WO2019051546A1

WO2019051546A1 - Methods and markers for assessing a response to a drug

Info

Publication number: WO2019051546A1
Application number: PCT/AU2018/050993
Authority: WO
Inventors: Andrew Rowland; Michael Joseph Sorich
Original assignee: The Flinders University Of South Australia
Priority date: 2017-09-13
Filing date: 2018-09-13
Publication date: 2019-03-21

Abstract

The present disclosure relates to methods and markers for assessing a response to a drug. In certain embodiments, the present disclosure provides a method of assessing a response of a subject to a drug, the method comprising assessing the response of the subject to the drug on the basis of the level and/or activity of a factor involved in processing the drug determined in extracellular vesicles from the subject.

Description

METHODS AND MARKERS FOR ASSESSING A RESPONSE TO A DRUG PRIORITY CLAFM

[001] This application claims priority to Australian Provisional Patent Application 2017903719 filed on 13 September 2017, the content of which is hereby incorporated by reference.

FIELD

[002] The present disclosure relates to methods and markers for assessing a response to a drug.

BACKGROUND

[003] The efficacy and tolerability of a drug in an individual is proportional to that individual's drug exposure, which for chronically administered drugs is primarily determined by the rate of drug removal from the body.

[004] Inadequate exposure to a drug may result in a lack of efficacy ("therapeutic failure"), while excessive exposure increases the risk of toxicity and reduces tolerability. For approximately 75% of chronically administered drugs, exposure is primarily determined by the rate of removal by the liver. Understanding the determinants of exposure is particularly critical for drugs that exhibit substantial variability in hepatic clearance and drugs where minor differences in exposure have a large impact on efficacy or tolerability (narrow therapeutic index drugs).

[005] In addition, substantial inter- and intra- individual variability also exists in the activity of factors that are involved in drug processing which govern the rate of removal of drugs from the body.

[006] As a consequence, administering a standard dose of a drug may result in differences in exposure and hence either a lack of efficacy or increased risk of toxicity. [007] Selecting an optimal drug dose for each individual maximises efficacy, minimises toxicity, and improves cost-effectiveness. Multiple studies have demonstrated the benefit of utilising strategies such as pharmacogenetic testing, reaction phenotyping and therapeutic drug monitoring to optimise drug dosing. However, each of these strategies have specific and significant issues that limit their use in clinical practice.

[008] As such, there is a need for new strategies that provide information as to how a subject may respond to exposure to a drug, such as an individual's drug clearance capacity, information on intra- and inter- individual variability in drug clearance, and to inform clinical practice by improving dose selection of a drug.

SUMMARY

[009] The present disclosure relates to methods and markers for assessing a response to a drug.

[0010] Certain embodiments of the present disclosure provide a method of assessing a response of a subject to a drug, the method comprising assessing the response of the subject to the drug on the basis of the level and/or activity of a factor involved in processing the drug determined in extracellular vesicles from the subject.

[0011] Certain embodiments of the present disclosure provide a method of assessing response of a subject to a drug, the method comprising:

determining the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject; and

assessing the response of the subject to the drug on the basis of the level and/or activity of the factor involved in processing the drug determined in the extracellular vesicles.

[0012] Certain embodiments of the present disclosure provide use of extracellular vesicles to assess a response of a subject to a drug. [0013] Certain embodiments of the present disclosure provide use of a biomarker in extracellular vesicles to assess a response of a subject to a drug.

[0014] Certain embodiments of the present disclosure provide a method of selecting a dose of a drug to be administered to a subject, the method comprising selecting the dose of the drug to be administered to the subject on the basis of the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

[0015] Certain embodiments of the present disclosure provide a method of selecting a dose of a drug to be administered to a subject, the method comprising:

determining the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject;

determining the response of the subject to the drug on the basis of the level and/or activity of the factor involved in processing the drug determined in the extracellular vesicles; and

selecting the dose of the drug to be administered to the subject on the basis of the response of the subject to the drug.

[0016] Certain embodiments of the present disclosure provide a method of administering a drug to a subject, the method comprising administering the drug to the subject at a dose selected on the basis of the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

[0017] Certain embodiments of the present disclosure provide a method of administering a drug to be administered to a subject, the method comprising:

administering the drug to the subject at a dose selected on the basis of the response of the subject to the drug. [0018] Certain embodiments of the present disclosure provide a method of treating a subject with a drug, the method comprising treating the subject with the drug at a dose selected on the basis of the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

[0019] Certain embodiments of the present disclosure provide a method of treating a subject with a drug, the method comprising:

treating the subject with the drug at a dose selected on the basis of the response of the subject to the drug.

[0020] Certain embodiments of the present disclosure provide a kit for performing a method as described herein.

[0021] Certain embodiments of the present disclosure provide use of a factor involved in the processing of a drug in extracellular vesicles as a biomarker for the response of a subject to the drug.

[0022] Certain embodiments of the present disclosure provide a method for identifying an agent that induces a factor involved in processing a drug, the method comprising: determining the ability of a candidate agent to induce the level and/or activity of the factor involved in processing a drug in extracellular vesicles; and identifying the candidate agent as an agent that that induces a factor involved in processing a drug.

[0023] Certain embodiments of the present disclosure provide a method of assessing the ability of an agent to induce a factor involved in processing a drug, the method comprising determining the ability of the agent to induce the level and/or activity of the factor involved in processing a drug in extracellular vesicles.

[0024] Certain embodiments of the present disclosure provide a method of screening a drug, the method comprising determining the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

[0025] Certain embodiments of the present disclosure provide a method of identifying a marker for assessing the response to a drug, the method comprising identifying a factor for which the level and/or activity in extracellular vesicles is indicative of the response of the drug in a subject.

[0026] Certain embodiments of the present disclosure provide a method of identifying a marker for assessing the response to a drug, the method comprising:

determining whether the level and/or activity of a candidate marker in extracellular vesicles is indicative of a response to the drug in a subject; and identifying the candidate marker as a marker for assessing the response to a drug.

[0027] Other embodiments are disclosed herein. BRIEF DESCRIPTION OF THE FIGURES

[0028] Certain embodiments are illustrated by the following figures. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the description.

[0029] Figure 1 shows a transmission electron microscopy image of microvesicles isolated from the extracellular media of HepG2 cells.

[0030] Figure 2 shows the total particle count and size distribution of exosomes isolated from plasma, as determined by nanoparticle tracking analysis (NTA). [0031] Figure 3 shows TsglOl expression in exosomes isolated from human plasma (A), exosomes isolated from the extracellular media of HepG2 cells (B), and HepG2 cells (C).

[0032] Figure 4 shows the concordance of exosome derived CYP3A4 biomarkers and midazolam CL/F in a cohort of healthy males (n=6). Panel A: Exosome derived CYP3A4 protein expression vs midazolam CL/F, Panel B: Exosome derived CYP3A4 mRNA expression vs midazolam CL/F, Panel C: Ex vivo CYP3A4 activity (rate of 1- hydroxymidazolam formation) vs midazolam CL/F, Panel D: Exosome derived CYP3A4 mRNA expression vs exosome derived CYP3A4 protein expression, Panel E: Ex vivo CYP3A4 activity vs exosome derived CYP3A4 protein expression, Panel F: Ex vivo CYP3A4 activity vs exosome derived CYP3A4 protein expression. correlations of exosomal CYP3A4 protein expression and midazolam CL/F pre- (diamonds) and post- squares) rifampicin dosing (A), and fold change in exosomal CYP3A4 protein expression and midazolam CL/F (post- / pre-) rifampicin dosing (B).

[0033] Figure 5 shows the concordance of the change in (Δ) exosome derived CYP3 A4 biomarkers and Δ midazolam CL/F in a cohort of healthy males (n=6) post- / pre- rifampicin dosing. Panel A: Δ Exosome derived CYP3A4 protein expression vs Δ midazolam CL/F, Panel B: Δ Exosome derived CYP3A4 mRNA expression vs Δ midazolam CL/F, Panel C: Δ Ex vivo CYP3A4 activity (rate of 1-hydroxymidazolam formation) vs Δ midazolam CL/F, Panel D: Δ Exosome derived CYP3A4 mRNA expression vs Δ exosome derived CYP3A4 protein expression, Panel E: Δ Ex vivo CYP3A4 activity vs Δ exosome derived CYP3A4 protein expression, Panel F: Δ Ex vivo CYP3 A4 activity vs Δ exosome derived CYP3 A4 protein expression.

[0034] Figure 6 shows CYP3 A4 mRNA expression in HepaRG cells versus exosomes.

[0035] Figure 7 shows induction CYP3A4 mRNA expression in human plasma pre- and post- rifampicin dosing (300mg QD PO for 7 days).

[0036] Figure 8 shows UGT2B7 protein expression in exosomes isolated from human plasma pre- (A) and post- (B) rifampicin dosing. [0037] Figure 9 shows data of a western blot of exosomal UGT2B7 protein (at ~50kD) isolated from two separate plasma samples from 6 healthy males.

[0038] Figure 10 shows the kinetics of 4-MU glucuronidation (pan UGT substrate) and midazolam 1-hydroxylation (CYP3A substrate) by exosome- and FILM- derived proteins. Panel A: 4-MU glucuronidation by exosomes, Panel B: 4-MU glucuronidation by HLM, Panel C: midazolam hydroxylation by exosomes, Panel D: midazolam hydroxylation by HLM.

DETAILED DESCRIPTION

[0039] The present disclosure relates to methods and markers for assessing a response to a drug.

[0040] In certain embodiments, the present disclosure relates to methods and markers for assessing variability in exposure and/or variability in response caused by differences in exposure for any drug where metabolism or protein mediated transport is a driver of exposure.

[0041] In certain embodiments, the present disclosure provides a method of assessing response of a subject to a drug.

[0042] In certain embodiments, the present disclosure provides a method of assessing a response of a subject to a drug, the method comprising assessing the response of the subject to the drug on the basis of the level and/or activity of a factor involved in processing the drug determined in extracellular vesicles from the subject.

[0043] The term "extracellular vesicles" as used throughput this specification refers to membrane enclosed structures derived from cells, and encompasses a variety of different terms such as exosomes, ectosomes, microparticles, microvesicles, membrane particles, separated microvesicles, exosome-like particles, apoptotic vesicles, promininosomes, prostasomes, texosomes, epididimosomes, migrasomes, and oncosomes, for examples as described in Tkach et al. Philos Trans R Soc Lond B Biol Sci. 2018 Jan 5;373(1737), herein incorporated by reference. In this regard, it will be appreciated in particular that the terms "extracellular vesicles", "exosomes" and "microvesicles" as used herein are used interchangeably.

