In another embodiment, two minipigs are used, using the same general steps as above, wherein one minipig is treated with the inhibitor(s) and the other minipig is treated with the control.

[00111] In a preferred embodiment, the transporter is MRP1 and the location is skin; the transporter is MRP2 and the location is liver and/or kidney; the transporter is MATE1 and the location is skin; the transporter is OCT1 and the location is liver; and/or the transporter is an OAT3 and the location is kidney.

[00112] In another embodiment, mRNA and/or protein expression of the transporter is compared. An overexpression of an uptake transporter is indicative that the modulator is an activator of the transporter gene and can increase the absorption, toxicity, and efficacy, reduce drug tolerance etc. of a substrate of the transporter. In contrast, a reduced expression caused by the modulator is indicative of the opposite. In contrast, an under expression of an efflux transporter is indicative that the modulator is an activator of the transporter gene and can reduce the absorption, toxicity, and efficacy, and increase drug tolerance etc. of a substrate of the transporter. In contrast, a reduced expression is indicative of the opposite.

A novel method to screen effective therapeutics using a minipig

[00113] The inventors have described a novel method of using a minipig, preferably a Gottingen ^® minipig, to screen effective therapeutics for an animal, preferably a mammal, and further preferably a human. The method can be used to screen a therapeutic for any diseases, including cancer, in any organ. Preferably, the method is used to screen a therapeutic for diseases related to the skin, liver, and/or kidney. Advantageously, the method can be used to screen effective therapeutics in vivo, in situ, ex vivo, and/or in vitro.

[00114] In one embodiment, the method includes the steps of:

(a) administering or exposing a therapeutic candidate to a minipig, preferably a Gottingen ^® minipig; and

(b) determining the pharmacokinetics of the therapeutic candidate in the minipig;

wherein, the pharmacokinetic of the therapeutic candidate in the minipig is indicative of the pharmacokinetic of the therapeutic in a subject, preferably a mammal, and further preferably a human.

[00115] This embodiment can predict one or more pharmacokinetics of the therapeutic candidate, including absorption, toxicity, efficacy, drug tolerance, and metabolism; and is particularly useful if the therapeutic candidate is (1 ) a substrate of the ABCC1 (MRP1 ) and targets a skin, preferably a human skin; (2) a substrate of the ABCC2 (MRP2) and targets a liver, preferably a human liver; (3) a substrate of the ABCC2 (MRP2) and targets a kidney, preferably a human kidney; (4) a substrate of the SLC47A1 (MATE1 ) and targets a skin, preferably a human skin; (5) a substrate of the SLC22A1 (OCT1 ) and targets a liver, preferably a human liver; and/or (6) a substrate of the SLC22A8 (OAT3) and targets a kidney, preferably a human kidney. Accordingly, the therapeutic with the desired pharmacokinetic can be screened using this method.

[00116] In another embodiment, another modified therapeutic candidate is administered or exposed to a homologous location of the same or different minipig. The pharmacokinetics of the therapeutic candidate is compared with that of the modified therapeutic candidate. The candidate with the more desirable pharmacokinetic is selected. This embodiment can be used to select the therapeutic candidate with more desirable pharmacokinetic characteristics. Some of the pharmacokinetic characteristics include absorption, toxicity, efficacy, drug tolerance, and metabolism. The embodiment is also particularly useful if the therapeutic candidate is (1 ) a substrate of the ABCC1 (MRP1 ) and targets a skin, preferably a human skin; (2) a substrate of the ABCC2 (MRP2) and targets a liver, preferably a human liver; (3) a substrate of the ABCC2 (MRP2) and targets a kidney, preferably a human kidney; (4) a substrate of the SLC47A1 (MATE1 ) and targets a skin, preferably a human skin; (5) a substrate of the SLC22A1 (OCT1 ) and targets a liver, preferably a human liver; and/or (6) a substrate of the SLC22A8 (OAT3) and targets a kidney, preferably a human kidney.

A novel method of in vivo visualization of transporters using minipig

[00117] The inventors have described a novel method of using a minipig, preferably a Gottingen ^® minipig, to visualize activities of transporters in vivo, in situ, ex vivo and/or in vitro. The method can identify the innate localization and activity(ies) of a transporter in minipigs and predict the innate localization and activity(ies) of a transporter in a subject, preferably a mammal and further preferably a human. The method can allow for real time visualization of substrate-transporter interaction, co-localization, and evaluate transporter activity. In one embodiment, the method includes the steps of:

(a) labeling one or more transporter(s) of a minipig, preferably a Gottingen ^® minipig; and;

(b) labeling one or more substance(s);

(c) administering or exposing the labeled substance(s) to the minipig; and

(d) visualizing the labeled substance(s) and labeled transporter(s).

