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CHAPTER 2

Functional Studies withHuman Isolated Tissues toBetter Predict Clinical Safetyand Efficacy

DAVID C. BUNTON

Biopta Ltd, Weipers Centre, Bearsden Road, Glasgow G61 1QH, UKE-mail: davidbunton@biopta.com

2.1 IntroductionResearch using human fresh tissue represents one of the fastest growingareas of drug discovery and development. There are two key drivers in the useof human tissue: rstly, the failure of the current approach to drug devel-opment which demands new approaches to reduce clinical attrition, andsecondly, the drive towards biomarkers for personalised medicine.The dominant approach to drug development, based on primary screening

in high-throughput models and secondary screening in animals, has previ-ously produced numerous ‘blockbuster’ drugs, but clinical attrition rates of95% are no longer viewed as sustainable. Human disease-relevant tissue isincreasingly viewed as a way to decrease clinical failures, particularly duringphase II and III where poor efficacy has been partly attributed to an over-reliance on animal models. The second major factor is the drive towards theuse of biomarkers and personalised medicines; as the search for

RSC Drug Discovery Series No. 41Human-based Systems for Translational ResearchEdited by Robert Coleman© The Royal Society of Chemistry 2015Published by the Royal Society of Chemistry, www.rsc.org

17

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blockbusters diminishes, the need for targeted therapies based on predictivenon-clinical and clinical human data increases (Table 2.1).Fresh, intact, functional human tissue assays aim to bridge the gaps

between in vitro cell-based studies, in vivo animal studies and clinicaltrials. Such tissues offer advantages over simpler cell-based models, avoidspecies differences and truly reect the diverse patient population. Forexample, cell-based assays lose functional relevance and do not retainimportant cell-to-cell relationships in a 3-D structure. Reconstructed orengineered 3-D tissues produced from stem cells or as reconstructedorganoids fail to reect the actual disease phenotype and diversity ofresponses found in healthy and diseased tissues obtained directly frompatients.Functional tissue studies also offer advantages over animal experiments.

Clearly, animal in vivo experiments provide important information on drugbehaviour at a system level, and the industry will continue to rely on thesemodels to investigate central nervous system control of the cardiovascular,respiratory and gastrointestinal systems. Animal models do, however,present one major problem. Few, if any, can truly be considered humandisease models that accurately and comprehensively reect human path-ophysiology. At best they are complex models representing some mecha-nistic features of a disease process, typically with articial initiators of the‘disease’ process (e.g. chemical irritation) and certainly not the chronictimeframe of most major diseases. Fresh human tissues, obtained directlyfrom the target patient population, avoid many of these problems andwhile there are some limitations in the range of experiments that can beconducted, there appears to be an urgent need for human tissue studies tobridge the gap between cell-based approaches, in vivo animal experimentsand clinical trials.Human tissue studies are also of value for non-clinical safety pharma-

cology studies in support of ICHS7A and ICHS6 guidelines on non-clinicalsafety studies for pharmaceuticals and biotechnology products, respectively.Information generated by human tissues may even be included in a clinicaltrial application or marketing authorisation application. For example, theFDA review of gastrointestinal drugs used studies in fresh human coronaryarteries to evaluate the safety of 5-HT4 agonists (Table 2.2).1

Despite these encouraging developments, functional human tissue assaysrepresent something of a conundrum: fresh intact human tissues aregenerally accepted as the gold standard non-clinical models; however, thetests are not yet deemed an essential part of the non-clinical development ofmost drugs.The present chapter describes the wide range of uses of human isolated

fresh tissues throughout drug development, including their traditional rolein target discovery and validation, through to their increasing use as a func-tional model during lead optimisation, safety pharmacology and preclinicalstrategies for stratied medicines. The chapter also describes some of thespecic challenges in acquiring and using fresh tissues.

Tab

le2.1

Sourcesof

hum

anfreshisolated

tissue

forresearch

Sourcesof

human

freshisolated

tissue

Organ

isation/Provider

Typical

applications

Advantages

Disad

vantages

Post

mortem

Patholog

yde

partmen

tor

biob

ank

Targetiden

tication

andvalida

tion

rather

than

functional

stud

ies

Awiderange

oftissue

sareavailable

whicharedifficu

ltor

impo

ssible

toob

tain

from

surgery

Post

mortem

interval

(PMI)