[0044] In certain embodiments, the extracellular vesicles have a size less than 100 nm. In certain embodiments, the extracellular vesicles have a size between 30 to 100 nm. Methods for determining the size of extracellular vesicles are known in the art.

[0045] In certain embodiments, the present disclosure provides a method of assessing a response of a subject to a drug by determining the level and/or activity of a factor involved in processing the drug using extracellular vesicles isolated from the subject.

[0046] In certain embodiments, the present disclosure provides a method of assessing a response of a subject to a drug, the method comprising:

[0047] In certain embodiments, the method provides the ability to assess the exposure of a drug to a subject. For example, the method may be used to directly or indirectly inform risk of drug toxicity and/or efficacy, and may be used to predict drug efficacy and/or toxicity directly as a surrogate for exposure and hence without measuring exposure to the drug directly.

[0048] In certain embodiments, the subject is a human subject. In certain embodiments, the subject is a subject suffering from, or susceptible to, a condition that requires treatment with a drug.

[0049] In certain embodiments, the subject is a subject for which the genotype of the subject does not provide clinical meaningfulness for how the subject may respond to a drug.

[0050] In certain embodiments, the subject is an animal subject. In certain embodiments the subject is a livestock animal (such as a horse, a cow, a sheep, a goat, a pig), a domestic animal (such as a dog or a cat) and other types of animals, such as monkeys, rabbits, mice, rats and laboratory animals. Veterinary applications of the present disclosure are contemplated.

[0051] In certain embodiments, the response to the drug comprises one or more of drug clearance, drug toxicity, drug efficacy, drug metabolism, drug distribution, drug absorption, and interaction of the drug with another drug. Other types of responses are contemplated, including a therapeutic and/or adverse response of a subject to a drug. Methods for assessing a response in a subject are known in the art.

[0052] The term "drug" as used herein refers to any agent exposed to a subject, including via administration routes that are systemic (e.g., via injection such as intravenous injection, orally in a tablet, pill, capsule, or other dosage form useful for systemic administration of pharmaceuticals), and topical (e.g., creams, solutions, pastes, ointment, including solutions such as mouthwashes, for topical oral administration), or any other route of administration. The term also includes prodrugs, agents that are used in complementary medicines (eg herbal medicines), and agents that are in foods, drinks and the diet.

[0053] The term "a factor involved in processing a drug" as used herein refers to a molecule, protein, enzyme, or a nucleic acid which is either involved directly or indirectly in processing the drug, metabolising the drug, converting the drug to another form, uptake or efflux of a drug, or may be a factor whose level or activity is associated with processing the drug, metabolising the drug, uptake or efflux of a drug, or converting the drug to another form, such a chemical product associated with processing the drug, metabolising the drug, uptake or efflux of the drug, or converting the drug to another form.

[0054] In certain embodiments, the determining of the level and/or activity of the factor involved in processing the drug comprises determining the level of the factor in the extracellular vesicles and/or determining the level of RNA encoding the factor in the extracellular vesicles. Methods for determining the level of the factor and the level of the RNA are known in the art. [0055] In certain embodiments, the determining of the level and/or activity of the factor involved in processing the drug comprises determining the activity of the factor in the extracellular vesicles. Methods for determining the activity of a factor are known in the art.

[0056] In certain embodiments, the factor involved in processing the drug comprises an enzyme involved in metabolising the drug. Methods for assessing the level and/or activity of enzymes involved in metabolising drugs are known in the art.

[0057] In certain embodiments, the enzyme involved in metabolising the drug comprises a cytochrome P450 enzyme.

[0058] Cytochromes P450 (CYPs) are proteins of a superfamily containing heme as a cofactor. CYPs use a variety of small and large molecules as substrates in enzymatic reactions. CYPs are the major enzymes involved in drug metabolism, and account for about 75% of the total metabolism (for example as described in Guengerich FP "Cytochrome p450 and chemical toxicology". Chemical Research in Toxicology. 21(1): 70-83). Many drugs undergo deactivation by CYPs, either directly or by facilitated excretion from the body. Many substrates are also bio-activated by CYPs to form their active compounds in vivo.

[0059] In certain embodiments the enzyme involved in metabolising the drug comprises one or more of CYP 1A1 (GeneCard GCID:GC15M074719), 1A2 (GeneCard GCID:GC15P074748), 2A1 (NCBI 24894), 2A6 (GeneCard GCID:GC19M040843), 2B3 (NCBI 286953), 2B6 (GeneCard GCID:GC19P040991), 2C8 (GeneCard GCID:GC10M095038), 2C9 (GeneCard GCID:GC10P094938), 2C11 (NCBI 29277), 2C18 (GeneCard GCID:GC10P094684), 2C19 (GeneCard GCID:GC10P094762), 2D1 (GeneCard GCID:GC22M042126), 2D3 (NCBI 24303), 2D6 (GeneCard GCID:GC22M042126), 2D10 (NCBI 13101), 2D18 (Kawashima et al. (1996) J. Biol. Chem. 271(45): 28176-28189) , 2E1 (GeneCard GCID:GC10P133520), 3A4 (GeneCard GCID:GC07M099759) and 3A5 (GeneCard GCID:GC07M099648) or a combination of one or more of the aforementioned enzymes. The related genes and proteins in other species may be readily identified by a person skilled in the art. [0060] Methods for assessing the level of such enzymes include immunological methods utilising antibodies to the proteins (such as ELISA), and determination of RNA levels, such as qPCR utilising appropriate primers, although other methods are contemplated. For example, ELISA kits for measuring the level of the following proteins are commercially available from LifeSpan Biosciences Inc. (Human CYP1A1 ELISA Kit (Sandwich ELISA) - LS-F6928; Human CYP1A2 ELISA Kit (CLIA) - LS- F29329; Human CYP2B6 ELISA Kit (Cell-Based ELISA) - LS-F3291; Human CYP2C8+9+18+19 ELISA Kit (Cell-Based ELISA) - LS-F3292;; Human CYP2D6 ELISA Kit (Sandwich ELISA) - LS-F 14051; Human CYP2E1 ELISA Kit (Sandwich ELISA) - LS-F9041; Human CYP3A4 / Cytochrome P450 3A4 ELISA Kit (Sandwich ELISA) - LS-Fl 1200; Human CYP3A4+5 ELISA Kit (Cell-Based ELISA) - LS-F3301) and from MyBioSource (CYP2A6 Elisa kit : : Human Cytochrome P450 2A6 (CYP2A6) ELISA Kit MBS7237397). Antibodies are also known to the enzymes or commercially available, and can be used to determine the level of the proteins.

[0061] Methods for assessing activity of enzymes involved in metabolising the drug are also known in the art and/or commercially available. For example, an assay kit for CYP3A4 is available from Abeam (CYP3A4 Activity Assay Kit (Fluor ometric) (ab211076)), which allows rapid measurement of CYP3A4 activity in biological samples. The assay utilizes a non-fluorescent CYP3A4 substrate that is converted into a highly fluorescent metabolite detected in the visible range (Ex/Em = 535/587 nm), ensuring a high signal-to-background ratio with little interference by autofluorescence. CYP3 A4 specific activity is calculated by running parallel reactions in the presence and absence of the potent inhibitor Ketoconazole and subtracting any residual activity detected with the inhibitor present.

[0062] Methods for determining the activity of other enzymes involved in metabolising the drug are known in the art or commercially available, for example: CYPlAl - Mohammadi Bardbori, Afshin. (2014). Assay for quantitative determination of CYPlAl enzyme activity using 7-Ethoxyresorufin as standard substrate (EROD assay). Protocol Exchange. 10.1038/protex.2014.043; CYP1A2 - CYP1A2 Activity Assay Kit (Fluorometric) (ab211074); CYP2A6 - Lavkekar et al (2007) - Indian Journal of Pharmaceutical Sciences May- June 2007 pp 448-451; CYP2B6 - Promega P450-Glo™ CYP2B6 Assay and Screening Systems; CYP2C8 - Promega P450-Glo™ CYP2C8 Assay; CYP2C9 - Promega P450-Glo™ CYP2C9 Assay and Screening Systems; CYP2C11 - Caliper Cat#400-0882; CYP2C18 - Cypex CYP2C18 QC Assays; CYP2C19 - BioVision Incorporated - Cytochrome P450 2C19 (CYP2C19) Activity Assay Kit (Fluorometnc) # K848; CYP2D6 - BioVision Incorporated Cytochrome P450 2D6 (CYP2D6) Activity Assay Kit (Fluorometnc) #: K703; and CYP2E1 - Cederbaum A. et al. (2014) Redox Biology 2: 1048-1054.

[0063] In certain embodiments, the enzyme involved in metabolising the drug comprises a UDP-glucuronosyl transferase. The UDP-glucuronosyl transferases are responsible for the process of glucuronidation, a major part of phase II (conjugation) drug metabolism.

[0064] In certain embodiments, the enzyme involved in metabolising the drug comprises one or more of an enzyme from UGT1A1 (GeneCard GC02P233760), UGT1A3 (GeneCard GC02P233729), UGT1A4 (GeneCard GC02P233718), UGT1A6 (GeneCard GC02P233691), UGT1A7 (GeneCard GCID:GC02P233681), UGT1A8 (GeneCard GCID:GC02P233618), UGT1A9 (GeneCard GCID:GC02P233671), UGT1A10 (GeneCard GCID:GC02P233636), UGT2B4 (Genecard GCID:GC04M069484), UGT2B7 (GeneCard GCID:GC04P069051), and UGT2B15 (GeneCard GCID:GC04M068646) or a combination of one or more of the aforementioned enzymes.

[0065] Methods for determining the level and /or activity of such enzymes are known in the art, and include for example immunological methods utilising antibodies to the proteins (such as ELISA), determination of RNA levels, such as qPCR utilising appropriate primers, the use of peptides to detect UGT enzymes by mass spectrometry. Other methods are contemplated.

