pH-dependent ileocolonic drug delivery, part II: Preclinical evaluation of novel drugs and novel excipients

(1)

University of Groningen

pH-dependent ileocolonic drug delivery, part II

Broesder, Annemarie; Kosta, Anne-Marijke M A C; Woerdenbag, Herman J; Nguyen, Duong

N; Frijlink, Henderik W; Hinrichs, Wouter L J

Published in:

Drug Discovery Today

DOI:

10.1016/j.drudis.2020.06.012

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from

it. Please check the document version below.

Document Version

Publisher's PDF, also known as Version of record

Publication date:

2020

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):

Broesder, A., Kosta, A-M. M. A. C., Woerdenbag, H. J., Nguyen, D. N., Frijlink, H. W., & Hinrichs, W. L. J.

(2020). pH-dependent ileocolonic drug delivery, part II: Preclinical evaluation of novel drugs and novel

excipients. Drug Discovery Today, 25(8), 1374-1388. https://doi.org/10.1016/j.drudis.2020.06.012

Copyright

Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

(2)

REVIEWS DrugDiscoveryTodayVolume25,Number8August2020

Teaser

Novel

drugs

and

novel

excipients

in

pH-dependent

ileocolonic

drug

delivery

systems

have

to

be

tested

in

animals.

Which

animal

species

are

suitable

and

what

in

vivo

methods

are

used

to

verify

ileocolonic

drug

delivery?

pH-dependent

ileocolonic

drug

delivery,

part

II:

preclinical

evaluation

of

novel

drugs

and

novel

excipients

Annemarie

Broesder

1

,

Anne-Marijke

M.A.C.

Kosta

2

,

Herman

J. Woerdenbag

1

,

Duong

N. Nguyen

1

,

Henderik

W. Frijlink

1

and

Wouter

L.J.

Hinrichs

1

1_University_of_Groningen,_Groningen_Research_Institute_of_Pharmacy,_Department_of_{Pharmaceutical}_Technology

andBiopharmacy,AntoniusDeusinglaan1,9713AVGroningen,TheNetherlands

2_University_of_Groningen,_University_Medical_Center_Groningen,_Department_of_Biomedical_Sciences_of_Cells_and

Systems,AntoniusDeusinglaan1,9713AVGroningen,TheNetherlands

Introduction

InpartIofthisreviewserieswedescribedinvitroandclinicalmethodstoinvestigateandverify

colonicdrugdeliveryofnovelpH-dependentsystems[1].Thesesystemsutilizethesharpbut

shortpHpeakof7.4(range7.2–7.7)intheileumforileocolonicdrugtargeting.Whenevaluating

theileocolonictargetingabilityofasystemorthetherapeuticefficacyofanileocolonicdelivered

drug,itis preferabletotestitdirectlyin humans[1].However,a noveldrug and/ora novel

excipientcannotbetestedinhumansifnosafetydatafromanimalstudiesareavailableforthe

drugorexcipientusedfortheileocolonicdrugdeliverysystems[2].Guidelinesconcerningsafety

testing have been provided by the International Council for Harmonisation of Technical

Requirements for Pharmaceuticals forHuman Use (ICH) [3]. According to these guidelines,

safety pharmacology studies have to be conductedwith the final formulation in laboratory

animalsiftheformulationsubstantiallyaltersthepharmacokineticsand/orpharmacodynamics

of the drug compared with previously tested formulations [4]. Given that ileocolonicdrug

deliverysystemstargetthedrugto thelowerpartsofthe gastrointestinal(GI)tract,boththe

pharmacokineticsandpharmacodynamicsofthedrugwillbealtered,thusnecessitatingsafety

testing with the finaldrug delivery system in laboratory animals. To achievepH-dependent

ileocolonicdrugdeliveryinanimals,theanimalspecieschosenshouldhaveasharpdistinctpH

peakinitsterminalileum.Ideally,theintestinalpHvaluesoftheanimalshouldbecomparable

withthosefoundinhumans,includingthepHpeakabovepH7.2intheterminalileum.This

wouldallowtheuseofestablishedpH-dependentileocolonictargeteddrugdeliverysystemsto

testnoveldrugs.TotestnovelpH-dependentexcipients,thepHoftheGItractofthechosen

animalspecieshastobesimilartothatofhumanstoobtainileocolonictargetinginhumans.