[00118] Labeling can be achieved using any fluorescent, luminescent and/or radioactive dye. The substance can be directly labeled and/or indirectly labeled using an antibody.

Visualization can be achieved using a microscope or any other detection apparatus. This embodiment can predict one or more behavior(s) of the transporter(s) in a subject, preferably human, if the substrate is (1 ) a substrate of the ABCC1 (MRP1 ) and targets a skin, preferably a human skin; (2) a substrate of the ABCC2 (MRP2) and targets a liver, preferably a human liver; (3) a substrate of the ABCC2 (MRP2) and targets a kidney, preferably a human kidney; (4) a substrate of the SLC47A1 (MATE1 ) and targets a skin, preferably a human skin; (5) a substrate of the SLC22A1 (OCT1 ) and targets a liver, preferably a human liver; and/or (6) a substrate of the SLC22A8 (OAT3) and targets a kidney, preferably a human kidney. The use of human transporter as biomarker for disease in a minipig.

[00119] Certain human transporter are biomarkers for minipig diseases. For example, the human SLC transporter MATE1 , which is predominantly expressed in human skin has been indicated as a biomarker for skin cancer (See WO 2014/184265A1 (2014)). The inventors have described a novel method of using human transporter as biomarkers for minipig disease, such as minipig skin cancer.

[00120] In a preferred embodiment, a human transporter biomarker, preferably an ABC and/or SLC transporter, further preferably a MATE1 , is used as a biomarker in minipig;

wherein a change in the expression of the homologous transporter in minipig is indicative of a minipig disease, preferably cancer, further preferably skin cancer. Some of the skin diseases detected by this embodiment include skin disease such as inflammatory skin disease, viral skin disease, fungal skin disease, bacterial skin disease, cancerous skin disease, and combinations thereof. Other skin disease detected by this embodiment include cancerous skin disease such as, actinic keratosis, basal cell carcinoma, melanoma, Kaposi's carcinoma, squamous cell carcinoma, and combinations thereof.

[00121] The examples that follow illustrate exemplary embodiments without limiting the scope of the application.

Example 1.

Measurement of constitutive mRNA expression of CYPs, ABC and SLC transporters in minipig liver, kidney and skin

[00122] mRNA expression levels of four cytochrome genes (CYP1A 1 (extrahepatic), CYP1A2 (hepatic), CYP2B22 and CYP3A29), four ABC transporter genes (ABCB1, ABCC1, ABCC2 and ABCG2), and four SLC transporter genes {SLC22A 1, SLC22A2, SLC22A6 and SLC22A8) were measured in the liver, kidney and skin of male and female Gottingen ^® minipigs. Collection of minipig liver, skin and kidney samples.

[00123] Samples of liver and skin collected from 4 male and 4 female minipigs were received fresh on dry ice. At reception, samples were immediately homogenized and lysed. Kidney total RNA from one female minipig was provided by amsbio, Ref. NR-901.

Liver homogenization.

[00124] Each liver sample was thoroughly washed in ice cold PBS solution. 120 mg (±10 mg) of liver sample were weighed and transferred into 2 mL CK28-R tissue

homogenization vial containing 6 ceramic balls of 2.8 mm diameter. One (1 ) mL of RNA lysis buffer (Promega) was added to the vial. The samples were homogenized using the tissue homogenizer system Precellys ^® during 30 seconds at 6000 rotations per minute using the cooling system. The tissue homogenates were then centrifuged at 12000 g at 4°C for 10 minutes. 175 μί of lysate were collected for total RNA extraction (for each liver sample).

Skin homogenization.

[00125] Five skin biopsies were performed on each skin sample using a biopsy punch device (6 mm of diameter) then transferred into 7 mL tissue homogenization CK28 tissue homogenization vial containing 36 ceramic balls of 2.8 mm diameter. One mL of RNA lysis buffer (Promega) was added to the vial. The skin samples were homogenized using the tissue homogenizer system Precellys ^® using six cycles of 23 seconds at 6300 rotation per minute using the cooling system. The skin homogenates were then centrifuged at 12000 g at 4°C during 10 minutes. The total lysate was collected for total RNA extraction (for each skin sample).