[0066] For example, ELISA kits are available as follows: UGT1A1 (MyBioSource UGT1A1 elisa kit : : Human UDP Glucuronosyltransferase 1 Family, Polypeptide Al (UGT1A1) ELISA Kit MBS2023366); UGT 1 A3 (MyBioSource UGT 1 A3 elisa kit : : Human UDP glucuronosyltransferase 1 family, polypeptide A3 ELISA Kit #MBS9316479); UGT1A4 (MyBioSource UGT1A4 elisa kit : : Human UDP glucuronosyltransferase 1 family, polypeptide A4 ELISA Kit # MBS9323957); UGT1A9 (MyBioSource UGT1A9 elisa kit : : Human UDP glucuronosyltransferase 1 family, polypeptide A9 ELISA Kit# MBS9316073); UGTIAIO (MyBioSource UGTIAIO elisa kit : : Human UDP glucuronosyltransferase 1 family, polypeptide A10 ELISA Kit #MBS9325822)), UGT2B4 (UGT2B4 elisa kit : : Human uridine diphosphate glucuronosyltransferase 2 family polypeptide B4, UGT2B4 ELISA Kit #MBS9304126), UGT2B7 (MyBioSource UGT2B7 elisa kit : : Human UDP glucuronosyltransferase 2 family, polypeptide B7 ELISA Kit #MBS9331876), and UGT2B15 (MyBioSource UGT2B15 elisa kit : : Rat UDP glucuronosyltransferase 2 family, polypeptide B15 ELISA Kit #MBS9341759).

[0067] Methods for assessing activity of UGT enzymes are also known in the art and/or commercially available, for example as described in Strassburg et al. (1998) J. Biol. Chem. 273 : 8719-8726, or using the UGT-Glo Assays are also available commercially from Promega.

[0068] Methods for determining the activity of various specific enzyme are known in the art or commercially available, for example: UGT1A1 (Promega UGT1A1 assay #V2121); UGT 1 A3 (Bertin Pharma UGT 1 A3 (human) MS2Plex® assay kit Cat No: T05016); UGT1A6 (Bertin Pharma UGT1A6 (human) MS2Plex® assay kit Cat No: T05017); and UGT2B7 (Promega UGT1B7 assay V2131), and UGT2B15).

[0069] In certain embodiments, the enzyme involved in metabolising the drug comprises one or more of an enzyme from an acetyltransferase, a glutathione S- transferases and a sulfotransferase. Acetyltransferases are enzymes that are involved in conjugation of acetyl groups and are important in the conjugation of metabolites from the liver involved in phase II metabolism. Glutathione S-transferases comprise a family of phase II metabolic isozymes which catalyse the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for the purpose of detoxification. Sulfotransferases are transferase enzymes that catalyse the transfer of a sulfo group from a donor molecule to an acceptor alcohol or amine.

[0070] Methods for determining the level and/or activity of these enzymes are known in the art, and include for example immunological methods utilising antibodies, or assays measuring their enzymatic activities. For example, the activity of glutathione - transferases may be determined using a commercially available kit from Sigma (Catalog number CS0410), acetyltransferase activity determined using a commercially available kit from Abeam (Acetyltransferase Activity Assay Kit (Fluorometric) #ab204536), and for sulfotransferases as described for example in Paul et al (2012) Anal. Bioanal. Chem. 403(6): 1491-1500, and available commercially from R&D Systems (Cat # EA003).

[0071] In certain embodiments, an increase in the level and/or activity of the enzyme involved in metabolising the drug is indicative of one or more of increased drug clearance, reduced drug toxicity, and reduced drug efficacy.

[0072] In certain embodiments, a decrease in the level and/or activity of the enzyme involved in metabolising the drug is indicative of one or more of an increased drug clearance, reduced drug toxicity, and reduced drug efficacy.

[0073] In certain embodiments, the drug is a prodrug and an increase in the level and/or activity of the enzyme involved in metabolising the drug is indicative of one or more of increased drug toxicity and increased drug efficacy.

[0074] In certain embodiments, the factor involved in processing the drug comprises a factor involved in drug uptake and/or drug efflux.

[0075] In certain embodiments, the factor involved in processing the drug comprises one or more of an ATP -binding cassette (ABC) transporter and a solute carrier (SLC) transporter.

[0076] ATP -binding cassette (ABC) transporters are described, for example, in Vasiliou V. et al (2009) Hum. Genomics 3(3):281-90. These pumps can move substrates in (influx) or out (efflux) of cells. Solute carrier transporters are described, for example, in Perland, Emelie; Fredriksson, Robert (2016-12-08). "Classification Systems of Secondary Active Transporters". Trends in Pharmacological Sciences. 38 (3): 305-315.

[0077] Methods are known in the art for determining the level or activity of ATP- binding cassette (ABC) transporters and solute carrier (SLC) transporters. [0078] For example, immunological detection methods using antibodies to the proteins, or determining the level of RNA encoding the transporters, may be used. Other methods are contemplated.

[0079] In certain embodiments, the factor involved in processing the drug comprises one or more of ABCBl (p-glycoprotein, MDR1) (GeneCard GCID:GC07M087504), ABCBl l (Genecard GCID: GC02M168922), ABCCl (MRP1) (GeneCard; GCID: GC16P015949), ABCC2 (MRP2) (GeneCard GCID: GC10P099782), ABCC3 (MRP3) (GeneCard GCID:GC17P050634), ABCC4 (GeneCard GCID: GC13M095019, ABCC5 (MRP5) (GeneCard GCID: GC03M183919), ABCG2 (BCRP) (GeneCard GCID: GC04M088090), SLC10A1 (Genecard GCID: GC14M069775), SLC10A2 (GeneCard GCID: GC13M103043), SLC22A1 (GeneCard: GC06P160121), SLC22A2 (GeneCard GCID: GC06M160173), SLC22A3 (GeneCard: GCID: GC06P160348), SLC22A4 (GeneCard GCID: GC05P132294), SLC22A5 (GeneCard GCID: GC05P132369), SLC22A6 (GeneCard GCID: GCl 1M062994), SLC22A7 (GeneCard GCID: GC06P043298), SLC22A8 (GeneCard GCID: GCl 1M063012), SLC22A11 (GeneCard GCID: GC11P064573), SLC47A1 (GeneCard GCID: GC17P019495), SLC47A2 (GeneCard GCID: GC17M019678), SLC01A2 (GeneCard GCID: GC12M021191), SLCOIBI (OATP1B1) (GeneCard GCID: GC12P021058), SLC01B3 (OATP1B3) (GeneCard GCID: GC12P020736), and SLC02B1 (GeneCard GCID: GC11P075267), or a combination of one or more of the aforementioned factors.

[0080] In certain embodiments, the method comprises assessing the level and/or activity of a factor involved in processing of a different drug which is processed in a similar manner to the drug. In this way, for example, response to exposure of new drugs may be determined.

[0081] In certain embodiments, the method comprises assessing the level and/or activity of a factor involved in processing of the drug when the subject has been exposed to an inducing agent. In certain embodiments, the inducing agent is the same as the drug.

[0082] In certain embodiments, the extracellular vesicles comprise extracellular vesicles from an organ or tissue. In certain embodiments, the extracellular vesicles comprise hepatic extracellular vesicles, although other types of extracellular vesicles are contemplated. Methods for isolating and identifying extracellular vesicles, including hepatic extracellular vesicles, are known in the art.

[0083] In certain method comprises enriching for hepatic extracellular vesicles. In certain embodiments, the method comprises enriching for hepatic extracellular vesicles using an antibody to a hepatic marker. In certain embodiments, the method comprises enriching for hepatic extracellular vesicles using an anti-ASGR antibody. Anti-ASGR antibodies are known in the art or commercially available.

[0084] In certain embodiments, the extracellular vesicles comprise extracellular vesicles from a biological fluid. In certain embodiments, the extracellular vesicles comprise extracellular vesicles from plasma. In certain embodiments, the extracellular vesicles comprise extracellular vesicles from one or more of the blood, plasma, serum, urine and saliva. Methods for isolating extracellular vesicles from biological fluids are known in the art. In certain embodiments, the extracellular vesicles comprise extracellular vesicles isolated from one or more of the blood, plasma, serum, urine and saliva.

[0085] In certain embodiments, the method comprises obtaining a sample from a subject and processing the sample to isolate/enrich extracellular vesicles. In certain embodiments, the method comprises obtaining a sample from a subject and isolating/enriching extracellular vesicles from the sample.

[0086] In certain embodiments, the method comprises assessing one or more other markers in the subject. In certain embodiments, the one or more other markers comprise a genetic marker. Methods for determining a genetic marker are known in the art.

[0087] In certain embodiments, the method comprises assessing the genotype of a subject. Methods for determining genotype are known in the art, and include next generation sequencing (NGS) sequencing. [0088] In certain embodiments, the method is used to assess or determine intra- individual variability of response to the drug and/or inter- individual variability of response to the drug.

[0089] In certain embodiments, the method is used to select a dose of a drug to be administered to a subject, to assist with administering a drug to a subject, to assist with treating a subject, or to assess exposure of a drug to a subject.

[0090] In certain embodiments, the method is used to assess or determine the correlation between the level and/or activity of a factor involved in processing a drug and the clearance of the drug.

[0091] In certain embodiments, the present disclosure provides use of the various factors involved in processing a drug as described herein as biomarkers.

[0092] In certain embodiments, the present disclosure provides use of the various factors involved in processing a drug as described herein as extracellular vesicle biomarkers.

[0093] In certain embodiments, the present disclosure provides use of extracellular vesicles to assess a response of a subject to a drug, as described herein.

[0094] In certain embodiments, the present disclosure provides use of extracellular vesicles to assess a response of a subject to a drug.

[0095] In certain embodiments, the present disclosure provides use of a biomarker in extracellular vesicles to assess a response of a subject to a drug.

[0096] In certain embodiments, the present disclosure provides a method of selecting a dose of a drug to be administered to a subject by assessing the response of the subject to the drug (or a proxy for the drug). [0097] In certain embodiments, the present disclosure provides a method of selecting a dose of a drug to be administered to a subject, the method comprising assessing the response of the subject to the drug according to a method as described herein.

[0098] In certain embodiments, the present disclosure provides a method of selecting a dose of a drug to be administered to a subject, the method comprising selecting the dose of the drug to be administered to the subject on the basis of the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

[0099] In certain embodiments, the present disclosure provides a method of selecting a dose of a drug to be administered to a subject, the method comprising:

[00100] In certain embodiments, the present disclosure provides a method of administering a drug to a subject.

[00101] In certain embodiments, the present disclosure provides a method of administering a drug to a subject by assessing the response of the subject to the drug as described herein and administering the drug to the subject at a dose selected on the basis of the response of the subject to the drug.

[00102] In certain embodiments, the present disclosure provides a method of administering a drug to a subject, the method comprising administering the drug to the subject at a dose selected on the basis of the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject. [00103] In certain embodiments, the present disclosure provides a method of administering a drug to a subject, the method comprising:

administering the drug to the subject at a dose selected on the basis of the response of the subject to the drug.