Variousspecieshavebeenusedforpreclinicaltestingofnewdrugsornovelexcipients,including

rats, mice,dogs,and rabbits.However, littleemphasishas beenput onthe translationfrom

animalspeciestohumansregardingthepHvaluesoftheGItract.

Inthisreview,we provideanoverviewofthepHvaluesofvariouspartsoftheGItractof

frequentlyusedlaboratoryanimalspeciesandofhumans.Weaimtodeterminewhichanimal

species, if any,could best beused to test novel drugs or novel excipients in pH-dependent

Reviews KEYNO TE REVIEW AnnemarieBroesder receivedherMScin

pharmacyfromthe

UniversityofGroningen

(TheNetherlands).

Currently,sheispursuing

herPhDintheDepartment

ofPharmaceutical

Technologyand

BiopharmacyattheUniversityofGroningenunder

thesupervisionofProf.DrH.W.FrijlinkandDr.W.L.

J.Hinrichs.Herresearchfocusesonoral

pH-dependentileocolonicdrugdeliveryandthesuitability

ofthesesystemsfordifferentdosageforms.

WouterL.J.Hinrichs

studiedchemistryatthe

UniversityofGroningenand

receivedhisPhDfromthe

UniversityofTwente(The

Netherlands)in1993.

Thereafter,heworkedasa

postdocintheUniversityof

TwenteandUtrecht

University(TheNetherlands)until1998.Sincethen,

hehasbeenanassistantprofessorattheDepartment

ofPharmaceuticalTechnologyandBiopharmacyat

GroningenResearchInstituteofPharmacy(The

Netherlands).Oneofhisresearchinterestsisoral

dosageforms.Hehas(co-)authoredmorethan140

originalarticlesinpeer-reviewedjournalsandbook

chapters.

HenderikW.Frijlinkhas

beenaprofessorand

chairmanoftheDepartment

ofPharmaceuticalTechnology

andBiopharmacyatthe

GroningenResearchInstitute

ofPharmacysince1998.Heis

interestedinsolidoraldosage

forms,theirtechnologicaland

biopharmaceuticalaspectsandgastrointestinal

targetingstrategy.Furtherinterestsofhiscurrent

researcharetechnicalandbiopharmaceuticalaspects

ofpulmonarydrugdelivery.Theformulationof

biopharmaceuticals,suchaspeptides,proteins,

vaccines,andgene-basedproducts,usingstabilizing

sugarglassesisafurtherresearchtopicinhis

department.Hehaspublishedover200

peer-reviewedpapersinthefield.

Correspondingauthor:Hinrichs,WouterL.J. (w.l.j.hinrichs@rug.nl)

1374 www.drugdiscoverytoday.com

1359-6446/ã2020TheAuthor(s).PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

(3)

ileocolonicdrugdeliverysystems.Subsequently,invivomethods

usedinpreclinicalevaluationstoinvestigateandverifyileocolonic

drugdeliveryarediscussed.

Luminal

pH

in

the

gastrointestinal

tract

of

animals

compared

with

humans

ThepHvaluesoftheGItractlargelydetermineinwhichpartofthe

GItractadrugisreleasedfromapH-sensitivedrugdeliverysystem.

Forileocolonicdrugdelivery,itisimportanttoselectananimal

speciesthathasasharpanddistinctpHpeakintheterminalileum,

asis foundin thehumanGItract[1].Inaddition topH,other

factors can have a role in the performance of pH-dependent

ileocolonic drug delivery systems, including intestinal length,

buffer capacity, fluid volume, motility, and transit time. These

factors have been extensively described by Hatton et al. and

Sjo¨grenetal.[5,6],andarenotfurtherdiscussedinthisreview.