forcolle

ctionof

tissue

istypically

hou

rsrather

than

minutes;tissu

equ

alitymay

becomprom

ised

.Not

suitab

lefor

mostfunctional

assessmen

tsbu

tmay

betheon

lyavailable

access

route

Residua

lsu

rgical

Surgical

depa

rtmen

t,pa

tholog

yor

biob

ank

Targetiden

tication

andvalida

tion

;stud

iesin

target

diseasetissue

;functional

stud

ies

ofsafety

andeffi

cacy

Rap

idcolle

ctionof

tissue

s;widerange

ofdiseased

tissue

savailablewhichare

avaluab

lemod

elfor

drug

Mainpu

rposeof

colle

ctionis

diag

nosis,a

lign

ingcolle

ction

proced

urewithrequ

irem

ents

forresearch

canbe

difficu

lt–

oen

obtain

leover

tissue

samplefollo

wingpa

tholog

ist’s

analysis

Non

-transp

lantable

orga

ns

Organ

procurem

ent

orga

nisation(O

PO)

orbiob

ankthat

liaiseswithOPO

Functional

stud

iesof

safety

andeffi

cacy;

may

also

beus

edfor

developm

entof

surgical

implan

tsor

med

ical

devices

Freshnessan

dvolume

oftissue

;ability

toreceivehealthycontrol

tissue

s

Freq

uency

oftissue

ismuc

hless

than

forresidu

almaterialan

dcostsaremuc

hgreater

Clinical

biop

sies

Clinical

research

orga

nisation;c

linical

research

depa

rtmen

tof

somehospitals

Biomarke

rstud

ies

aspa

rtof

aclinical

trial;colle

ctionof

diseased

tissue

sfrom

specicpa

tien

tgrou

ps,

e.g.

psoriasisor

atop

icde

rmatitis

Collectioncondition

scanbe

specied

exactly.

Allo

wsmoreextensive

investigationsof

safety

andeffi

cacy

inpa

tien

tsdu

ringaclinical

trial,

e.g.

measu

remen

tof

surrog

atemarke

rsof

clinical

effects

Tim

ean

dcostsinvolved

inorga

nisingan

dplan

ningthe

stud

y,very

smallam

ounts

oftissue

may

beavailablean

dmay

not

bepracticalto

retrieve

thetissue

ofinterest

Functional Studies with Human Isolated Tissues 19

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Tab

le2.2

Hum

antissue

testsus

edin

supp

ortof

safety

pharmacolog

ysu

bmission

s

Hum

antissue

Endpo

int

Regulatorytest

Regulatorydo

cument

Cardiac

muscle/

Purkinje

bres

Actionpo

tential

measu

remen

ts,

espe

cially

duration

andrhythm

Cardiovascu

lartelemetry

core

batteryin

vivo

anim

alICH

S7A

Section2.7.2

Coron

aryartery

Coron

aryartery

vasosp

asm

lead

ing

tomyocardialinfarction

Cardiovascu

lartelemetry

core

batteryin

vivo

anim

al–

butwou

ldon

lypick

this

upindirectly

throug

hch

ange

inbloo

dpressu

rean

dheart

rate

ICH

S7A

Section2.7.2

Resistance

arteries

Vasocon

striction/dilatation

Con

trol

oforga

nbloo

dow

andmain

determ

inan

tof

periph

eral

vascular

resistan

ce(m

eanarterial

bloo

dpressu

re¼

cardiacou

tput

�total

periph

eral

resistan

ce)

Cardiovascu

lartelemetry

core

batteryin

vivo

anim

al–measu

res

ofbloo

dpressu

rean

dbloo

dow

(byplethysmog

raph

y)

ICH

S7A

Section2.7.2

Bronch

iBronch

ocon

striction/dilatation

Respiratory

core

batteryin

vivo

anim

alICH

S7A

Section2.7.3

Stom

achor

intestinal

smoo

thmus

cle

GImotility–de

tectionof

unde

sirable

side

effects

ontran

sittimesu

chas

diarrhoe

aan

dconstipation

Recom

men

dedas

asu

pplemen

tary

stud

yas

part

ofICHS7

ASection2.8.2.3–tran

sittime

ICH

S7A

Section2.8.2.3

Skin

inam

mationtests

Measu

remen

tof

cytokineprod

uction

Not

part

ofph

armaceu

ticalregu

lation

sbu

tsimilar

assays

recommen

dedforman

ybiolog

icalsus

ingin

vivo

mod

elsof

immun

esystem

REACH

EU

directive

Gut

inam

mationtests

Measu

remen

tof

cytokineprod

uction

Recom

men

dedas

asu

pplemen

tary

stud

yas

part

ofICHS7

ASection2.8.2.3–

gastrointestinal

injury

potential

ICHS7

ASection2.8.2.3

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Functional Studies with Human Isolated Tissues 21