[00104] In certain embodiments, the present disclosure provides a method of treating a subject with a drug. Drugs suitable for treating a subject may be selected by a medical practitioner.

[00105] In certain embodiments, the present disclosure provides a method of treating a subject with a drug, the method comprising assessing the response of a subject to the drug as described herein and treating the subject with the drug at a dose selected on the basis of the response of the subject to the drug.

[00106] In certain embodiments, the present disclosure provides a method of treating a subject with a drug, the method comprising treating the subject with the drug at a dose selected on the basis of the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

[00107] In certain embodiments, the present disclosure provides a method of treating a subject with a drug, the method comprising:

treating the subject with the drug at a dose selected on the basis of the response of the subject to the drug. [00108] In certain embodiments, the present disclosure provides a kit for performing a method as described herein.

[00109] In certain embodiments, the kit comprises one or more reagents as described herein, and/or instructions for performing a method as described herein.

[00110] In certain embodiments, the kit comprises one or more reagents for isolating and/or enriching extracellular vesicles. Methods for enriching and/or isolating extracellular vesicles are as described herein.

[00111] In certain embodiments, the kit comprises one or more reagents for detecting a factor involved in processing a drug and/or determining the level and/or activity of a factor involved in processing a drug. Reagents are as described herein.

[00112] In certain embodiments, the kit comprises instructions for using the components of the kit.

[00113] In certain embodiment, the kit comprises one or more of the following components:

(i) one or more reagents for isolating and/or enriching extracellular vesicles;

(ii) one or more reagents for detecting the level and/or activity of a factor involved in the processing a drug; and optionally

(iii) instructions for using the components of the kit.

[00114] In certain embodiments, the present disclosure provides use of a factor involved in the processing of a drug in extracellular vesicles as a biomarker for the response of a subject to the drug.

[00115] In certain embodiments, the present disclosure provides use of a factor involved in the processing of a drug in extracellular vesicles as a biomarker for the response of a subject to the drug. [00116] In certain embodiments, the present disclosure provides a method for screening for an agent that induces a factor involved in processing a drug.

[00117] In certain embodiments, the present disclosure provides a method for identifying an agent that induces a factor involved in processing a.

[00118] In certain embodiments, the present disclosure provides a method for identifying an agent that induces a factor involved in processing a drug, the method comprising:

determining the ability of a candidate agent to induce the level and/or activity of the factor involved in processing a drug in extracellular vesicles; and identifying the candidate agent as an agent that that induces a factor involved in processing a drug.

[00119] In certain embodiments, the candidate agent is a drug.

[00120] Factors involved in processing a drug, and methods for determining their level and/or activity, are described.

[00121] In certain embodiments the method is used to identify the agent as a moderate or strong inducer of the factor involved in processing a drug.

[00122] In certain embodiments, the method comprises use of animals or an animal model. In certain embodiments, the method comprises use of clinical studies in humans.

[00123] In certain embodiments, the present disclosure provides a method of assessing the ability of an agent to induce a factor involved in processing a drug.

[00124] In certain embodiments, the present disclosure provides a method of assessing the ability of an agent to induce a factor involved in processing a drug, the method comprising determining the ability of the agent to induce the level and/or activity of the factor involved in processing a drug in extracellular vesicles. [00125] In certain embodiments, the present disclosure provides a method of screening a drug.

[00126] In certain embodiments, the present disclosure provides a method of screening a drug, the method comprising determining the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

[00127] Factors involved in processing the drug are described herein. Methods for determining the level and/or activity of a factor are described herein.

[00128] In certain embodiments, the present disclosure provides a method of identifying a marker for assessing the response to a drug.

[00129] In certain embodiments, the present disclosure provide a method of identifying a marker for assessing the response to a drug, the method comprising identifying a factor for which the level and/or activity in extracellular vesicles is indicative of the response of the drug in a subject.

[00130] Factors involved in processing the drug are described herein. Methods for determining the level and/or activity of a factor are described herein.

[00131] In certain embodiments, the present disclosure provides a method of identifying a marker for assessing the response to a drug, the method comprising:

[00132] In certain embodiments, the present disclosure provides a marker identified by a method as described herein.

[00133] Standard techniques may be used for cell culture, recombinant DNA technology, oligonucleotide synthesis, enzyme assays, antibody production, peptide synthesis, tissue culture and transfection. Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.

[00134] The following references provide directions to assist with performing one or more methods described herein, and are hereby incorporated by reference: Molecular Cloning: A Laboratory Manual, 3rd ed., Vols 1, 2 and 3 J.F. Sambrook and D.W. Russell, ed., Cold Spring Harbor Laboratory Press, 2001; Laboratory Manual on Biotechnology (2008); Practical Immunology - A Laboratory Manual, Balakrishnan, Senthilkumar & Karthik, Kaliaperumal & Duraisamy, Senbagam. (2015) 10.13140/RG.2.1.4075.4728; Cell Biology (Third Edition) A Laboratory Handbook, 2006 Edited by Julio E. Celis; ISBN: 978-0-12-164730-8, Academic Press; and "Antibodies: A Laboratory Manual" Edward A. Greenfield; Cold Spring Harbor Laboratory Press, 2014; Lin, M. Z., Martin, J. L. and Baxter, R. C. (2015).

[00135] The present disclosure is further described by the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

EXAMPLE 1 - Isolation of exosomes

[00136] To isolate exosomes, a membrane affinity chromatography method has been developed which utilises biochemical features to selectively isolate exosomes from the sample matrix.

[00137] This approach reproducibly isolates high purity vesicles of comparable or superior yield to ultracentrifugation (for example as described in Enderle D, Spiel A, Coticchia CM, et al. Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method. PLoS One, 2015; 10: e0136133). [00138] As demonstrated in Figure 1, the membrane affinity chromatography protocol was used to isolate high purity microvesicles from plasma/serum or the extracellular media of cultured cells (HepRG cells).

[00139] Intact exosomes may be isolated from plasma/serum stored at -80°C. Isolation of exosomes from plasma post-storage does not significantly affect exosome integrity (for examples as described in Ge Q., et al. Molecules, 2014;19: 1568-75). This robustness reaffirms the capacity for retrospective exosomal marker analyses using archived samples. The capacity to extract exosomes from urine and saliva has also been extensively demonstrated (for example as described in Keller S, et al. J Transl Med, 2011 : 9:86).

[00140] For the isolation of exosomes from pre-filtered plasma/serum or cell culture supernatant, 1 mL of Buffer 1 (loading buffer) was added to 1 mL of sample and mixed by gently inversion. The sample/buffer mix was loaded onto the reservoir compartment of an exoEasy spin column, centrifuged at 500 x g for 1 min and the filtrate (flow- through) was discarded. Ten millilitres of Buffer 2 (wash buffer) was added to the reservoir compartment and centrifuged at 5000 x g for 5 min. The column was moved to a fresh collection tube and 400μ1 of Buffer 3 (elution buffer) was added to the membrane and incubated at room temperature for 1 min after which time the column was centrifuged at 500 x g for 5 min and the elutant was collected. When isolating from plasma/serum, the sample is centrifuged at 16,000g prior to isolation to remove larger EVs.

EXAMPLE 2 - Isolation of exosomes utilising immunocapture

[00141] A protocol to be used to isolate exosomes from plasma using immunocapture is set out below.

[00142] To specifically isolate (enrich for) circulating exosomes from a single origin, microbeads coated with an antibody against a cell surface antigen that is unique to an organ or tissue may be used (for example as described in Hong CS, Muller L, Boyiadzis M, et al. Isolation and Characterization of CD34⁺ Blast-Derived Exosomes in Acute Myeloid Leukemia. PLoS One, 2014; 9: el 03310). [00143] For example, the asialoglycoprotein receptor (ASGR) is a lectin that binds and removes targeted glycoproteins from the circulation and is exclusively expressed on the surface of hepatocytes. Anti-ASGR antibodies are available from suppliers including Aviva (Cat# OACD01809) and Santa Cruz (Cat# sc-393849). Each recognises an external-facing epitope on ASGR, and is validated for immunoprecipitation.

[00144] An immunocapture protocol utilising microbeads coated with an anti-ASGR antibody to selectively isolate and enrich for hepatocyte-derived exosomes from human plasma is as follows:

[00145] For the isolation of hepatocyte specific exosomes from pre-filtered plasma or cell culture supernatant, 1 mL of sample is loaded onto the reservoir compartment of a Dynabead column with an anti-ASGR antibody with an external-facing epitope on ASGR1. The sample is then passed down the column using a μϊ ΑΧ^ Separator.

EXAMPLE 3 - Characterisation of exosomes

[00146] Figure 1 demonstrates the ability to isolate microvesicles using membrane affinity chromatography from HepG2 cells.

[00147] We have also successfully isolated microvesicles from the extracellular media of cultured immortalised human hepatocytes (HepaRG cells), and human plasma and human serum.

[00148] It was found that the diameter (30 to 100 nm) and round appearance of the isolated microvesicles were consistent with established morphological criteria for exosomes (for example as described in Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol, 2002; 2: 569-79).

[00149] The yield and size distribution of exosomes and other microvesicles isolated by membrane affinity chromatography and precipitation are demonstrated in Figure 2. In both cases, the mean particle size was -100 nm. [00150] Furthermore, western blot analysis (shown Figure 3) probing with an antibody raised against TsglOl, a validated exosome marker (for example as described in Vancells JC, Suarez ER, Embade N, et al. Characterization and Comprehensive Proteome Profiling of Exosomes Secreted by Hepatocytes. J Proteome Res, 2008; 7: 5157-66) supported the identity of the isolated microvesicles as exosomes. Specifically, the observed enrichment of TsglOl in microvesicles (Lane B) compared to cells (Lane C) is an established characteristic of exosomes.

EXAMPLE 4 - Circulating exosome-derived CYP3A4 protein expression predicts midazolam clearance

[00151] 1. Methods

[00152] (i) Study Protocol

[00153] Study Cohort and Medications. Healthy male participants (n = 30) aged 21 to 35 years old were enrolled into the study following screening by physical examination. Participants were required to refrain from use of drugs and herbal products, including tobacco and alcohol, grapefruit juice and consuming large amounts of cruciferous vegetables prior to and for the duration of the study. Participant characteristics are summarised in Table 1. Midazolam (Pfizer midazolam for injection) was purchased from Pfizer, NSW, Australia; rifampin (Rifadin) was purchased from Sanofi, NSW, Australia; clarithromycin (Klacid) was purchased from Abbott Laboratories, NSW, Australia.