Methods

to

determine

the

pH

in

the

gastrointestinal

tract

in

laboratory

animals

Similartohumanstudies,pHvaluesintheGItractofanimalshave

beendeterminedwithaspirationvia theoralroute,tetheredpH

electrodes, and with pH-sensitive radio telemetry capsules. In

humans,aspirationviacolonoscopyhasalsobeenusedtomeasure

thepHofthelowerGItract,buttoourknowledgethismethodhas

neverbeen usedin animals [1].Aspiration via the oralroute is

generallylimitedtotheupperGItractandcanbeusedto

deter-minethemeanpHofthecollectedstomachorduodenalfluidex

vivo[7,8].IfthepHofthe entireGItractduringtransitistobe

measured,radiotelemetrycapsulescanbeused[9–11].Adrawback

ofthesecapsulesistheirlargesize(usuallyaround1020mm),

whichlimits theiruse to larger animals,suchasdogs and pigs

(Table1) [12–15].With laboratoryanimals,exvivopH

measure-mentsoftheintestinalcontentsaftersurgicalcollectionofsamples

from different segments of the GI tract is possible [16–23]. A

drawbackofthismethodisthatonlythemeanpHofthecollected

fluid is determined. In addition, the pH should be measured

immediately after collection to prevent possible postsampling

pHchanges. Thisis particularlyrelevant forthecontent ofthe

colon,becausebacterialfermentationofpolysaccharidesresultsin

the formationofacidic products whichcanlower the pHafter

collection[24].AnotheroptiontomeasurethepHinanimalsisto

opentheGItractbysurgeryandtomeasurethepHinsituwitha

pHelectrode[25–32].WithinsitupHmeasurementsand witha

pH-sensitiveradiotelemetrycapsulepossiblepHalterationsafter

samplingareexcluded,becausethepHvaluesaremeasured

im-mediately.

pH

values

in

the

gastrointestinal

tract

of

laboratory

animals

The mean or median pH values found in various studies for

differentsegmentsoftheGItract(stomach,duodenum,jejunum,

ileum,cecum,andcolon)ofrabbits,pigs,dogs,rats,mice,guinea

pigs,andmonkeysareshownasdotsinFig.1,togetherwiththe

valuesforhumansasreviewedin partI ofthis series[1].From

publicationsinwhichboththemedianandmeanpHvalueswere

given,themeanvalueswereusedinthisreview.Whenmultiple

pHvalueswerereportedforacertainGItractregion,forinstance

the fundus and antrum of the stomach, the mean value was

calculatedand used.InFig.1,theminimumandmaximumpH

valuesareindicatedbybarsandthemeanpHvalues,calculated

fromalldifferentstudiescombined,byaverticalline.Thestudy

sizewasnottakenintoaccountforthecalculationofthemean.In

thisoverview,nodifferentiationismadebetweenthefastedand

fed states,becauseof alackofsufficientdata onthis point.To

enablecomparison,thepreviouslyreportedvaluesforhumansin

thefastedandfedstateswerealsocombined.Table1providesan

overview of the studies used, with information about the pH

measurementmethodand thestate(fastedor fed)underwhich

theexperimentswerecarriedout.

RabbitsandpigshavepHvaluesinthestomachthatarewithin

the range of the human values (Fig. 1A,B, respectively) [16–

18,25,33]. Themean pHvaluesof theduodenum and jejunum

intheseanimalspeciesarebelowtheminimumpHof7.2thatis

foundinthehumanileum,whereasthepHvaluesintheileum

werehigher[16–18,25,33].

AbroadervariationwasfoundforthepHinthestomachofdogs

(pH 1.1–6.8; Fig. 1C) and rats (pH 3.2–6.7; Fig. 1D) [7–

11,16,18,21,23,26,28–32].Intheseanimals,theminimumpHof

7.2ofthehumanileumisalreadysurpassedintheduodenumand

jejunuminsomeofthestudies.Additionally,themeanpHinthe

ileumofbothdogsandratswasbelowthisminimumpHof7.2

[7,16,18–21,23,26,28–30].

MicehaveahigherpHinthe stomachbuta lowerpHinthe

small and large intestine compared with humans (Fig. 1E)

[16,22,23,28]. The minimum pH of 7.2 found in the ileum of

humanswasnotreachedinanypartofthemurineGItract.

Inguineapigs,the pHvaluesofthe GItractarehigherthan

thoseofhumans(Fig.1F).TheminimumpHof7.2wasreachedin

theduodenuminsomeofthestudiesandwasabovepH7.2inthe

jejunuminallstudiesuntilthececum,wherethepHdropsuntil

6.7[16,25].

Inmonkeys,themeanpHinthestomachwashigherthanin

humans(Fig.1G)[16,34].ThepHincreasestopH6.0intheileum

andthendropsto5.0inthececumandcolon[16].However,care

mustbeexercised todrawdefinite conclusionsfrom thesedata

becausethepHvaluesofthesmallintestineandcolonarebasedon

onlyonestudy.