Extraction of total RNA.

[00126] Extraction of total RNA from each sample was performed using SV total RNA Isolation System Kit (Promega), according to the instructions provided by the manufacturer. Concentration of total RNA in each sample was determined by measuring the absorbance at 260 nm using Take 3 plate on Synergy 2 plate reader (BioTek ^® ). The quality of total RNA in each sample was checked by measuring the absorbance at 230 nm and 280 nm. The data were analyzed using validated Gen5 Secure software (BioTek ^® ). The acceptance criteria are:

• the absorbance value measured at 260 nm should be < 2, and

• the ratio 260/230 should be > 1.8 and/or

• the ratio 260/280 should be > 1.8.

Total RNA samples were stored at -80°C until use.

Reverse transcription reactions.

[00127] 500 ng of total RNA were reverse transcribed using high capacity cDNA Reverse Transcription Kit (Applied Biosystems, Appendix 1 ) in a final volume of 100 μΙ_ in 96-well format plate (MicroAmp Optical 96-well reaction plate). The reverse transcription reactions were held on 2720 Thermal Cycler instrument (Applied Biosystems) or 7500 Real Time PCR (Applied Biosystems) using the following temperature program:

• 10 minutes at 25°C

• 120 minutes at 37°C

• 5 minutes at 85°C

• 4°C for infinity.

The Reverse transcription product cDNA was transferred to small tubes and stored at -80°C. Real-time PCR reactions.

[00128] Real time PCR reaction was performed according to the instructions provided by the manufacturer. The target genes were amplified by real-time-PCR using five (5) μΙ_ of the reverse transcription product (cDNA) and a specific CYP, ABC and SLC transporter TaqMan ^® gene expression assays (Applied Biosystems™). The housekeeping gene GAPDH was similarly amplified using a GAPDH TaqMan ^® gene expression assays (Applied Biosystems™) and was used as reference gene in order to normalize the target transcript. [00129] Target and reference gene sequences were amplified independently in separate reaction wells in triplicate. The TaqMan ^® gene expression assays containing both reverse and forward oligonucleotide sequences and the TaqMan ^® probe with FAM as dye label were used. The PCR reactions were performed in a final volume of 25 μΙ_ containing 5 μΙ_ of cDNA, 12.5 μΙ_ TaqMan ^® Universal PCR Master Mix, no AmpErase ^® UNG (Applied Biosystems™), 1 .25 μΙ_ TaqMan ^® gene expression assays (Applied Biosystems™) and 6.25 μΙ_ RNase free water (Promega) in 96-well format plate (MicroAmp Optical 96-well reaction plate, Applied Biosystems™). The PCR reaction was held in Applied Biosystems ^® 7500 Real-Time PCR System, as follows:

50°C during 2 minutes

95°C during 10 minutes

95°C during 15 sec and 60°C during 1 minute x 40 cycles

The PCR fluorescence data (provided by FAM probe) were analyzed with a validated 7500 System SDS software (version 1 .4.0.25, 21 CFR Part 1 1 Module, Applied Biosystems™). The results were expressed as Ct (cycle threshold), which is inversely proportional to the amount of gene in the sample. The acceptance criteria were coefficient of variation CV (%) between triplicate PCR reactions were below 0.5%. mRNA expression analysis.

[00130] The quantification approach used for determining the constitutive (basal) expression of a given gene is termed the comparative. The C _t values of each target gene were normalized by GAPDH housekeeping gene used as reference gene or endogenous control as follow:

AC _t = Ct target gene - C _t housekeeping gene

The constitutive gene expression for all samples was calculated using the formula:

constitutive gene expression = 2 ^"AC _t

[00131] For CYPs, we used mean and SEM of 4 animals per sex. For transporters, inventors used one animal per sex. ABC transporter expression in minipig.

[00132] Our analysis of the ABC transporter expression showed that ABCC1 (MRP1) had the highest constitutive mRNA expression in minipig skin, with more MRP1 expressed in males than in females. In contrast, the constitutive mRNA expression of ABCC2 (MRP2) in minipig skin was very low (See for example, Figure 6Erreur ! Source du renvoi

introuvable.). Accordingly, inventors' results showed that MRP1 is the main ABC transporter in minipig skin.