Participant characteristics

[00154] Study Design. Exposure to a 1 mg oral dose of midazolam was assessed at baseline (Day 1) following a 7 day course of rifampin (Day 8) and following a 3 day course of clarithromycin (Day 15). Timed blood samples were collected prior to and for up to 6 hrs post midazolam dosing. Between day 1 and 8 participants self-administered rifampin (300mg QD PO), then between day 12 and day 14 participants self- administered clarithromycin (250 mg BD). Rifampin and clarithromycin plasma concentrations were determined pre-midazolam dosing on day 8 and 15, respectively.

[00155] Determination of midazolam exposure. 100 μΐ. of plasma sample was diluted in 300 μΐ. of methanol containing 0.1% formic acid and 7.5 ng/mL d₆-midazolam (assay internal standard) then vortexed for 3 min, then centrifuged at 16,000 g for 5 min.

[00156] Analytes in a 2.5 μΐ. aliquot of the resulting supernatant were separated from the sample matrix by ultra-performance liquid chromatography (UPLC) performed on a Waters ACQUITYTM BEH C18 column (100 mm χ 2.1 mm, 1.7 μιη; Waters Corp., Milford, USA) using a Waters ACQUITYTM UPLC system. Column elutant was monitored by mass spectrometry (MS), performed on a Waters Q-ToF Premier TM quadrupole, orthogonal acceleration time-of-flight tandem mass spectrometer (Q-ToF- MS) operating in positive electron spray ionisation (ESI+) mode. Selected ion data was extracted at the analyte [M+H]+ precursor m/z. Resulting pseudo-MRM spectra were analysed using Waters TargetLynx software. Plasma analyte concentrations were determined by comparison of normalised peak areas in participant samples to those of calibrators.

[00157] Determination of CYP3A4 protein expression in circulating exosomes. Exosomes were isolated from pre-dose plasma samples collected on day 1, 8 and 15 by membrane affinity chromatography using exoEasy isolation columns (Qiagen). Exosomal CYP3A4 protein was extracted post exosome isolation by lysing exosomes with PathScan® sandwich ELISA lysis buffer (Cell Signalling Technologies). Expression of exosomal CYP3A4 protein was quantitatively assessed by sandwich ELISA (Company).

[00158] The ex vivo activity of exosomal CYP and UGT proteins were examined using metabolite formation assays developed for the assessment of human liver microsomal enzyme kinetics. Exosomes were activated by incubating on ice for 30 min in the presence of the pore forming peptide alamethicin (50 μg/mg protein).

[00159] 4MU glucuronidation assay: Incubations in 200μΙ. contained 0.1M phosphate buffer (pH 7.4), 4mM MgC12, exosomes (O. lmg protein) and 4MU (5 to 300μΜ). Following a 5 min pre-incubation, reactions were initiated by the addition of 5mM UDPGA. Incubations were performed at 37°C in a shaking water bath for 120 min. Reactions were terminated by the addition of 2μΙ. of perchloric acid (70%). Samples were centrifuged at 4000g for 10 min. A 2.5μΙ. aliquot of the supernatant fraction was analysed by LC-MS.

[00160] Midazolam 1-hydroxylation assay: Incubations in 200μΙ. contained 0.1M phosphate buffer (pH 7.4), exosomes (O. lmg protein) and midazolam (1 to 50μΜ). Following a 5 min pre-incubation, reactions were initiated by the addition of ImM NADPH generating system (ImM NADP, lOmM glucose-6-phosphate, 2R7/mL glucose-6-phosphate dehydrogenase, and 5mM MgC12). Incubations were performed at 37°C in a shaking water bath for 45 min. Reactions were terminated by the addition of 400μΙ. of ice cold methanol containing 0.1% formic acid. Samples were centrifuged at 4000g for 10 min. A 7.5μΙ. aliquot of the supernatant fraction was analysed by LC-MS. [00161] Data analysis. Non-compartmental methods were used to estimate the area under the plasma-concentration time curve (AUC) and maximal concentration (C_max) for midazolam at baseline (Day 1), following a 7-day course of rifampin (Induction (IND) phase; Day 8) and following a 3-day course of clarithromycin (Mechanism based inhibition (MB I) phase; Day 15).

[00162] A multivariable linear regression model was used to predict midazolam AUC based on exosomal CYP3A4 protein expression with adjustment for factors associated with probe clearance (e.g. established genotypes, BMI, ethnicity). Leave-one-out cross- validation was utilised to evaluate the predictive performance of these variables which are summarised as the percent of variability explained (R ), mean squared error and bias.

[00163] 2. Results: [00164] (i) Trial Conduct

[00165] Thirty male subjects completed the study. Three subjects experienced minor adverse events that were attributed to study interventions, but that did not affect their completion of the study (one participant experienced discomfort at cannulation site on Day 8, one experienced dry mouth during the rifampin dosing phase, and one experienced stomach discomfort during clarithromycin dosing phase).

[00166] Adherence to rifampin and clarithromycin dosing was determined by assessment of analyte concentrations in pre-midazolam dosing blood samples on Day 8 and Day 15, respectively. Mean (± standard deviation; S.D.) plasma rifampin and clarithromycin concentrations were 513 ± 146 μg/L and 334 ± 81 μg/L, respectively. Observed concentrations were consistent with predicted exposure profiles for these drugs with the respective dosing regimens, and are indicative of good adherence in all participants.

[00167] (ii) Baseline midazolam exposure, as per Table 2, the mean AUC and Cmax values defining baseline midazolam exposure were 1029μg/L/hr and 6.58 μg/L, respectively. [00168] (iii) Induction and inhibition of midazolam exposure, following rifampicin dosing (300mg QD) for 7 days, the mean AUC for midazolam was reduced by 3.2-fold and following clarithromycin dosing (250mg BD) for 3 days, the mean AUC for midazolam was increased by 74%.

Table 2 - Pharmacokinetic parameters describing midazolam exposure

[00169] (iv) Capacity to predict midazolam AUC using exosomal CYP3A4 protein expression, mRNA expression and ex vivo activity.

[00170] Figure 4 shows the concordance between exosome derived CYP3A4 biomarkers (mRNA expression, protein expression, and ex vivo activity) and midazolam apparent oral clearance (CL/F). R² values for the correlations of exosome derived CYP3A4 protein expression (Figure 4 A), exosome derived CYP3A4 mRNA expression (Figure 4B), and ex vivo CYP3 A4 activity (Figure 4C) with midazolam CL/F were 0.905, 0.787, and 0.832, respectively. The concordance between the different exosome derived CYP3A4 biomarkers was also assessed; R2 values for the correlation of ex vivo CYP3A4 activity (Figure 4D) and exosome derive CYP3A4 mRNA expression (Figure 4E) with exosome derived CYP3A4 protein expression were 0.928 and 0.794, respectively, while the R2 value for the correlation of ex vivo CYP3A4 activity with exosome derived CYP3A4 mRNA expression (Figure 4F) was 0.638.

[00171] Figure 5(A-C) demonstrates the concordance between the change in (Δ) exosome derived CYP3A4 biomarkers and the change in midazolam CL/F post- / pre- rifampicin dosing. R2 for the correlation of the change in exosome derived CYP3A4 biomarkers and the change in midazolam CL/F was invariably >0.828. The R2 value for the correlation of the change in exosome derived CYP3A4 biomarkers post- / pre- rifampicin dosing (Figure 5D-F) was invariably > 0.959.

[00172] To place this in context, despite extensive study there are no genotypes that are known to explain any meaningful proportion of inter-individual variability in CYP3 A4 activity and existing endogenous markers are only able to explain a small proportion (R2 0.1 to 0.4) of CYP3A4 inter-individual variability.

[00173] In contrast, these data indicate that assessment of exosomal CYP3A4 protein can define the majority of inter-individual variability in the activity of this enzyme. In Figure 3b we further demonstrate the ability of exosomal CYP3A4 expression to define the majority (>90%) of the variability in the extent of CYP3A4 induction following treatment with rifampicin.

[00174] The prediction performance of the exosomal CYP3A4 protein biomarker shown in Figure 4 and 5 appears to be significant and importantly does not require the trial subject to be administered a restricted drug.

[00175] Using Taq-man quantitative reverse transcriptase PCR (q-RT-PCR) we have also confirmed the presence of CYP3A4 mRNA in exosomes isolated from the extracellular media of cultured untransfected HepaRG cells and human plasma pre- and post- rifampicin dosing (300mg QD PO for 7 days).

[00176] HepaRG cells were treated either with DMSO or 15 μΜ Rifampicin for 96 hours in exosome depleted medium and exosomal RNA were isolated from the culture medium 96 h post-treatment, using the ExoRNeasy serum/plasma maxi kit (Qiagen). Total RNA from cells were also harvested at the same time point, using Trizol (Invitrogen). All steps were performed according manufacturers' instructions. Spectrophotometry (GeneQuant II spectrophotometer) was used to determine the concentration and purity of all RNA samples. The RNA was reverse transcribed to cDNA using the Superscript™ VILO™ cDNA Synthesis Kit according to manufacturer's protocol (Invitrogen, Cat no: 11754050) and the CYP3A4 mRNA in exosomes were then quantified.

[00177] Firstly, the reverse-transcribed cDNA was pre-amplified by PCR using the CYP3A4 TaqMan® Gene Expression Assay (Life Technologies, Assay ID:Hs00604506_ml, Cat no: 4453320) and TaqMan™ PreAmp Master Mix (Life Technologies, Cat no: 4391128). Briefly, the 50 μΐ reaction mixture contained l-250ng cDNA, 25ul Taqman PreAmp Mater mix (2x), Taqman CYP3 A4 gene expression assay consisting the primer and probe (20x). The PCR reaction was initially incubated at 95°C for 10 minutes, followed by 14 cycles of 95°C for 15 seconds and 60°C for 4 minutes.

[00178] The pre-amplified PCR product was then diluted in 1 :5 ratio with lxTE buffer, and quantified using quantitative reverse transcription PCR (qRT-PCR) with the TaqMan™ Gene Expression Master Mix (Life Technologies, Cat no: 4369016) and the same Taqman CYP3A4 gene expression assay primers and probes used for pre- amplification. The 20 μΐ reaction mixture contained 5 μΐ pre-amplified cDNA product, 1 μΐ Taqman Gene expression Assay (20x), 10 μΐ Taqman Gene Expression Master mix (2x). The PCR reaction was initially incubated at 50°C for 2 minutes and then 95 °C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The expression level of mRNA was analysed using the Rotor-Gene 6 software (Corbett Life Science). 18S and GAPDH were used as normalizing controls (TaqMan® Gene Expression Assay IDs Hs99999901_sl and Hs02786624_gl respectively). The quantification of CYP3A4 mRNA in cells was measured by qRT-PCR using the same Taqman gene expressions assays without the pre-amplification step.