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2.2 Sourcing, Storing and Transporting Human FreshTissues: The First Step is the Most Important

Human fresh, intact isolated tissues appear an obvious choice of testsystem, yet the use of such tissues has until recently remained the preserveof a small number of specialist academic centres willing and able to buildtheir lab activities around the irregular and oen unpredictable availabilityof fresh tissue specimens from surgery or transplants. In this chapter‘human tissue’ is dened as those samples which contain groups ofdifferent cell types that together form a working tissue, e.g. an isolatedblood vessel, a blood sample, a section of gastrointestinal mucosa or anisolated airway.Functional studies are almost always carried out in fresh tissue samples

and only rarely in samples that have been frozen, allowing biologicalmechanisms to be challenged by test compounds. This type of study istechnically challenging and with current preservation techniques, the studiesare oen of short duration, typically hours or days. For non-clinical func-tional studies to be of translational value to clinical effects, the relevance ofthe tissue is dependent on the quality of the collection, storage and prepa-ration of the tissue prior to experiments. This is carried out in such a wayas to minimise functional and/or structural changes within the tissue withthe aim of retaining the in vivo properties. Extensive characterisation of thetissue is oen carried out prior to or during experiments to ensure that therelevance has been retained, for example a RIN score (RNA Integrity Number)of 7.0 or higher may be appropriate to ensure the quality of RNA isolatedprior to any investigation into gene expression levels in healthy and diseasedtissue samples. Sample quality has been found to vary quite markedly, insome cases with a low percentage of samples being considered suitable foruse in research.2

In many instances, the rapid turnaround of tests using fresh tissue meansthat some sort of functional qualication of tissue viability must be used asthe primary inclusion/exclusion criterion, with follow-up by conventionalmeans such as RIN scores or histology being conducted at a later time. Forexample, functional pharmacology experiments in blood vessels may includean assessment of the responses from cell types most sensitive to damagesuch as endothelial release of nitric oxide.The provision of human tissue samples for research is now much easier

and generally of a higher quality than 10 years ago. Numerous organisationscan carry out customised collections from specic patient groups.3

There remains, however, a strong perception that insufficient tissue isavailable to research, when in fact the problem is one of coordination andawareness. In the UK there are approximately 2 million animals used inexperiments each year, in contrast to only a few thousand human tissuespecimens collected for research (estimated from comparisons between therelative number of peer-reviewed publications in animals and human

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tissues). Yet the problem is not one of tissue availability – in fact there areover 600 000 surgical residual human tissues generated annually in the UK4

and over 95% of patients are happy to donate their tissues to medicalresearch, including commercial research. The under-utilisation of humantissues stems from logistical difficulties in collecting tissue, lack of incentiveon the part of healthcare staff to carry out additional duties on top of busyworkloads and the need for exible working patterns by researchers makinguse of fresh tissue. Animal experiments are more convenient but this isoen at the expense of biological relevance. In the UK, there are approxi-mately 700 000 surgical procedures each year (according to NHS HospitalStatistics), but tissue is made available from a tiny fraction of this, perhapsless than 1–2%. Patients are very supportive of human tissue research, withone survey suggesting that over 95% of patients are willing to donateresidual tissues.5

While access to xed and frozen tissues continues to become easier, accessto fresh human tissue continues to be difficult, and the costs, time andlogistical problems that exist when using fresh tissue have not yet beenovercome. The provision of surplus tissue for drug discovery is correctly ofsecondary importance to the surgeon or pathologist; therefore, there can bea conict of interest between the pathologist and the researcher that ulti-mately limits the availability of fresh tissue for researchers and tissuesuppliers. On a positive note, the recent increase in the number of tissuebanks, although primarily focused on creating banks of frozen or xedtissues, might, in the near future, provide a more structured system for thesupply of fresh tissues. Although several commercial companies offer tosupply processed, xed or frozen human tissue, those seeking fresh humantissue for their research oen need to create a bespoke tissue supply networkor outsource the experimental work to a contract research organisation(CRO) with a developed tissue network.Research teams must be exible, to use tissue as and when it becomes

available. Several studies have examined the use of various transplant solu-tions for the preservation of tissue function; however, little improvement hasbeen made on standard physiological solutions. Tissue >2 mm3 exceeds thelimits of diffusion and, therefore, supplementary methods, such as contin-uous gassing, perfusion or cooling at 4–8 �C, are oen necessary. Recentinnovations, such as articial haemoglobin and haemoglobin crosslinked tosuperoxide dismutase, might help to prolong tissue function but this has notyet been demonstrated. Optimisation of cryopreservation procedures forfresh tissues offers one further potential route for increasing the experi-mental window. Lung and vascular tissues retain many of their functionalresponses when thawed.There is clearly sufficient fresh tissue and public support to allow its use as

an essential and routine element of drug development; however, greatercooperation between biobanks, regulatory bodies, CROs and pharmaceuticalcompanies can make the collection of fresh tissues for research ‘the norm’rather than ‘the exception’.