[00133] In addition, inventors' analysis showed that ABCC2 (MRP2) had the highest constitutive mRNA expression in minipig liver, with more MRP2 expressed in females than in males. In contrast, the constitutive mRNA expression of ABCC1 (MRP1) in minipig liver was very low (See for example, Figure 7). Accordingly, inventors' results showed that MRP2 is the main ABC transporter in minipig liver.

[00134] Finally, inventors' analysis showed that ABCC2 (MRP2) had the highest constitutive mRNA expression in minipig kidney. In contrast, the constitutive mRNA expression of ABCB1 (MDR1) in minipig kidney was very low (See for example, Figure

8Erreur ! Source du renvoi introuvable.). Accordingly, inventors' results showed that MRP2 is the main ABC transporter in minipig kidney.

SLC transporter expression in minipig.

[00135] Inventors' analysis of the SLC transporter expression showed that none of the four SLC transporter genes tested in minipig skin showed any detectable mRNA expression.

SLC47A 1 (MATE1) was not cloned in minipig skin (See for example, Figure 5). Accordingly, inventors' results showed that none of the SLC transporter tested is the main SLC transporter in minipig skin.

[00136] In addition, inventors' analysis showed that SLC22A 1 (OCT1) was the only constitutive mRNA expression in minipig liver, with more OCT1 expressed in females than in males (See for example, Figure 9). Accordingly, inventors' results showed that OCT1 is the main SLC transporter in minipig liver.

[00137] Finally, inventors' analysis showed that SLC22A8 {OAT3) had the highest constitutive mRNA expression in minipig kidney. In contrast, the constitutive mRNA

expression of the other SLC transporter tested in minipig kidney were very low (See for example, Figure 10). Accordingly, inventors' results showed that OAT3 is the main SLC transporter in minipig kidney.

CYP expression in minipig.

[00138] Inventors' analysis of the CYP expression showed that CYP1A1 had the highest constitutive mRNA expression in minipig skin, with more CYP1A 1 expressed in males than in females. In fact, in general, the CYPs tested were expressed in higher amounts in males than in females. Of the four CYPs tested, the constitutive mRNA expression of CPY1A2 was very low in minipig skin (See for example, Figure 1 1 ). Accordingly, inventors' results showed that CYP1A1 is the main cytochrome P450 in minipig skin.

[00139] In addition, inventors' analysis of the CYP expression showed that CYP1A2 and CYP3A29 had high constitutive mRNA expression in minipig liver, with females expressing more CYP1A2. Of the four CYPs tested, the constitutive mRNA expression of CPY2B22 was very low in minipig skin (See for example, Figure 12). Accordingly, inventors' results showed that CYP1A2 and CYP3A29 are the main cytochrome P450 in minipig liver.

[00140] Finally, inventors' analysis showed that CYP2B22 had the highest constitutive mRNA expression in minipig kidney. Of the four CYPs tested, the constitutive mRNA expression of CPY1A2 was very low in minipig skin (See for example, Figure 13).

Accordingly, inventors' results showed that CYP2B22 is the main cytochrome P450 in minipig liver.

[00141] Comparing the constitutive mRNA expression of CYPs in skin, liver and kidney, inventors found that in general, expression is higher for the CYPs tested in liver than in skin or kidney. In addition, expression for CYP2B22 and CYP3A29 is higher in the kidney than in the skin. Finally, expression for CYP1A 1 is higher in the skin than in the kidney. (See for example, Figure 1 1 -Figure 13).

Example 2.

Measurement of constitutive mRNA expression of ABC and SLC transporters in human liver, kidney and skin

[00142] Human skin in organ-culture, hepatocytes in primary culture and kidney total RNA were used to analyze constitutive gene expression by TaqMan Real-time RT-PCR essentially as described in Example 1. Fifteen (15) drug transporter genes were selected for the study based on the recent EMA guidance on drug-drug interactions as the most likely clinical sources of drug interactions. (CPMP/EWP/560/95/Rev.1 Corr. 2 ^** ). Among them included eleven (1 1 ) uptake transporter genes that belonged to the SLC transporter family. They are SLC01B1 (OATP1B1), SCL22A8 (OAT3), SCL01B3 (OATP1B3), SLC22A6 (OAT1),

SLC22A2 (OCT2), SCL22A 1 (OCT1), SLC03A1 (OATPD), SLC02B1 (OATPB), SLC47A2 (MATE2), SLC04A1 (OATPE), and SLC47A 1 (MATE1). Also included were four (4) efflux transporter genes that belonged to the ABC transporter family. They are ABCB1 {P-gp or MDR1), ABCC1 (MRP1), ABCC2 (MRP2 or cMOAT), and ABCG2 (BCRP or MXR). The mRNA expression levels of the transporter genes were in the liver, kidney and skin of human samples.