Selection

of

an

appropriate

animal

species

Forthepreclinicalevaluationofnoveldrugsornovelexcipients

applied in pH-dependentsystems, we foundthat no particular

animalspeciesiscommonlyused(Table2).Itisremarkablethatin

moststudiesnoinformationisgivenabouttherationalebehind

thechosenanimalspecies.Inviewoftheworkingprincipleof

pH-sensitiveileocolonictargeteddrugdeliverysystems,apHpeakin

theterminalileumoftheanimalshouldbeconsideredasthemost

importantfactor.Furthermore,pHvaluessimilartothehumanGI

tract wouldbeideal,becausein thatcase anexistingand

well-validatedpH-dependentsystemcanbeusedtoobtainileocolonic

drugdeliveryinthechosenanimalspeciesornovelpH-dependent

excipientscanbeevaluated.

ThepHprofileoftheGItractofmonkeys,mice,andguineapigs

differsfromthatinhumansandnodistinctivepHpeakhasbeen

foundinthe terminalileum.Therefore,theselaboratoryanimal

speciesshouldbeconsideredunsuitablefortestingnoveldrugsin

pH-dependentileocolonicdrugdeliverysystems(Fig.1).Dogsand

ratsmightbesuitablebecausethemeanpHvaluesinthevarious

DrugDiscoveryTodayVolume25,Number8August2020 REVIEWS

www.drugdiscoverytoday.com 1375

Reviews

KEYNO

TE

(4)

REVIEWS Drug Discover y Today V olume 25, Number 8 August 2020 TABLE 1

Overview of studies investigating the pH values of the GI tract in laboratory animals and in healthy human individuals

Animal Segment gastrointestinal tract Method Refs

Species Breed Stomach Duodenum Jejunum Ileum Cecum Colon pH measurement Fasted/fed

Monkey Cynomolgus X X X X X X Ex vivoa Fed ad libitum [16]

X BravoTMcapsuleb Fasted and fed [34]

Pig From farms X X X X X X Ex vivoa Fed ad libitum [16]

Crossbreed of large white and Landrace X X X X X X In situa _{Fed ad libitum} _[25]

X X X X X X Ex vivoa _{Fed ad libitum} _[18]

Large white, Landrace, and Essex X X X X X X Ex vivoa _{Fasted for 1 hour} _[17]

Mouse Balb/c X X X X X X Ex vivoa _{Fasted and fed} _[23]

House X X X X X In situa _N/Ae _[27]

X X Ex vivoa _{Fed ad libitum; high and low fiber diet} _[22]

White X Ex vivoa _{Fed ad libitum} _[16]

Rat Albino Norwegian X X X X X In situa _{Fasted and fed ad libitum} _[28]

Porton-Wistar X X X X In situa _{Fasted, fed ad libitum and fed standardized} _[29]

X X X X In situa Fed [26]

White X X X X X X Ex vivoa Fed ad libitum [16]

Wistar X X X X X X Ex vivoa Fasted and fed [23]

X X X X X X Ex vivoa Fed ad libitum [18]

X X X X In situa Fasted [30]

Rabbit New Zealand Whites X X X X X X Ex vivoa Fed ad libitum [16]

X X X X X X Ex vivoa Fed ad libitum [18]

X X X X X X In situa Fed ad libitum [25]

X X N/Ae _{Fed ad libitum} _[33]

Guinea pig N/Ae _X _X _X _X _X _X _{Ex vivo}a _{Fed ad libitum} _[16]

Dunkin-Hartley White X X X X X X In situa _{Fed ad libitum} _[25]

Dog Beagle X Heidelberg capsule Fasted [9]

X Aspiration Fasted [8]

X In situa _Fasted _[31]

X BravoTM_capsule _{Fed big or small meal before capsule ingestion} _[10]

X BravoTM_capsule _{Fasted, fed standard meal and fed slurry meal} _[11]

X In situa _Fed _[32]

From domestic households X X X X X X Ex vivoa Fed ad libitum [16]

Labrador X X Aspiration Fasted [7]

X Ex vivoa Fasted and fed [20]

Mixed X Ex vivoa Fasted [19]

N/Ae X X X X X X Ex vivoc Fasted [21]

Human X X X X X Telemetry capsule Fasted; food when capsule left stomach [105]

X X X X X BravoTMcapsule Fasted; food 30 min or 4 h after ingestion [106]

1376 www .drugdisco verytoday .com Reviews KEYNOTE REVIEW

(5)

Drug Discove ry Toda y V olume 25, Number 8 A ugust 2020 REVIEWS TABLE 1 (Continued )