Functional Studies with Human Isolated Tissues 23

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2.3 Common Experimental Approaches toInvestigations in Isolated Fresh Human Tissues

2.3.1 Tissue Baths and Wire Myographs

Tissue baths and wire myographs are the most commonly used experimentalmethod to study function in intact fresh tissues (Figure 2.1). Classicalfunctional assays are strain-gauge systems that measure the contraction andrelaxation of isolated muscle segments following application of a testcompound.6 In combination with imaging systems, a range of useful data(e.g. force, tension, pressure, volume, growth) can be collected in all types ofhuman muscle tissue including smooth muscle (blood vessels, gut, airways,renal and reproductive tubules), cardiac muscle and skeletal muscle.Early development of antihypertensive drugs relied heavily on assays of

isolated large blood vessels usually removed during surgery or post-mortem,but the application of such techniques in drug development has until recentlybeen fairly sporadic. With the discovery that small resistance arteries(between 150 mm and 300 mm) are primarily responsible for the control oforgan-specic blood ow and overall vascular regulation of blood pressure, ithas been possible to screen greater numbers of compounds for their vasculareffects. Sufficient numbers of isolated functional blood vessels with measur-able responses to pharmacological agents can be obtained from very smallbiopsies of tissue from a range of organs including the skin, kidneys, lung,heart and skeletal muscle.7–9 Investigations in small arteries have not onlycontributed to our understanding of vascular biology (e.g. the importance ofthe vascular endothelium as a major modulator of vascular function,10 thenitric oxide pathway,11 the endothelin pathway,12 vascular endothelial growthfactor13 and endothelium-derived hyperpolarising factor,14 but also representa valuable screening tool for the prediction of vascular safety concerns orefficacy. The detection of vascular effects in resistance arteries representsa potential safety concern, because even small changes in systemic bloodpressure can be large enough to increase mortality and morbidity.These same techniques can be applied to other tissue types. Respiratory

tissue is signicantly more delicate than blood vessels and therefore providesa major technical challenge; however, the same sensitive techniques used forblood vessels have been adapted to small airways.15 Application of test drugson respiratory tissue from patients suffering from chronic obstructive airwaydiseases and asthma have allowed bronchodilators to be assessed, providingsubtle mechanistic insight in living human respiratory tissue that could nototherwise be revealed.16 Lung parenchymal tissue also has contractile prop-erties and the application of drug candidates to tissue strips can provideinsight into drug effects.17

In addition, human cardiac muscle (donated from recipient heartsremoved during transplant procedures, or from transplant donor hearts thatare not suitable for transplant) and atrial sites are available (atrial append-ages may be removed during cardiac catheterisation).18

Figure 2.1 Isolated tissue specimens mounted in (a) tissue (‘organ’) baths and(b) wire myographs for measurement of force generation bycontractile tissues. (c) shows a strip of male urethra mounted in anorgan bath; contractions or relaxations of the tissue can be measuredand graphed to model concentration–response relationships. (d)shows data generated from contractile responses of circular andlongitudinal urethral smooth muscle in the presence of increasingconcentrations of the b1-adrenergic agonist phenylephrine (PE).

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Electrical activity, recorded by means of tiny microelectrodes applied tostimulated tissue, can monitor the effect of applied drugs on various loca-tions on isolated cardiac segments such as Purkinje bres, allowingmeasurements of cardiac action potentials.19 The race between the growinguse of functional intact human tissues and efforts to recreate such

Functional Studies with Human Isolated Tissues 25

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phenotypically relevant cardiac tissue through stem cell derived car-diomyocytes will be intriguing.Although most experiments in tissue baths and wire myographs are short-

lived, lasting perhaps 8–10 hours, some researchers have sustained freshintact cultured tissues allowing the measurement of not only contractileeffects but also the release of endogenous mediators such as cytokines20 orchanges in growth or atrophy. This creates opportunities for researchers toinvestigate toxic effects of compounds following repeated exposures ratherthan ‘single shot’ safety pharmacology end points.