[00179] The data is shown in Figure 6, which shows CYP3A4 mRNA expression in HepaRG cells versus exosomes.

[00180] Quantification of CYP3A4 mRNA in cells and exosomes isolated from the extracellular media of HepaRG cells similarly demonstrated that when treated with rifampicin (15μΜ) for 96 hours, CYP3A4 expression in cells and exosomes increased 18- and 16- fold, respectively. [Figure 4] These data demonstrate the presence of DME and transporter mRNA and protein in circulating exosomes, and the fundamental capacity of exosomes to track induction of DME expression.

[00181] The expression of CYP3A4 in human plasma at day 7 was detectable in the patient sample (Ct value 33). However, it was undetectable at the day 0 time-point. Since the fold change could not be calculated, the resulting PCR products were separated by gel electrophoresis to visualize the increase in CYP3A4 expression (see image below). CYP3A4 amplicon length was 119bp. The data are visually represented in the Western Blot shown in Figure 7.

[00182] Our analysis demonstrated that the abundance of CYP3A4 mRNA in exosomes isolated from plasma post-rifampicin dosing was 8-fold higher than expression in matched pre- rifampicin dosing samples.

[00183] Furthermore, there is potential to enhance the prediction performance of exosomal biomarkers by understanding and adjusting time lag between hepatic expression and circulating exosomal protein levels, by combining potential predictors such as exosomal mRNA and protein, and by specifically isolating hepatic exosomes.

[00184] Figure 8 shows UGT2B7 protein expression in exosomes isolated from human plasma pre- (A) and post- (B) rifampicin dosing.

[00185] The western blot shown in Figure 8 demonstrates protein expression for another important DME (UGT2B7) in exosomes isolated from the plasma pre- and post- rifampicin dosing (300 mg QD for 7 days). Non-quantitative densitometry analysis of resulting bands is consistent with an increase in UGT2B7 expression post rifampicin dosing. Importantly, although these data relate primarily to CYP3A4, analysis of exosomal DME and transporter biomarkers represents a general method that should be equally applicable across all DMEs and transporters.

[00186] There are currently no endogenous markers to assess induction for most DMEs and transporters, and probes are not available for a number of key DMEs which restricts the ability to evaluate the clinical importance of some DDIs. The generalisability of our data are further supported by a proteomic screening of exosomes isolated from rat hepatocytes, which demonstrated an abundance of xenobiotic metabolising CYP (2A1, 2B3, 2C11, 2D1 2D3, 2D10, 2D18, 2D26) and UGT (2B2, 2B3 and 2B5) enzymes, and the co-enzyme NADPH-cytochrome P450 reductase.

[00187] In contrast to previous attempts to correlate the activity of DMEs and transporters with peripheral markers of expression isolated from whole blood, our data presented in Figure 4a unexpectedly demonstrate that exosomes provide a representative snapshot of DME activity. Furthermore, the data for CYP3 A4 and UGT2B7 presented in Figures 5b and 6 support the ability of tracking induction of DME and transporter expression using exosome-derived mRNA and/or protein biomarkers.

[00188] Figure 9 shows data of a western blot of exosomal UGT2B7 protein (at ~50kD) isolated from two separate plasma samples from 6 healthy males. This western blot demonstrates highly reproducible isolation of UGT2B7 protein from all paired samples.

[00189] The ex vivo kinetics of UGT and CYP3A catalysed metabolism by exosomes isolated from plasma were assessed using 4-methylumbeliferone (4MU) and midazolam as probe substrates, respectively. Parallel experiments were performed using pooled HLM as the enzyme source for comparison of exosome- and liver- derived enzyme kinetics. Kinetic data for the UDPGA-dependent conversion of 4-MU to 4-MU glucuronide and the NADPH-dependent oxidation of midazolam to 1 -hydroxy midazolam by exosomes and HLMs were well described by the Michaelis-Menten equation (Figure 3). Mean kinetic parameters (Km, Vmax and CLint) describing 4-MU glucuronide and 1 -hydroxy midazolam formation by exosomes and HLMs are reported in in Table 3. Table 3

[00190] No metabolite formation was observed when incubations were performed in the absence of added cofactors. The rate of metabolite formation was diminished by >90% when incubations were performed using exosomes that were not activated by prior incubation with alamethicin on ice; the mean (±SD) maximal rates of 4-MU glucuronide formation by activated and native exosomes were 150 ± 7.6 and 6.5 ± 0.4 pmol/min/mg, respectively, while the mean (±SD) maximal rates of 1 -hydroxy midazolam formation by activated and native exosomes was 14.3 ± 0.7 pmol/min/mg and 0.35 ± 0.07 pmol/min/mg.

EXAMPLE 5 - Study for inter-individual variability in drug metabolising enzymes (DME) and transporter expression

[00191] While potentially of equal importance in terms of their impact, intra- and inter- individual variability have distinct clinical implications; inter-individual variability may necessitate different baseline dose requirements between individuals, while intra- individual variability may necessitate changes in dose requirement for an individual over time.

[00192] Similarly, when undertaking clinical studies to assess the capacity of a biomarker to define or track inter- or intra- individual variability in hepatic clearance, different protocols are required; assessment of intra-individual variability requires sampling on multiple visits, pre- and post- an intervention to perturb activity, typically in a smaller cohort of highly controlled individuals (e.g. 18 to 35 y/o healthy males of a single ethnicity), while assessment of inter-individual variability requires sampling at a single visit, typically in a larger more diverse cohort in terms of age, gender and ethnicity.

[00193] A study to evaluate the capacity of exosomal biomarkers to define inter- individual (population) variability in DME and transporter activities is set out below.

[00194] Clinical study protocol: Healthy male and female participants (n = 120) will be administered an oral drug cocktail comprising caffeine (lOOmg; CYP1A2), dextromethorphan (30mg; CYP2D6), fexofenadine (30mg; P-gp), losartan (25mg; CYP2C9), midazolam (lmg; CYP3A4) and omeprazole (20mg; CYP2C19) and timed blood samples will be collected. Blood, urine and saliva samples collected prior to the administration of the cocktail will be used to evaluate exosomal DME and transporter mRNA and protein expression.

[00195] Participants: As the intent in the study is to maximise inter-individual variability within a healthy population, this study will be undertaken in a cohort of healthy adult males and females aged 18 to 60 year with a BMI in the range 18 to 40. Study participants will need to be able to provide informed consent, and female participants will be required to provide a negative pregnancy test at the time of screening to avoid any potential complications associated with the administration of study drugs.

[00196] Sample Size: 120 participants are required to achieve a sufficiently narrow confidence interval for measuring the smallest association of potential interest. Based on preliminary evidence suggesting an R²>0.7 is achievable and clinical opinion, an R² of at least 0.5 (i.e. exosomal biomarkers define at least 50% of inter-individual variability of probe clearance) is considered to be of clinical value. In order to influence clinical practice the R² estimated should be sufficiently precise (i.e. confidence interval width less than 0.2) such that smaller correlations that are unlikely to be clinically meaningful are excluded. Based on a linear regression model a sample size of 120 will detect an expected R² value of at least 0.5 (alternative hypothesis) and to rule out an R² value of 0.25 or less (null hypothesis) with a power of 0.90 and alpha of 0.01.

[00197] In vitro studies: Complementary in vitro analyses will be performed using exosomes isolated from the extracellular media of HUH7, HepG2 and HepaRG cells grown in exosome depleted media (Thermo Fisher). These analyses will utilise established in vitro probes for a broader panel of DMEs including 7 CYP (1A2, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4) and 7 UGT (1A1, 1A4, 1A6, 1A9, 2B7, 2B10, 2B15) to evaluate the strength of the correlation between activity and exosomal mRNA and protein expression. Corresponding studies will also be performed to correlate exosomal and cellular mRNA and protein expression for other transporters (eg OATP 1B1 and 1B3, P-gp and BCRP). Preliminary data presented in Figures 3 and 4 support the capacity of exosomal biomarkers to define expression CYP and UGTs.

EXAMPLE 6 - Induction of DME and transporter expression

[00198] A study may be undertaken evaluate the capacity of exosomal markers to track induction as an important source of intra-individual variability in DME and transporter activity.

[00199] Clinical study protocol: CYP and P-gp activities will be assessed baseline (Day 1) and over a 10 day period around a 7 day course of the non-selective CYP and P-gp inducer rifampicin (600mg QD PO) dosed on days 2 to 8). On Day 1 and Day 8, healthy male participants (n = 24) will be administered the oral drug cocktail described in Example 5 and timed blood samples will be collected. Blood, saliva and urine sample collected prior to administration of the drug cocktail will be analysed to quantify exosomal CYP and P-gp mRNA and protein expression. Blood samples will also be collected and analysed to quantify exosomal CYP and P-gp mRNA and protein expression on Days 4, 6 and 10 to track the time course over which CYP and P-gp induction is reflected in circulating exosomes.

[00200] Participants: To minimise inter-individual variability, the study will be undertaken in healthy adult males aged 21 to 35 with a BMI in the range 18 to 30. Study participants must be able to provide informed consent and may be excluded from either study on the basis of established criteria.

[00201] Sample Size: Accounting for confounding variables, measurement error and population variability, and applying an intervention effect size of 1.5 (based on the reported effect of rifampicin on CYP and P-gp expression) a sample size of 24 will provide > 83 % power to demonstrate significant correlation of exosomal biomarkers with changes in CYP and P-gp activities.

[00202] In vitro studies: Complementary in vitro analyses will be performed using exosomes isolated from the extracellular media of HepaRG cells. These analyses will utilise a selection of established in vitro modulators of CYP 1A2, 2C9, 2C19, 2D6 and 3A4 expression that act via different mechanisms to evaluate the capacity of exosomal biomarkers to track different sources and magnitudes of modulation of CYP expression. Preliminary data presented in Figures 3b and 4 support the capacity of exosomal biomarkers to track induction of CYP expression caused by rifampicin. These studies will efficiently evaluate the capacity of exosomal biomarkers to track different types of modulation of CYP expression and will underpin future clinical studies.

[00203] Common methods relating to Examples 5 and 6.

[00204] DME and transporter activities: Reaction phenotyping will be undertaken to quantify DME and transporter activities. Timed samples analysed using a Waters AcquityTM ultra-performance liquid chromatography - quantitative time-of-flight mass spectrometry platform will be used to establish probe concentrations time profiles according to an established protocol.