Animal Segment gastrointestinal tract Method Refs

Species Breed Stomach Duodenum Jejunum Ileum Cecum Colon pH measurement Fasted/fed

X X X In situa Fasted [38]

X X X X X Telemetry capsule Fasted; food allowed when capsule left stomach [40]

X X X X Intellicap1 capsule Fasted; food allowed 4 h after ingestion [107]

X X Heidelberg capsule Fasted and fed [108]

X X Aspiration Fasted [109]

X X X SmartPill Fed; FDA standard breakfast [111,112]

X X X SmartPill Fasted; after 4.5 h standardized lunch [111]

X X X X X Telemetry capsule Fed; food allowed after ingestion of capsule [36]

X X X IntelliCap1 Fasted; food 3, 6 and 10 h after ingestion [92]

X X SmartPill Fed; gastric-emptying meal [113]

X Heidelberg capsule Fasted; food 3 h after capsule left stomach [9]

X In situa _{Fasted and fed standard meal} _[114]

X In situa _Fasted _[116]

X In situa Fasted and fed [118]

X Heidelberg capsule Fed; standardized breakfast [119]

X X X X X Telemetry capsule Fasted; food allowed when capsule left stomach [122]

X X X X Telemetry capsule Fed; normal diet [123]

X X X X Telemetry capsule Fasted; food allowed when capsule left stomach [37]

X X Heidelberg capsule Fed [125]

X Aspiration Fasted, fed and fed fat-enriched meal [126]

X BravoTM_capsuled _{Fed; standardized meal twice daily} _[127]

X Aspiration Fasted and fed [130]

X X X Telemetry capsule Fasted; food allowed when capsule left stomach [131]

X X X SmartPill Fasted; food allowed 6 h after ingestion [132]

X X SmartPill Fasted; standardized meal when capsule left stomach [133]

X X SmartPill Fed; test meal, after 6 h normal diet [39]

a

Measured with a pH electrode.

b

Capsule attached to stomach.

c

Colorimetric and with a pH electrode.

d

Capsule attached to duodenal bulb.

e Not available. www .drugdisco verytoday .com 1377

(6)

REVIEWS DrugDiscoveryTodayVolume25,Number8August2020 (a) (c) (e) (g) (b) (d) (f) Stomach Duodenum Jejunum IIeum Cecum Colon Stomach Duodenum Jejunum IIeum Cecum Colon Stomach Duodenum Jejunum IIeum Cecum Colon Stomach Duodenum Jejunum IIeum Cecum Colon Stomach Duodenum Jejunum IIeum Cecum Colon Stomach Duodenum Jejunum IIeum Cecum Colon Stomach Duodenum Jejunum IIeum Cecum Colon 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Luminal pH Luminal pH Luminal pH Luminal pH Luminal pH Luminal pH Luminal pH

Drug Discovery Today

FIGURE1

Luminalgastrointestinal(GI)pHvaluesoflaboratoryanimals(dark-green,orange,orredbars)andhealthyhumanindividuals(light-greenbars).ThedifferentpH valuesoftheGItractofthe(a)rabbit[16,18,25,33],(b)pig[16–18,25],(c)dog[7–11,16,19–21,31,32],(d)rat[16,18,23,26,28–30],(e)mouse[16,22,23,27], (f)guineapig[16,25],and(g)monkey[16,34]aregivenagainstthevaluesinhealthyhumanindividuals[9,36–40,92,105–133].TheGIpHrangesareindicatedby bars,theverticallineinthebarsindicatesthemeanpH.ThemeanormedianpHvaluesofthedifferentsegmentsoftheGItractfoundinthedifferentstudiesare indicatedbytheblackdots.OnlyonestudywasfoundforpHvaluesfortheduodenum,ileum,cecum,andcoloninthemonkeyand,therefore,onlythisvalueis givenasadotandaverticalline.Table1inthemaintextdetailsthestudiesthatwereusedtoobtaintheminimum,maximum,andmeanpHvaluesforthe differentsegmentsoftheGItract.

1378 www.drugdiscoverytoday.com Reviews KEYNO TE REVIEW

(7)

segmentsoftheir GItract aregenerallycomparable tothosein

humans, including a pH peak in the terminal ileum (Fig. 1).