2.3.2 Perfusion Myographs

Perfusion systems aim to closely mimic in vivo physiological conditions andare used where dynamic interactions between the tissue, blood ow, nutri-ents, metabolites or gas exchange are required to properly model drug–tissueinteractions. For example, perfusion myography is a technique where iso-lated tubular tissues such as blood vessels can be exposed to test drugsapplied either to the endothelial surface or adventitial surface (Figure 2.2).Physiological pressures and ow rates can be applied to the tissue, activatingmechanisms in the endothelium that are silent in static in vitro systems, suchas endothelial shear stress and myogenic tone.21 Sophisticated imaginganalysis allows measurements not only of vessel diameter but also intracel-lular signalling and changes in vascular permeability. In addition, theinteraction between isolated tissues and other cell types can be examined.A similar ex vivo perfusion technique in mouse carotid arteries has been usedto investigate monocyte adhesion and rolling, a crucial step in atheroscle-rosis,22 and it seems feasible that a similar approach could be applied tohuman fresh arteries.

2.3.3 Organoculture Systems and Precision-Cut Tissue Slices

A number of organ culture systems have been used to study local inam-matory processes in tissues such as skin,23 gut mucosa,24 synovium, carti-lage25 and adipose tissue.26 Accurate assessment of inammation using theculture method critically depends on careful handling and preparation oftissue. Because these methods retain the normal behaviour and growth ofprimary human tissue they provide a more relevant screening system thanavailable articial tissue models27 and up to 40–50 individual ‘biopsies’ oftissue per donor can be cultured, given the volume of fresh tissue potentiallyavailable from surgery or transplant.In vitro application of various challenges, e.g. ischaemia, lipopolysac-

charide, or drug candidates, have been assessed by measuring the releaseof cytokines, inammatory mediators or metabolites by radioimmuno-assay following culture of tissue slices.28 These can be compared to theresponses in other important cellular components of respiratory tissuessuch as mast cells or pulmonary epithelial tissue.29 In vitro assays that

Figure 2.2 Perfusion myograph systems allow dynamic control or measurementsof pressure and ow within isolated tubular tissues; in addition tosimple visualisation (a) and measurements of tissue dimensions (b),such systems may also allow imaging of tissue permeability andintracellular signalling through confocal microscopy.

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evaluate pulmonary toxicity have been developed using pulmonary alve-olar macrophages to provide fairly simple and inexpensive screens. Thesemeasure deleterious changes in parenchymal cell populations as a markerof a toxic response and can even be combined with tracheal organ culturesto produce brosis aer exposure to inorganic and organic brogens likesilica or asbestos.30

Standardising complex human tissue assays can be challenging; oneapproach is to use precision-cut tissue slices of between 250 and 1000 mmthickness, which are uniform in their dimensions and minimise intra-patient variation in drug responses. Slices are typically prepared by infusionof low melting point agarose into the tissue or organ, which then solidiesupon cooling and allows slices to be prepared using equipment such asa Krumdieck tissue slicer.31 As the temperature is increased to 37 �C, the

Figure 2.3 Tissue biopsies and slices can be maintained in culture for hoursto days, allowing a range of functional end points to be measured.A series of images can be captured by video microscopy and imageanalysis allowing measurements of lumen diameter; here, a time-capture series of human isolated bronchus demonstrates contractionupon exposure to carbachol (1 � 10�5 M) at 0, 8, 16, 16, 24 and 40minutes aer drug addition (images courtesy of Graeme Macluskie,Biopta Ltd).

Functional Studies with Human Isolated Tissues 27

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agarose is removed, leaving a functional tissue preparation that retains the3D complexity of the native organ but which can be maintained in culture fordays (Figure 2.3).Probably the most difficult human tissue from which to obtain functional

data is neural tissue. This is from the perspective of supply as well as thetechnical difficulty in preparation; however, there are some in vitro functionalbioassays available. These use tiny thin brain slices or preparations ofganglia kept in specialised media that allow electrophysiological measures ofsynaptic activity to be made following pharmacological or physical stim-ulus.32 The most desired assay for investigations into chronic pain within thepharmaceutical industry is the dorsal root ganglion (DRG) assay. A majorobstacle is the vulnerability of neural tissue to time-dependent failure andthe difficulty in sourcing DRG tissue within a short time frame. While thereare a number of assays in use in academic institutions these are oen notcommercially useful. Moreover, assays of cultured neural tissue are difficultto validate because they only remain functional for a few hours. Findingfunctional human tissue assays that correlate to diseases such asAlzheimer’s, Parkinson’s, psychosis or depression has become a major

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target. It appears likely that this is an area where advances in stem celltechnologies will make a signicant impact, if measures to collect andtransport fresh neural tissue for longer periods are not developed.