Collection of human liver, skin and kidney samples.

[00143] Fresh human skin samples from 3 different donors were used and maintained in organoculture. Four skin biopsies of 6 mm diameter were used per well of 6-well plate, filled with long term skin culture medium (Biopredic, France). The culture plates were kept in a cell culture incubator set at 37°C, 5% C0 ₂ and saturated hygrometry. A pool of cryopreserved human hepatocytes were used as liver samples. Human kidney cells were extracted from 2 different donors. Skin and Liver homogenization.

[00144] Human skin and hepatocyte samples were homogenized and total RNA were extracted essentially as described in Example 1 . Briefly, after homogenization of skin samples or hepatocytes in lysis buffer (Promega).

Extraction of total RNA.

[00145] Total RNA was isolated from cell lysate essentially as described in Example 1 . Briefly, total RNA was extracted using SV Total RNA Isolation System (Promega), in accordance with the instructions provided by the constructor. Total RNA concentration was quantified by a spectrophotometer.

Reverse transcription reactions.

[00146] Constitutive mRNA expression was quantified by reverse transcription reactions essentially as described in Example 1 . Briefly, quantification of human transporter mRNA was quantified using TaqMan PCR techniques (Applied Biosystems). Experiments were carried out on a 7500 real time PCR System (Applied Biosystems) using Assay-on-Demand gene expression products. For this, 500 ng of total RNA were reverse-transcribed using the High Capacity RNA to cDNA Master Mix kit (Applied Biosystems). PCR amplifications were performed in a total volume of 25 μί using the TaqMan Universal Master Mix (Applied

Biosystems). Real-Time PCR was performed by denaturation at 95°C for 10 min, followed by 40 PCR cycles with the following specifications:

• 95°C for 15 seconds and

• 60°C for 60 seconds.

Real-time PCR reactions.

[00147] Real-time PCR reactions were performed essentially as described in Example 1. Briefly, TaqMan Gene Expression Assays from Applied Biosystems were used in the expression profiling experiments. GAPDH was used as a reference gene for normalization in each sample. All real-time PCR measurements were performed in triplicates. mRNA expression analysis.

[00148] Relative quantification of the expression level of each transcript in each sample was calculated using the C _t method, also called AAC _t method. (Livak et ai, Methods 25, 402 (2001 )). Briefly, expression values for target genes were normalized to the concentration of GAPDH, which showed the least variation among reference genes in our biological systems. Gene expression values were calculated based on the C _t method, in which RNA samples were designated as calibrators to which the other samples were compared. The Ct data for the transporters and GAPDH in each sample were used to create AC _t values, where

AC _t = Ct transporter - C _t GAPDH. The RQs were calculated using the formula: RQ = 2 ^"ΔΔα . The results are expressed as 2 ^"Δα , or as 2 ^{" Ct} .

ABC transporter expression in humans.

[00149] Inventors' analysis of the ABC transporter expression showed that ABCC1 (MRP1) had the highest constitutive mRNA expression in human skin, similar to that of the minipig; and ABCC2 (MRP2) had the highest constitutive mRNA expression in human liver, similar to that of the minipig. (See for example, Figure4). In addition, inventors' analysis showed that unlike minipig kidneys, where the predominant ABC transporter expressed was ABCC2 (MRP2), in humans kidneys, the predominant ABC transporter expressed constitutively was ABCB1 (MDR1) followed by ABCC2 (MRP2) (See for example, Figure 4). Accordingly, inventors' results showed that MRP1 is the main ABC transporter in human skin, MRP2 is the main ABC transporter in human liver, and MDR1 and MRP2 are the main ABC transporters in human kidney. SLC transporter expression in humans.

[00150] Inventors' analysis showed that the expression profiles of SLC transporters are specific to the three organs studied, skin, liver and kidney, with different expression profiles in each of the organs tested (See for example, Figure 5). Interestingly and surprisingly, the expression profile in minipig liver matched that of human.