However,theirmeanpHvalueintheileumisbelowtheminimum

value in humans. Furthermore, the pH values in the GI tract

substantiallyvaried amongthedifferent studies.Therefore,itis

advised tocheck the inter-andintraindividual variation in pH

valuesoftheGItractofthedogsorratsusedintheexperiments.

GiventherelativelybroadrangeofgastricpHvaluesindogs,itis

recommendedtopretreattheanimalswitha0.1MHCl-KCl

solu-tion,viaanorogastrictube,tolowerthepHofthestomach[35].

RabbitsandpigshavepHvaluescomparabletothoseinhumans,

includingthepHpeakintheileumabovepH7.2(Fig.1).ThepH

valuesinthedifferentsegmentsoftheGItractshowedrelatively

littlevariationbetweenthedifferentstudies.Forrabbits,themean

pHinthejejunumwasslightlyhigher(7.1)thaninhumans,which

mightresultinprematuredrugrelease.However,therabbitisthe

onlyanimalspecieshavingadistinctpHpeakintheileum,with

theminimumpHabove7.0.Forpigs,theminimumpHfoundwas

6.6,whichisconsiderablylower.Infouroutoffivepigstudies,a

pHintheileumabove7.0wasfound,whereasthiswasthecasefor

allthreerabbitstudies.Thus,whenusingpigs,itisadvisedtocheck

thepHvaluesoftheGItractoftheindividualanimalstoverify

whetherthey arecomparable tothoseofhumans andconstant

overtime.

Overall,the rabbitappearsto bethe mostreliablespecies for

testingnoveldrugswithestablishedpH-dependentileocolonicdrug

deliverysystemsbecauseofthelowpHvariabilityandthedistinct

pHpeakabove7.0intheileum.Whentheinter-andintraindividual

variationsinpHvaluesofthedifferentregionsoftheGItractare

checkedforrabbits,ileocolonicdrugtargetingmightbeprovedwith

evenmorecertainty.IncaseswherethepHvaluesoftheGItractfor

individualpigs or dogsarecomparableto thoseinhumans, are

constantovertime,andifthepHthresholdofthesystemisreached,

these animal species might be good alternatives to the rabbit,

becauselargerdrugdeliverysystemscanbeadministered. When

multipleanimalspeciesarefoundtohaveappropriatepHvaluesin

theGItract,pilotstudiescouldbeperformedtodeterminewhich

speciesismostoptimaltoobtainileocolonicdrugdeliverywiththe

chosendrugdeliverysystem.Totestthetherapeuticeffectofanovel

druginpreclinicalstudies(Table2),diseasedratherthanhealthy

animalsshouldbeused.However,adiseasedstatecouldchangethe

pHvaluesintheGItractinanimals,eventhoughitwasfoundthat

colonicdiseasesdidnotnegativelyaffectthepHvaluesinhumans

[1,36–40].However,despitetheseresults,itisstillrecommendedto

checkthepHvaluesoftheGItractofdiseasedanimals,becausethe

effectsofacolonicdiseaseontheintestinalpHhavenotyetbeen

describedforanimals.

It should also be possible to mimic a specificdisease in the

chosenanimalspeciestoenablemeasurementofthetherapeutic

effect ofa noveldrug. In the literature,several colonic disease

models have been described for the rabbit (Table 3), which,

accordingtoourreview,isthemostreliableanimalspeciestotest

noveldrugsinapH-dependentileocolonicdrugdeliverysystem.

Anotherimportant aspecttobetakenintoconsiderationisthat

thepharmacokineticsand/orpharmacodynamicsofadrugmight

bedifferentinananimalcomparedwithhumans.Thus,ananimal

speciesshouldbeselectedwithcare,toavoidfalsenegativeorfalse

positiveresults.

In

vivo

methods

to

investigate

or

verify

ileocolonic

targeting

in

laboratory

animals

Beforeperforminginvivostudies,thepHdependencyofachosen

deliverysystemshouldbeverifiedinachallenginginvitro

disso-lutiontest.ThedissolutiontestshouldmimicthepHprofileand

preferablythebuffercapacity,buffertype,andionicstrengthof

thehumanGItract,asdescribedinpartIofthisseries[1].Todraw

conclusionsfromtheinvivoefficacydataofanoveldrugorthe

targeting ability of a drug delivery system containing a novel

pH-dependentexcipient,itisimportanttoverifyileocolonicdrug

delivery in the chosen animal species.Different methods have

been used toverifyileocolonicdrug delivery in animals.These

methodsincludeinvestigationoftissuesamplesandutilizationof

imaging techniques, such as X-ray imaging (radiography and

fluoroscopy),

g

-scintigraphy,fluorescencemicroscopy,and

near-infrared(NIR)fluoroscopy.Furthermore,drugplasma

concentra-tionsandtherapeuticeffectshavebeenused.Thesemethodsare

discussedinmoredetailinthefollowingsections.