[00205] Healthy participant ADME genotype: DME and transporter genotypes will be determined for all study participants using the iPLEX® ADME PGx Pro panel (Agenda Bioscience). This approach utilises a MassARRAY MALDI-TOF mass spectrometry platform for genotype detection (single nucleotide polymorphism and insertion/deletion) and quantification (copy number variation). The iPLEX® ADME PGx Pro panel defines 266 haplotypes for 36 key genes known to influence drug exposure, including all DMEs and transporters investigated in this project. Participant genotype will be included as a covariate to account for genetic polymorphisms known to affect DME and transporter function.

[00206] Exosomal DME and transporter biomarkers: Exosomes will be isolated from the extracellular media of cultured untransfected cells (HepG2, HUH7, HepaRG) or human biological samples (plasma, urine or saliva) by membrane affinity chromatography using exoEasy isolation columns (Qiagen). Exosomal RNA will be extracted by in situ exosome lysis during the isolation process using Qiazol lysis reagent (Qiagen). Exosomal protein will be extracted post isolation by lysing exosomes with PathScan® sandwich ELISA lysis buffer (Cell Signalling Technologies). RNA concentration will be determined using a NanoDrop 8000 UV-Vis spectrophotometer and integrity will be evaluated using an Agilent 2100 Bioanalyzer. Based on the DME or transporter's mRNA abundance, expression will be quantified by either reverse transcription polymerase chain reaction (RT-PCR) or droplet digital PCR (ddPCR). For both systems TaqMan Gene Expression kits incorporating the Pre-Amp system (Thermo Scientific) will be used to maximise amplification of biomarker cDNA. DME and transporter mRNA expression will be normalised by correcting for mean cycle time of the endogenous housekeeping genes 18S ribosomal RNA (18S) and glyceraldehyde 3- phosphate dehydrogenase (GAPDH). Expression of exosomal DME and transporter protein will be analysed qualitatively by western blot or quantitatively by sandwich ELISA. Western blot and sandwich ELISA protocols will be optimised based on individual antibody and conjugate characteristics.

[00207] Statistical Analysis: For healthy participant studies a standard non- compartmental method will be used to calculate the area under the plasma-concentration time curve (AUC) and probe CL/F. A multivariable linear regression model will be used to predict probe CL/F based on exosomal protein/mRNA expression for the corresponding DME or transporter with adjustment for all other factors associated with probe clearance (e.g. established genotypes, weight, gender, ethnicity). Leave-one-out cross-validation will be utilised to evaluate the predictive performance of these variables which will be summarised in terms of the percent of variability explained (R²), mean squared error and bias. [00208] Application to clinical samples: In order to demonstrate the clinical translation of the approach, the capacity of exosomal biomarkers for CYP3A4 and P-gp expression to predict exposure to small molecule kinase inhibitors (KI) will be assessed using archived plasma samples from a cohort of patients (n = 50).

EXAMPLE 7 - Enrichment of hepatocyte derived exosomes

[00209] A study may be undertaken to develop and evaluate a protocol to selectively isolate hepatocyte-derived exosomes, facilitating future assessment of hepatic vs extra- hepatic DME and transporter activity.

[00210] Development of anti-ASGR conjugated microbeads: In order to facilitate the selective isolation of hepatocyte-derived exosomes, magnetic microbeads coated with an anti-ASGR (hepatocyte specific cell surface protein) antibody will be developed. This will be achieved by conjugating a biotinylated anti-ASGR antibody (Aviva Systems Biology) to MACSflex immunocapture microbeads (Miltenyi Biotec).

[00211] Validation of selective hepatocyte derived exosome isolation: The capacity of anti-ASGR conjugated microbeads to selectively isolate hepatocyte derived exosomes will be assessed in vitro using exosomes isolated from multiple hepatic and non-hepatic (renal, colon and breast) cell lines transfected with unique fluorescent tagged tsglOl proteins. Expression of the tagged-tsglOl in exosomes isolated from each cell line will be evaluated in separately isolated, crude pooled (exosomes isolated from pooled extracellular media by membrane affinity chromatography) and hepatocyte enriched pooled (exosomes isolated from pooled extracellular media using anti-ASGR conjugated microbeads) samples. The impact of immunocapture on hepatocyte-derived exosome yield will be assessed by measuring the difference in fluorescence intensity of the liver specific fluorescent tag (i.e. GFP tag) between the separately isolated hepatic exosomes and the hepatocyte enriched pooled exosomes. The purity and capacity for enrichment of hepatocyte exosomes will be assessed by measuring the fluorescence intensity of the non-liver fluorescent tags (i.e. the non-GFP tags BFP, CFP and YFP) between the crude pooled and the hepatocyte enriched pooled exosomes. EXAMPLE 8 - Significance

[00212] The significance of some embodiments of the present disclosure is set out below.

[00213] Some embodiments will yield key translational outcomes across three distinct tiers. The most immediate and clear translation relates to the detection of induction DDIs. The preliminary data presented in Figures 3 and 4 demonstrate a fundamental capacity of exosomal biomarkers to accurately track induction of DME expression. The capacity to track induction of DME and transporter expression using exosomal biomarkers will streamline the development process for all new drugs by facilitating the assessment of a drug's induction 'perpetrator' risk during standard phase I dosing studies, thereby eliminating the need to undertake additional explicit induction studies.

[00214] It is in the public interest to expedite drug development as current inefficiencies lead to fewer new drugs to treat unmet medical needs and drugs of higher cost that threaten the sustainability of the healthcare system. For DMEs and transporters that do not have validated probes, exosomal biomarkers will provide additional information on the potential for induction DDIs over what is currently possible and thereby contribute to improving the safety of drugs entering clinical use.

[00215] At a broader and more clinical level, demonstrating the capacity of exosomal markers to define inter-individual variability in DME and transporter expression, will establish a novel strategy that provides complementary and unique insights regarding the activity of DMEs and transporters to refine the prospective optimisation of drug dosing in a manner that addresses the limitations of existing strategies.

[00216] For a number of key DMEs and transporters the additional information provided by exosomal biomarkers has the potential to markedly improve our ability to predict the optimal dose for an individual prior to starting therapy. Particularly given the emergence of high cost medicines such as ivacaftor ($22,500/month) and KIs ($2500 to $6500/month) that rely on CYP for clearance, exhibit considerable variability in exposure and have demonstrated benefit from dose optimisation, and considering the substantial financial burden associated with hospitalization resulting from adverse drug reactions ($3475/ADR), there are considerable financial imperatives supporting the translation of this exosomal biomarkers strategy to optimise drug dose.

EXAMPLE 9 - Clinical guidance on dose selection

[00217] Reaction phenotyping is not used clinically to guide dosing as it requires the administration of one or more prescription drugs (with regulatory/safety considerations) and collection of timed blood samples over many hours. The use of therapeutic drug monitoring is limited to a small number of drugs in specific settings, therapeutic drug monitoring requires the patient have started treatment (and typically to have achieved steady state) prior to the collection of a blood sample at a specific time relative to a dose for analysis by a specific assay that must be individually validated for each drug. This is particularly relevant as many newer drugs (eg efavirenz (_tl/2 >40hrs), simeprevir (t ₂ >40hrs) and sunitinib (t ₂ ~60hrs) have a prolonged t ₂ that necessitates dosing for up to four weeks before TDM can be used. To contextualise the importance of this delay, while therapeutic drug monitoring is used to guide efavirenz dosing, the inability to define an early optimal dose creates the risk of a low resistance barrier (resistance can emerge within lwk of initiating therapy) that necessitates supra-therapeutic pre- therapeutic drug monitoring dosing and precludes the use of efavirenz as a sole agent.

[00218] Genotype defines variability in DMET activity caused by functional changes in protein structure, but often poorly accounts for variability in activity due to differences in expression (e.g. CYP3A4). Practically, genotype and exosomal markers can both be determined using the same sample. Importantly (i) only a single blood sample is required with no strict conditions (e.g. prior drug administration or time-sensitive collection), (ii) evaluated using a common assay, and (iii) can inform optimal dosing prior to commencing therapy. The capacity of exosomal markers in combination with genotype (study participants will be comprehensively genotyped using the iPLEX ADME PGx Pro panel) to define variability in non-hepatic drug metabolising enzymes and transporter (DMET) activity caused by either expression level or functional differences.

[00219] The greatest advantage of the combination of exosomal markers (and also in some in stances in combination with pharmacogenetics) over therapeutic drug monitoring is the ability to generate high quality evidence of clinical validity and utility. The preclusive cost and effort required to develop prospective high quality evidence for therapeutic drug monitoring continues to be a major barrier to clinical translation. Our strategy may use routinely archived samples from RCTs to establish robust evidence defining the effect of variability in DMET activity on clinical outcomes. Evidence for exposure effects based on the key RCTs for a drug is highly influential to clinicians, clinical guidelines, regulators, and payors. For example, Pfizer recently retrospectively genotyped patients in pivotal axitinib RCTs for CYP3A genotype using archived blood samples. The analysis was unsuccessful due to the poor ability of genotype alone to predict CYP3A activity but it clearly demonstrates both the ability to retrospectively and efficiently generate high-quality evidence and the interest of industry and trialists in identifying baseline predictors of exposure.

[00220] For example, using midazolam as an example drug. In order to achieve a consistent exposure of midazolam between individuals:

• If the exosome CYP3A4 protein level is less than 2.0 ng/mL for a specific individual, the dose of midazolam should be halved for that individual.

• If the exosome CYP3A4 protein level is greater than 3.1 ng/mL for a specific individual, the dose of midazolam should be doubled for that individual.

[00221] This refers to values based on total exosomes. Using exosomes enriched for hepatic origin, the value may differ and can be readily determined.

[00222] Midazolam is used as an example here because of the data presented above demonstrating the relationship between exosome CYP3A4 levels and the dose required to standardise exposure. Similar studies can be conducted for other drugs.

[00223] The thresholds for other drugs (in particular those that it is more clinically important to standardise exposure) can also be determined. EXAMPLE 10 - Identifying drugs that are likely to cause drug-drug interactions by inducing (i.e. increasing level s/activity) of CYP3A4

[00224] One example with respect to the use of the methods described herein is for identifying drugs that are likely to cause drug-drug interactions by inducing (i.e. increasing level s/activity) of drug metabolising enzymes such as CYP3 A4.

[00225] Specifically, the methods may be used to identify strong and moderate inducers of an enzyme such as CYP3 A4.

[00226] This method may be applied in drug development to understand the general risks associated with using the drug.