[00151] Of the eleven (1 1 ) SLC transporter genes tested in human skin, six (6) were not detected. They are SLC01B1 (OATP1B1), SLC01B3 (OATP1B3), SLC22A 1 (OCT1), SLC22A2 (OCT2), SLC22A6 (OAT1), and SLC22A8 (OAT3). The remaining five (5) SLC transporter genes were constitutively expressed in human skin. They are SLC02B1

(OA TPB), SLC03A 1 (OATPD), SLC04A 1 (OA TPE), SLC47A 1 (MATE1), and SLC47A2 (MA TE2). Of these, SLC47A 1 (MATE1) had the highest constitutive mRNA expression in human skin, supporting MATE1 to be important in topical drug exposure and in drug-drug interactions in dermatology. (See for example, Figure 5). The MATE1 transporter was not cloned in minipig. Further, none of the four SLC transporters analyzed in minipig (SLC22A6 (OA T1), SLC22A8 (OAT3), SLC22A 1 (OCT1), and SLC22A2 (OCT2)) showed any detectable constitutive mRNA expression in minipig skin.

[00152] Inventors' analysis also showed that SLC22A 1 (OCT1) had the highest constitutive mRNA expression in human liver, similar to that of the minipig (See for example, Erreur ! Source du renvoi introuvable.5). Inventors' analysis further showed that both SLC22A6 (OA T1) and SLC22A8 {OAT3) had high levels of constitutive mRNA expression in human kidney, however, only SLC22A8 {OAT3) had the highest constitutive mRNA expression in minipig kidney (See for example, Figure 5). Comparing human constitutive SLC transporter expression in liver to that of skin and kidney, they found SLC04A 1 (OA TPE) expression levels in skin were comparable to that of kidney. However, when compared to human liver, SLC04A 1 (OATPE) expression levels were 70 times lower (See Figure14). In general, inventors' results showed that MATE1 is the main SLC transporter in human skin, OCT1 is the main SLC transporter in human liver, and OAT1 and OAT3 are the main ABC transporters in human kidney.

Regulation of SLC transporters in human skin

Chemical Regulation of SLC transporters in ex vivo human skin and liver.

[00153] To study the chemical regulation of SLC transporters in the human, inventors treated their skin samples and hepatocyte primary culture with 20 μΜ Rifampicin (an antibiotic) for 3 days. Human skin samples were obtained from two donors. Untreated skin and liver samples were used as control. The culture plates were kept in a cell culture incubator set at 37°C, 5% CO2 and saturated hygrometry. At the end of treatment period gene expression of the transporters was measured as described herein.

[00154] Inventors' results showed that rifampicin down-regulated the expression of SLC47A 1 (MATE1) and SLC47A2 (MATE2) in skin and reduced the constitutive mRNA expression of these genes by 44% in SLC47A1 (MATE1) (p<0.05) and 30% in SLC47A2 (MATE2). (See Figure15). In addition, rifampicin reduced the constitutive mRNA expression of SLC47A 1 (MATE1 ) in human liver by 48%. (See Figure16). Taken together, their results showed that rifampicin regulates mRNA expression of SLC47A1 (MATE1) in human skin and fresh human hepatocytes, and rifampicin also regulates SLC47A2 (MATE2) mRNA

expression in human skin.

Physical Regulation of SLC transporters in ex vivo human skin.

[00155] To study the physical regulation of SLC transporters in the human skin, they exposed skin samples, for one hour every day for 3 days, to UV light via a solar simulator (UVA 1 10 W/m ² ; UVB 20 W/m ² ). Human skin samples were obtained from two donors. Control samples were not exposed to UV light. The culture plates were kept in a cell culture incubator set at 37°C, 5% CO ₂ and saturated hygrometry. At the end of treatment period gene expression of the transporters was measured, as described herein. [00156] Inventors' results showed that UV down-regulated the expression of SLC47A 1 (MA TE1) and SLC47A2 (MATE2) in skin and reduced the constitutive mRNA expression of these genes by 43% in SLC47A 1 (MATE1) (p<0.05) and 60% in SLC47A2 (MATE2). (See Figure 17). Taken together, inventors' results support that certain SLC transporters are sensitive to and can be regulated by UV.