2.3.4 Membrane Transport and Ussing Chambers: Skin, LungMucosa, Gastrointestinal Tract and Glandular Tissues

Tissue sheets such as skin and glandular tissue can be investigated in specialtissue culture techniques where a test compound is added to the apicalsurface of the tissue and its absorption or direct pharmacological activity ismeasured. Testing using skin biopsies has been common for many years andinvolves the application of a drug to a suspended portion of tissue; the activecompound passes through the dermal region as a measurement of skinpenetration (Franz cell technique).33 Subsequent testing of each tissue typeindividually can also act as a useful measure of toxicity and efficacy (fordermal medication) screening. Engineered ‘humanised’ skin is now a realityand commercially available for this approach to testing. Whether the engi-neered skin possesses all of the phenotypic characteristics required is not yetclear, however early studies of human epidermal tissues appear to showmorphology and phenotype that is quite similar to fresh human skin.34

Nonetheless, fresh normal human skin is perhaps the most readily accessedof human tissue specimens, with thousands of cosmetic procedures beingconducted in the UK each year, and remains the gold standard approach forstudies on dermal responses and drug absorption.An alternative to the Franz cell technique, which is useful for under-

standing not only permeability but also ion transport and physiologicalprocesses, is the Ussing chamber method (Figure 2.4). Voltage and currentelectrodes are placed on either side of the tissue allowing changes in iontransport to be measured in the presence of test drugs. One key differencebetween the Ussing and Franz techniques is the status of the tissue duringthe experiment. For Franz experiments the tissue is oen no longer ‘living’and may simply act as a physical barrier to drug penetration; however, inUssing chamber experiments a section of intact mucosal tissue carefullydissected from the trachea, bladder or gastrointestinal tract remains fullyfunctional for up to 16 hours, permitting the assessment of drug trans-porters, ion channels and secretory processes.Recent publications have highlighted the value of human fresh gastroin-

testinal tissues in Ussing chambers both for the prediction of fractionabsorbed (‘Fa’, the percentage of an orally delivered drug that achievessystemic absorption from the gastrointestinal tract)35 and as a way to betterunderstand the inuence of disease status on regional permeability.Although cell-based assays used to predict drug absorption are popular it isaccepted that there are differences in the expression of drug transporters(such as Pgp) and enzymes in the human small intestine, which is the site ofmost oral drug absorption; moreover, signicant differences exist between

Figure 2.4 Ussing chambers are used to measure drug absorption and metabolismin intact mucosal preparations from the trachea or gastrointestinaltract. Sensitive voltage and current electrodes allow measurements ofion ux across polarised membranes.

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species.36 Gastrointestinal diseases such as ulcerative colitis and Crohn’sdisease may alter drug absorption, which can be modelled using diseasedtissue from surgical resections.37

2.4 Applications of Functional Tissues in DrugDevelopment: Safety, Efficacy and PersonalisedMedicines

Perhaps the most important benet of fresh human tissue is to obtaindiseased specimens from the target patient population. The main output offunctional assays is to create concentration–response relationships in thistarget human tissue, with the goal of translating non-clinical data to relevantclinical end points that will de-risk the clinical development process(Figure 2.5).The use of functional tissue models is not yet routine. Instead, they are

oen used to compare human tissue derived data with outputs from othermore commonly used test systems such as cell-based assays or animal in vitroor in vivo models. Human tissue is generally considered the ‘gold standard’test system against which other models may be assessed; therefore, datafrom cell-based assays or animal models that is in concordance with humantissue derived data increases condence in the translation of non-clinicaldata to patients. In contrast, differences between human tissue derived data

Figure 2.5 Flow chart describing the processes involved in a functionalpharmacology study with the objective of translating preclinical datato relevant clinical end points such as blood pressure.

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and other non-clinical methods can act as an early detection system forspecies differences in drug effects, or may raise questions about thepredictive capacity of the cell-based assay or animal model. In either case, thegeneration of human data early in the drug development process increasesthe chances of later clinical success.Human tissues are not only employed during early discovery and devel-

opment (Figure 2.6), they may also be useful when troubleshooting unex-pected clinical ndings. Many drugs proceed to clinical trials with minimalhuman data; unanticipated species differences may lead to clinical obser-vations in humans that were not detected in preclinical animal species.