[00227] For example, a blood sample is to be taken before the commencement of the drug to be evaluated as an inducer of CYP3A4. A blood sample is also taken in the same individual after taking the drug for a number of days (typically at least 7 days). This is undertaken for a group of individuals - typically at least 10 individuals. The exosome level/activity of CYP3A4 is compared between the on-therapy sample and the sample collected prior to commencing the drug. Based on our preliminary evidence if the average increase in the level of the exosome CYP3A4 is 80% (i.e. 1.8 times the original level) this should identify a drug that will be classified as a 'strong inducer' of CYP3A4. If the increase is between 20% to 80%, the drug will be classified as a 'moderate inducer' of CYP3A4.

[00228] The above is expected to apply for other drug metabolising enzymes and transporters (although the actual values for the increase that identify strong and moderate inducers are likely to differ). The above is based on measuring levels in total exosomes extracted from blood. mRNA and activity of CYP3A4 in the exosomes (instead of protein levels) can also be measured and obtain similar information.

EXAMPLEl 1 - Evidence of translational principle

[00229] A recent study of 27 patients with metastatic melanoma reported that a dabrafenib steady-state trough concentration (Css trough) of >48 ng/mL predicted the incidence of Grade >2 adverse events requiring dose reduction (ROC AUC = 0.94; Accuracy = 79%).

[00230] CYP 2C8 and 3A4, and P-gp are the major proteins involved in the clearance of dabrafenib. While CYP2C8 and P-gp expression can be partially explained by pharmacogenomics (PGx), there is currently no marker that robustly explains variability in CYP3A4 expression.

[00231] Exosomes alone or in combination with PGx can define variability in the activity of these proteins and be applied to archived clinical trial samples.

[00232] In Australia between 2015 and 2017 dabrafenib was dispensed to 1,355 patients, 189 were hospitalised due to supra-therapeutic dosing. The average cost of hospitalisation for a cancer patient due to an adverse drug reaction is $7082 AUD. Accounting for the cost of analysis in all new patients, avoiding 70% of these hospitalisations equates to an average cost saving to the Australian healthcare system from reduced hospitalisations using exosome informed dabrafenib dosing of $446,000 AUD annually.

EXAMPLE 12 - Addressing the limitations of therapeutic drug monitoring (TDM)

[00233] The greatest advantage of exosomes over TDM is the ability to generate high quality evidence of clinical validity and utility. The preclusive cost and effort required to develop prospective high quality evidence for the TDM has, and continues to be, a major barrier to clinical translation for this approach. Our strategy uses routinely archived samples from randomly controlled trials (RCTs) to establish robust evidence defining the effect of variability in CYP, UGT or transporter activity on clinical outcomes.

[00234] Having evidence for exposure effects derived from key RCTs supporting the use of a drug is important in influencing regulators, clinicians and clinical guidelines. As an example, in 2014 Pfizer retrospectively genotyped patients in pivotal axitinib RCTs for CYP3A genotype using archived blood samples. The analysis was unsuccessful due to the poor ability of genotype alone to predict CYP3A activity but demonstrates both the ability to retrospectively generate high-quality evidence and the interest of industry and trialists in identifying baseline predictors of exposure.

[00235] Although the present disclosure has been described with reference to particular embodiments, it will be appreciated that the disclosure may be embodied in many other forms. It will also be appreciated that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[00236] Also, it is to be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise.

[00237] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[00238] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[00239] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[00240] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments. [00241] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[00242] Future patent applications may be filed on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and/or by claiming a continuation status. It is to be understood that the following claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Nor should the claims be considered to limit the understanding of (or exclude other understandings of) the present disclosure. Features may be added to or omitted from the example claims at a later date.

[00243] Although the present disclosure has been described with reference to particular examples, it will be appreciated by those skilled in the art that the disclosure may be embodied in many other forms.

Claims

1. A method of assessing a response of a subject to a drug, the method

comprising:

2. The method according to claim 1, wherein the response to the drug comprises one or more of drug clearance, drug toxicity, drug efficacy, drug distribution and drug absorption and interaction of the drug with another drug.

3. The method according to claims 1 or 2, wherein the factor involved in processing the drug comprises an enzyme involved in metabolising the drug.

4. The method according to claim 3, wherein the enzyme involved in metabolising the drug comprises a cytochrome P450 enzyme.

5. The method according to claims 3 or 4, wherein the enzyme involved in metabolising the drug comprises one or more of CYP 1A1, 1A2, 2A1, 2A6, 2B3, 2B6, 2C8, 2C9, 2C11, 2C18 2C19, 2D1, 2D3, 2D6, 2D10, 2D18, 2E1, 3A4 and 3A5.

6. The method according to claim 3, wherein the enzyme involved in metabolising the drug comprises a UDP-glucuronosyltransf erase.

7. The method according to claims 3 or 6, wherein the enzyme involved in metabolising the drug comprises one or more of an enzyme from the following families: UGT1, UGT2, UGT1A, UGT2A, and UGT2B.

8. The method according to claim 7, wherein the enzyme involved in metabolising the drug comprises one or more of an enzyme from UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGTIAIO, UGT2B4, UGT2B7, and UGT2B15.

9. The method according to claim 3, wherein the enzyme involved in metabolising the drug comprises one or more of an enzyme from an acetyltransferase, a glutathione S-transferases and a sulfotransferase.

10. The method according to any one of claims 3 to 9, wherein an increase in the level and/or activity of the enzyme involved in metabolising the drug is indicative of one or more an increased drug clearance, reduced drug toxicity, and reduced drug efficacy.

11. The method according to any one of claims 3 to 9, wherein the drug is a prodrug and an increase in the level and/or activity of the enzyme involved in metabolising the drug is indicative of one or more increased drug toxicity and increased drug efficacy.

12. The method according to claims 1 or 2, wherein the factor involved in processing the drug comprises a factor involved in drug uptake and/or drug efflux.

13. The method according to claim 1, 2 or 12, wherein the factor involved in processing the drug comprises one or more of an ATP -binding cassette (ABC) transporter and a solute carrier (SLC) transporter.

14. The method according to claim 13, wherein the factor involved in processing the drug comprises one or more of ABCBl (p-glycoprotein, MDR1), ABCB11, ABCC1 (MRPl), ABCC2 (MRP2), ABCC3 (MRP3), ABCC4, ABCC5 (MRP5), ABCG2 (BCRP), SLCIOAI, SLC10A2, SLC22A1, SLC22A2, SLC22A3, SLC22A4, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLC22A11, SLC47A1, SLC47A2, SLC01A2, SLC01B1 (OATP1B1), SLC01B3 (OATP1B3), and SLC02B1.

15. The method according to any one of claims 1 to 14, wherein the method comprises assessing the level and/or activity of a factor involved in processing of a different drug which is processed in a similar manner to the drug.

16. The method according to any one of claims 1 to 15, wherein the method comprises assessing the level and/or activity of a factor involved in processing of the drug when the subject has been exposed to an inducing agent.

17. The method according to claim 16, wherein the inducing agent is the drug.

18. The method according to any one of claims 1 to 17, wherein the extracellular vesicles comprise hepatic extracellular vesicles.

19. The method according to any one of claims 1 to 18, wherein the method comprises enriching for hepatic extracellular vesicles.

20. The method according to claim 19, wherein the method comprises enriching for hepatic extracellular vesicles using an anti-ASGR antibody.

21. The method according to any one of claims 1 to 20, wherein the extracellular vesicles comprise extracellular vesicles from one or more of blood, plasma, serum, urine and saliva.

22. The method according to any one of claims 1 to 21, wherein the determining of the level and/or activity of the factor involved in processing the drug comprises determining the level of the factor in the extracellular vesicles and/or determining the level of RNA encoding the factor in the extracellular vesicles.

23. The method according to any one of claims 1 to 22, wherein the method further comprises assessing one or more other markers in the subject.

24. The method according to claim 23, wherein the one or markers comprise a genetic marker.

25. The method according to any one of claims 1 to 26, wherein the method is used to assess intra-individual variability of response to the drug and/or inter- individual variability of response to the drug.

26. Use of extracellular vesicles to assess a response of a subject to a drug.

27. Use of a biomarker in extracellular vesicles to assess a response of a subject to a drug.

28. A method of selecting a dose of a drug to be administered to a subject, the method comprising assessing the response of the subject to the drug according to the method of any one of claims 1 to 25 and selecting the dose of the drug to be administered to the subject on the basis of the response of the subject to the drug.

30. A method of selecting a dose of a drug to be administered to a subject, the method comprising selecting the dose of the drug to be administered to the subject on the basis of the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

31. A method of selecting a dose of a drug to be administered to a subject, the method comprising:

32. A method of administering a drug to a subject, the method comprising assessing the response of the subject to the drug according to the method of any one of claims 1 to 25 and administering the drug to the subject at a dose selected on the basis of the response of the subject to the drug.

33. A method of administering a drug to a subject, the method comprising administering the drug to the subject at a dose selected on the basis of the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

34. A method of administering a drug to be administered to a subject, the method comprising:

35. A method of treating a subject with a drug, the method comprising assessing the response of a subject to the drug according to the method of any one of claims 1 to 25 and treating the subject with the drug at a dose selected on the basis of the response of the subject to the drug.

36. A method of treating a subject with a drug, the method comprising treating the subject with the drug at a dose selected on the basis of the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

37. A method of treating a subject with a drug, the method comprising:

38. A kit for performing a method according to any one of claims 1 to 25, 28 to 37.

39. The kit according to claim 38, wherein the kit comprises one or more of the following components:

(i) one or more reagents for isolating and/or enriching extracellular vesicles;

(iii) instructions for using the components of the kit.

40. Use of a factor involved in the processing of a drug in extracellular vesicles as a biomarker for the response of a subject to the drug.

41. A method for identifying an agent that induces a factor involved in processing a drug, the method comprising:

42. The method according to claim 41, wherein the method is used to identify the agent as a moderate or strong inducer of the factor involved in processing a drug.

43. A method of assessing the ability of an agent to induce a factor involved in processing a drug, the method comprising determining the ability of the agent to induce the level and/or activity of the factor involved in processing a drug in extracellular vesicles.

44. A method of screening a drug, the method comprising determining the level and/or activity of a factor involved in processing the drug in extracellular vesicles from the subject.

45. A method of identifying a marker for assessing the response to a drug, the method comprising identifying a factor for which the level and/or activity in extracellular vesicles is indicative of the response of the drug in a subject.

46. A method of identifying a marker for assessing the response to a drug, the method comprising:

47. A marker identified according to claim 46.