[00157] The extensive constitutive mRNA expression of the MATE1 transporter in human skin and its sensitivities to UV suggest that the MATE1 transporter can serve as biomarkers for skin cancers, particularly solar-induced skin cancer. Furthermore, inventors' results indicate that expression of MATE1 transporter in human skin can play an important role in chemo-sensitivity of cutaneous cancer cells, and thus can be further exploited for the discovery of novel agents for treatment of skin cancers. This is confirmed by investigating the expression of MATE1 in skin cancer from different origin. More precisely, by determining mRNA levels of MATE1 in paired cancerous and adjacent non-cancerous specimens from a large number of patients with different types of skin cancer.

Example 3.

Examples of common skin disease and examples of its treatment

[00158] Examples of inflammatory skin disease include eczema, dermatitis, psoriasis, diaper rash and acne. Treatments for inflammatory skin disease include topical ointments that reduce itching and swelling. Examples of topical treatment for acne include, tretinoin, benzoyl peroxide, clindamycin, doxycycline, isotretinoin, tetracycline, minocycline, salicylic acid, azelaic acid, erythromycin topical, drospirenone-ethinyl estradiol, tazarotene, benzoyl peroxide-clindamycin, ethinyl estradiolnorgestimate, sulfacetamide sodium, clindamycin- tretinoin, combinations thereof and the like. Examples of topical treatment for psoriasis include cortisone, retinoids derived from vitamin A, vitamin D analogues, salicylic, lactic acid, combinations thereof and the like.

[00159] Examples of a viral skin disease include, chicken pox, measles, herpes 1 , herpes 2, and shingles. Examples of treatment include topical prescription medications. [00160] Examples of a fungal skin disease include, Candida, athlete's foot, and ringworm. Examples of treatment include oral medications, topical ointments, powders, oral antiseptics, and a combination thereof.

[00161] Examples of a bacterial skin disease include, impetigo, cellulitis, MRSA, folliculitis, scabies, and necrotizing fasciitis. Examples of treatment include antibiotics, surgery and a combination thereof.

[00162] Examples of a cancerous skin disease include, basal cell cancer, squamous cell cancer, and melanoma. Treatments can include chemotherapy, radiation therapy, surgery and a combination thereof.

[00163] Other common skin diseases include rosacea, vitiligo, and impetigo. Examples of topical treatment for rosacea include metronidazole, azelaic acid, combinations thereof and the like. An example of a topical treatment for vitiligo includes hydroquinone. Examples of topical treatment for impetigo include erythromycin, mupirocin, combinations thereof and the like.

Example 4.

Examples of ABC transporter inhibitors

[00164] Inhibitors for MDR1 (ABCB1 or P-gp) include Ritonavir, Cyclosporine, Verapamil, Erythromycin, Ketoconazole, Itraconazole, Quinidine, Elacridar (GF120918), LY335979, and Valspodar (PSC833).

[00165] Inhibitors for MRP1 (ABCC1 ) include Probenecid, Cyclosporine, and MK571.

[00166] Inhibitors for MRP2 (ABCC2 or cMOAT) include Cyclosporine and Probenecid.

[00167] Inhibitors for BCRP (ABCG2 or MXR) include Oestrone, 17p-oestradiol,

Fumitremorgin C, Elacridar (GF120918), and Gefitinib. Example 5.

Using minipigs to predict transporter activity on target drugs pharmacokinetics in humans

[00168] The constitutive expression profiles similarities between minipig and human makes minipig a good large animal model to evaluate transporter activity on target drug

pharmacokinetics. For example, the pharmacokinetics of the target drug leukotriene-C4

("LTC4"), such as its absorption and distribution in human skin, can be predicted by evaluating the absorption and distribution of the target drug in minipig skin. Here, the target drug LTC4 is a specific substrate of the ABC transporter MRP1. As shown in Erreur ! Source du renvoi introuvable.4, the expression of MRP1 in minipig skin is similar to that of humans. Accordingly, the absorption and distribution profile of LTC4 in minipig skin and the presence or absence of a MRP1 inhibitor, such as MK571 , can predict the absorption and distribution profile of LTC4 in human skin.