2.4.1 Predicting Efficacy in Clinically Relevant Human Tissues

Failure to predict clinical efficacy is the single biggest cause of clinicalfailure, accounting for around 50–60% of phase II and III failures. Functionaltissues can be used to predict clinical efficacy by understanding the behav-iour of the human drug target in its natural environment.In order to properly estimate efficacy in a non-clinical setting, various

factors that can inuence in vivo efficacy need to be replicated in the in vitrosystem, in particular for drugs that act as agonists at their intended target.

Figure 2.6 Human functional tissue research and the drug development process.

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Although many drugs have been considered selective antagonists, it is nowrecognised that few compounds are without at least partial agonist effects,which may vary across tissue types, not least because agonist/stimulatorpotency is both a compound and a tissue-dependent output. It is thereforevaluable to investigate the effects of test compounds in the native tissue aswell as the more commonly used high receptor expression clone systems,which can make compounds look more potent than they truly are in nativetissue. For example, a natural lower expression system (native tissues) mayturn full agonists to partial agonists/effective antagonists, which in turn willinuence decisions on clinical effectiveness. Moreover, many drug receptorsare activated preferentially along various signalling pathways by differentagonists and the correct balance of signalling is tissue-dependent. Intracel-lular targets are also inuenced by cell penetration and native tissues havea balance of transporters that can change the primary pharmacodynamics oftissue and hence the potency and time course of activity of a compound.

2.4.2 Predicting Safety and Toxicology Risks UsingFunctional Tissues

Other than lack of efficacy, safety and toxicology represent the greatest risksof clinical failure, with approximately 30% of drugs failing during clinicaltrials.38 Human tissues are increasingly being used to investigate specicrisks and concerns are being raised about the translation of animal results tohumans, following the high-prole withdrawals and warnings associatedwith rofecoxib.39 Particular concerns exist around the prediction of cardio-vascular side effects, where small numbers of patients display signicantadverse reactions which are only apparent when tens of thousands ofpatients receive the drug. It is questionable whether any non-clinical modelexists that can predict such effects because the number of patients studied istypically quite small; however, regulators are increasingly requesting

Figure 2.7 Contractile responses to 5-hydroxytryptamine (‘5-HT’, serotonin) incanine and human isolated subcutaneous arteries. Canine arteriesare much less responsive to the effects of 5-HT and as such wouldunderestimate the vascular effects of 5-HT agonists.

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non-clinical human data to allow cross-comparisons with animal data, inparticular where there are known to be signicant differences between theresponses of humans and the species typically used in safety pharmacologystudies, such as rodents, dogs and non-human primates (Figure 2.7).In vitro tests on human tissue or cells do have limitations in their repre-

sentation of whole body drug response. A number of research groups andcompanies are developing ‘organ-on-a-chip’methods40 or in vitro recreationsof organ systems to allow the simultaneous study of multiple tissue typesand, more importantly, the interaction between various organs.Human tissue assays are useful in measuring the direct toxicity of drug

candidates and there are large economic and developmental benets fromobtainingmore human-based information prior to clinical trials with regardsto potential toxic effects. The metabolism of any chemical, inuenced bygene, cell and tissue mechanisms, can be studied using human-basedADMET (ADME and toxicology) tests such as the Ussing chamber technique.Many combine chemical affinity with functional measures of metabolism toderive algorithms that closely mimic biochemical effects, at least at themolecular level. The cytochrome (CYP) family of enzymes mediates >90% ofhuman drug metabolism and genetic expression of CYP proteins followexposure to toxic medication. Toxicity and metabolism, predicted usinghuman liver slices, primary hepatocytes and liver cell lines with cloned CYPenzymes have identied approved drugs (based on animal studies alone) thatwent on to harm humans.41

2.4.3 Functional Tissues and the Developmentof Personalised Medicines

It has been 12 years since the term ‘personalised medicine’ (or increasingly‘genomic medicine’) was coined following the mapping of the humangenome; however, with the exception of a small number of cancer therapies,

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the expected ow of drugs has not yet materialised. Pharmacogenomics nowappears to represent only a part of the process towards achieving this goaland having moved towards proteomics on the assumption that proteinexpression – not gene expression – is the key factor determining drugsensitivity, it is now recognised that a true prediction of drug effects inindividual patients, for the majority of diseases and chronic ailments, willrequire investigations that stretch from genomic level through to detailedhuman pharmacology at the organ/system level.42

It is known that inter-individual responses to drugs are variable and thata surprisingly high proportion of patients gain no benet. For example, 40–70% of patients are classied as non-responders to bronchodilator beta-2-adrenceptor agonists in the treatment of asthma.43