Harvesting

tissue

samples

After sacrificing an animal, tissue samples can be collected in

whichthedrugconcentrationisdeterminedorinwhichthedrug

deliverysystemand/orthedrugisdetectedwithmicro-or

macro-scopic techniques.Different sections of the GI tractcan be

re-moved fromthe animal, suchasthe stomach, smallintestine,

cecum, and large intestine. Four different methods have been

developed to analyzeharvested tissue samples(Table 4). Allof

thesemethodsareapplicablefordrugsthatareabsorbedintothe

systemiccirculation,but,undercertainconditions,threeofthem

canbeusedfordrugsthatarenotabsorbedfromtheGItract.

In the firstmethod (i) the intestinal content isimmediately

washedaway.Subsequently,thedrug,ifpresent,isextractedfrom

thetissuebyanappropriatemethodandquantified[41–45].With

thismethod,drugreleaseisindicatedbythepresenceofdrugin

the extract because onlyreleased drug can be absorbedby the

intestinaltissue.Inthe secondmethod(ii),the washingstepis

omittedandtheluminalfluidisincluded,nexttotheintestinal

tissue,intheextractionprocedure[46,47].Todrawconclusions

fromthedrugcontentdata,itisimportanttovalidatethe

extrac-tionprocedureandtodeterminewhetherthedrugiscompletely

extractedfromthedrugdeliverysystemornotatall.Ifthedrugis

indeed completely extracted (iia) from the dosage form, drug

releaseisindicatedbyincompleterecoverybecause,inthatcase,

partofthedrughasbeenabsorbedintothesystemiccirculation

[47].Ifthedrugisnotextractedfromthedosageform(iib),then

presenceofthedrugintheextractisindicativeofdrugrelease[46].

Inthe thirdmethod(iii),onlythedrugcontent inthe luminal

contentismeasuredwhilethetissueitselfisnotused[48].Forthis

method,itisalsoimportanttoknowwhetherthedrugis

complete-lyextractedfromthedrugdeliverysystemornotatall.Ifthedrug

iscompletelyextracted(iiia),incompleterecoveryindicatesdrug

release.Ifthedrugisnotextracted(iiib),thepresenceofthedrug

intheextractindicatesdrugrelease.Inthefourthmethod(iv),the

drug deliverysystem isretrieved fromtheluminalcontent and

onlythedrugcontentinthedeliverysystemitselfisdetermined

[49].Inthismethodincompleterecoveryindicatesdrugrelease.

Inthreeofthedifferentapproaches,thelocationofreleaseof

drugsthatarenotabsorbedintothesystemiccirculationcanbe

Reviews

KEYNO

TE

(8)

REVIEWS Drug Discover y Today V olume 25, Number 8 August 2020 TABLE 2

Overview of pH targeted ileocolonic drug delivery systems tested in laboratory animals

Animal Drug delivery system In vivo test Refs

Species Rationale pH values

GI tract mentioned

pH-dependent matrix (M) and/or coating (C)

Drug/marker Read out Colon arrival

determined with

Dog N/Aa Yes Eudragit S (C) Mesalazine; tegafur Plasma samples Plasma concentrations

compared with previous determined colon arrival time

[77]

Based on practical considerations Yes Eudragit FS 30 D (C) Meloxicam Plasma samples Plasma concentrations

and colon arrival time from literature

[78]

GI tract comparable to human Yes Eudragit S (C) Mesalazine Tissue sections

harvested; plasma samples

Plasma and tissue concentrations

[42]

Pharmacokinetics No Eudragit FS 30 D Lovastatin Plasma samples N/A [134]

Dog and rat N/A No Eudragit S (M) Insulin; salicylic acid Plasma samples and

tissue sections harvested

Plasma concentrations and visual examination of harvested tissue sections

[53]

GI tract comparable to human Yes Eudragit P-4135 F (C);

Eudragit L (C); Eudragit S (C)

Norfloxacin; fluorescein Tissue sections harvested; plasma samples

Sulfasalazine method [52]