[00169] For example, apply 10 μΙ_ of 2 mM of MK571 or buffer to 1 cm ² of ex vivo minipig skin for 30 minutes to produce a MRP1 ^" sample or a MRP1 ⁺ sample respectively. Then, contact the minipig skin sample with 10 μΙ_ of 68.5 nM (0.01 mCi/mL) tritium labeled LTC4 for 6 hours. The amount of LTC4 absorbed by the minipig skin can be measured by analyzing the amount of LTC4 that reached the receptor fluid using scintillation counter and the distribution of the LTC4 in the skin is analyzed by autoradiography. A significant reduction of the target drug (i.e. LTC4) in the receptor fluid in the ABC transporter negative sample (i.e. MRP1 ^" sample) as compared to the control, is indicative that the ABC transporter is an essential efflux transporter of target drug out of the skin. Thus, the result would predict that the ABC transporter is also an essential efflux transporter for target drug in human skin.

[00170] The distribution profile of TLC4 in minipig skin can also be analyzed by

autoradiography. A distribution profile that shows the migration of the target drug (i.e. TLC4) from epidermis to the dermis in the ABC transporter negative sample (i.e. MRP1 ^" samples) as compared to the control is indicative that the ABC transporter is an essential efflux transporter of the target drug out of the skin. Thus, this result predict the ABC transporters also as essential efflux transporters for target drug in human skin.

References

[00171] The following publications, references, patents and patent applications are hereby incorporated by reference in their entireties.

[00172] Thiebaut, F., Tsuruo, T., Hamada, H., Gottesman, M.M., Pastan, I., Willingham, M.C., (1987). Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proceedings of the National Academy of Sciences 84, 7735-7738.

a. Thiebaut et ai, Proc. Natl. Acad. Sci. 84, 7735 (1987)

[00173] Baron JM, Holler D, Schiffer R, Frankenberg S, Neis M, Merk HF, Jugert FK., (2001 ). Expression of multiple cytochrome p450 enzymes and multidrug resistance- associated transport proteins in human skin keratinocytes. The Journal of Investigative Dermatology 1 16, 541 -548.

a. Baron et al., J. Invest. Dermatol. 116, 541 (2001 )

[00174] Smith G, Dawe RS, Clark C, Evans AT, Comrie MM, Wolf CR, Ferguson J, Ibbotson SH., (2003). Quantitative real-time reverse transcription-polymerase chain reaction analysis of drug metabolizing and cytoprotective genes in psoriasis and regulation by ultraviolet radiation. The Journal of Investigative Dermatology 121 :390-398.

a. Smith et al., J. Invest. Dermatol. 121 , 390 (2003)

[00175] Shazik C., Wenzel J., Marquardt Y., Kim A., (201 1 ). P-Glycoprotein (ABCB1 ) expression in human skin is mainly restricted to dermal components. The Journal of

Experimental Dermatology 20:450-452.

a. Shazik et ai, J. Exp. Derm. 20, 450 (201 1 )

[00176] Ito K, Nguyen HT, Kato Y, Wakayama T, Kubo Y, Iseki S, Tsuji A., (2008). P- glycoprotein (ABCB1 ) is involved in absorptive drug transport in skin. Journal of controlled release 131 :198-204.

a. Ito et al., J. Control Release, 131 , 198 (2008) [00177] Stride, B.D., Grant, C.E., Loe, D.W., Hipfner, D.R., Cole, S.P., Deeley R.G., (1997). Pharmacological characterization of the murine and human orthologs of multidrug- resistance protein in transfected human embryonic kidney cells. Molecular pharmacology 52:344-353.

a. Stride et al., Mol. Pharmacol. 52, 344 (1997)

[00178] CPMP/EWP/560/95/Rev. 1 Corr. 2 ^** . Guideline on the investigation of drug interactions, (2012). European Medicines Agency.

a. CPMP/EWP/560/95/Rev.1 Corr. 2 ^**

[00179] Suenderhauf, C, Parrott, N., (2013). A physiologically based pharmacokinetic model of the minipig: data compilation and model implementation. Pharmaceutical research 30:1 -15.

a. Suenderhauf et al., Pharm. Res. 30, 1 (2013)

[00180] Livak, K.J. , Schmittgen, T.D., (2001 ). Analysis of relative gene expression data using real-time quantitative PCR and the 2 ^_ΔΔε _τ Method. Methods 25:402.

a. Livak et ai, Methods 25, 402 (2001 )

[00181] Mohsen A. Hedaya, (2012). Basic Pharmacokinetics, Second Edition. Taylor & Francis Group, LLC.

a. Hedaya, Basic Pharmacokinetics (2012)

[00182] Osman-Ponchet, H., (2014). Use of SLC mammalian skin transporter. WO 2014/184265A1.

a. WO 2014/184265A1 (2014)