The rst example of the power of personalised medicine was thedevelopment of herceptin; human tissue research was central to the devel-opment of a personalised approach to the treatment of breast cancers thatare HER-2 (human epidermal growth factor receptor) positive.44,45 Recentefforts have moved to other similar prospects, where a clear link betweena single gene mutation and the effectiveness of a drug can be reasonablypredicted.The development of a biomarker strategy has become widely accepted as

a critical part of the development process, with the hope that the correctselection of biomarkers ultimately serves several different purposesincluding improved diagnosis and screening of patients, evaluation of risk/predisposition, assessment of prognosis, monitoring (recurrence of disease),prediction of response to treatment, and, of most relevance to early devel-opment human tissue research, as a surrogate response marker. Thisapproach has led to a sharp increase in the number of drugs approved forcancer and HIV. Importantly, improved target selection has, under the FDAAccelerated Approval Program, brought forward the availability of 26 newchemical entities. Biomarkers have reduced the need to wait until a patient’slong-term survival has been established, which is not only a commercialupside, but more importantly it is an ethically superior method by which tobring new medicines to market.The use of biomarkers as a surrogate also has the potential to tackle the

low numbers of drugs in development for orphan diseases. Miyamoto andKakkis46 have proposed that the use of surrogate markers could lower thebarrier for accelerated approval of treatments for rare diseases, which couldmake such drugs commercially viable. In the vast majority of cases, access towell-characterised blood, serum, urine or sputum is needed for validation ofthe selected biomarker, but this can oen be difficult for rare diseases andthe key to future success appears to lie in networks of biobanks cooperatingto standardise their collection procedures for rare samples.47

Surrogates need not be limited to proteins extracted from blood, urineor sputum; solid tissue samples obtained from surgery or clinical biopsiescan represent an additional way to measure function in specic patientgroups. Perera et al.48 (Figure 2.8) collected gluteal biopsy samples from

Figure 2.8 Functional tissues can be useful in longitudinal studies providingclinical biomarkers of drug activity that are not possible with bio-uids. In this example, the effects of hormone replacement therapy(HRT) on the vasodilator responses of small arteries isolated fromgluteal biopsies of women with and without type 2 diabetes. Inhealthy women, acetylcholine causes a pronounced relaxation of thearteries; however, this effect is blunted in women with type 2diabetes. Aer 6 months of HRT, a second set of gluteal biopsieswere taken, revealing that the vasodilator responses of women withtype 2 diabetes was similar to that of healthy women.

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women with and without diabetes and compared the effects of a 6 monthcourse of hormone replacement therapy (HRT) on the responses of smallarteries in both patient groups. Women with diabetes were, unsurpris-ingly, found to have a severely blunted baseline response (pre-HRT group)to the vasodilator acetylcholine, when compared to healthy women(control group). Aer 6 months, the responses of the women with diabeteswere vastly improved (post-HRT group) and were comparable to theresponses of healthy women. Endothelial function served as a potentbiomarker of cardiovascular health and the efficacy of the HRT inimproving vascular function in a specic patient group. The use of func-tional studies is of course limited to tissues, such as gluteal biopsies thatcan be safely obtained during clinical trials.Complex data on the site-specic pharmacology of tissues is extremely

valuable for safety studies, for example the expression of the 5-HT1 receptortype varies greatly throughout the vascular system. The failure to recognisethe site-specic effects of the migraine treatment sumatriptan, whichconstricts cerebral blood vessels, led to concerns once it was recognised thatthe drug was also capable of constricting coronary arteries.49

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2.5 SummaryHuman functional tissue research is one of the fastest growing areas of drugdiscovery and development and is clearly aligned with the strategic shi ofthe pharmaceutical industry towards biomarkers and personalised medi-cine. In 10–15 years’ time it will probably be seen as a central pillar of all drugdevelopment programmes.There are, however, some major differences between functional tissue

research and other cornerstones of drug development. Firstly, successfulprovision of high-quality human tissues will be a signicant factor in theimpact the sector makes on development costs and timelines. Second, thedearth of human tissue-based studies in some therapeutic areas is stag-gering, requiring a sea change not only in pharmaceutical R&D but alsoacademic research to better understand biological mechanisms of humancells and tissues. Thirdly, the generation of evidence of the scientic andcommercial impact of functional tissue research will dramatically change thelandscape, accelerating the demand for fresh, functional disease-relevanttissues and leading to specic functional tissue assays becoming part of theregulatory landscape.

AcknowledgementsSincere thanks to Michael Finch for his help in preparing this chapter.

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