Rat N/A No Eudragit S (C) Mesalazine and/or

curcumin

Colitis severity N/A [135]

N/A No N-succinyl chitosan/Zn2+

(M)

Mesalazine and/or zinc Colitis severity Colitis severity and in vitro release

[99]

N/A No Acrylic acid and butyl

meth-acrylate polymers (M)

Aceclofenac Colitis severity N/A [136]

N/A No Eudragit S (M) Celecoxib and/or

curcumin

Colitis severity N/A [137]

N/A No Poly(starch/acrylic acid)

(M)

Rutin Colitis severity Colitis severity and in

vitro release

[100]

N/A No Eudragit P-4135 F (M) Tacrolimus Colitis severity, plasma

samples

N/A [138]

N/A No Eudragit P-4135 F (M) Tacrolimus Colitis severity Colitis severity and in

vitro release

[101]

N/A No Eudragit S (M) Aceclofenac Paw edema severity N/A [139]

N/A No Eudragit P-4135 F (M) Calcitonin;

carboxy-fluorescein

Plasma samples Plasma concentrations

and colon arrival time from literature

[79]

N/A No P(LE-IA-MEG) (M) Dexamethasone Plasma samples Plasma samples and in

vitro release

[81]

N/A No Eudragit S (C); Eudragit L

(C); Eudragit RS100 (C)

Insulin Plasma samples N/A [140]

N/A No Eudragit L100-55 (M);

Eudragit L (M); Eudragit S (M)

Celecoxib Plasma samples; colitis

severity N/A [141] 1380 www .drugdisco verytoday .com Reviews KEYNOTE REVIEW

(9)

Drug Discove ry Toda y V olume 25, Number 8 A ugust 2020 REVIEWS TABLE 2 (Continued )

GI tract mentioned

determined with

N/A No Eudragit S (C) Budesonide Tissue sections

harvested

Tissue concentrations [41]

N/A No Eudragit S (C); alginate

(M)

Ginger extract Tissue sections

harvested; clinical effects on colon cancer

Visual observation of ileocecal junction

[50]

N/A No Eudragit S/Compritol (M)

10-hydroxy-camptothecin; coumarin-6

Tissue sections harvested; microscopy; plasma samples

[46]

Disease model available No Eudragit S (M) Carboxy-fluorescein Tissue sections

Plasma and colon concentrations

[44]

Disease model available Yes Eudragit P-4135F (M) Ellagic acid with and

without fluorescein

Tissue sections harvested; plasma samples

Plasma concentrations and visual observation of lower part of small intestine

[51]

Based on practical considerations No Eudragit S (C) IgY Tissue sections

N/A No Eudragit S (M); Eudragit L

(M); Eudragit S/Eudragit L (M)

Insulin Tissue sections

Insulin content in microspheres from harvested tissue sections

[49]

Disease model available No Eudragit S/PLGAb_(M);

Eudragit S (M)

Budesonide; coumarin-6 Tissue sections harvested; plasma samples; microscopy; colitis severity

[47]

N/A Yes Eudragit S (M); Eudragit L

(M); Eudragit L100-55 (M)

Prednisolone Plasma samples Not determined because

of failure of system (pH threshold not reached in rat)

[143]

N/A No Eudragit S/L and

Surelease1 (C)

Capecitabine Tissue sections

harvested

N/A No Eudragit S (C) Curcumin and

cyclosporine

Colitis severity Colitis severity and in vitro release

[103]

N/A No Eudragit S (M) Tacrolimus Colitis severity In vitro release [145]

Rat and mouse N/A No Eudragit S (C) Budesonide; DiRc_;

coumarin-6

Tissue sections harvested; plasma samples

Visual observation of harvested tissue sections combined with plasma and colon

concentrations

[45]

N/A No Pluronic/Polyacrylic acid

(M)

Epirubicin; Toluidine Blue O

Tissue sections harvested; plasma samples; tumor size

Visual observation of harvested tissue sections

[54]

N/A No P(CFC-MAA-MEG) (M) Dexamethasone Tissue sections

Plasma and colon concentrations [43] www .drugdisco verytoday .com 1381

(10)

REVIEWS Drug Discover y Today V olume 25, Number 8 August 2020 TABLE 2 (Continued )

GI tract mentioned

determined with

Mouse N/A No Eudragit S/Eudragit L (C) Sulfasalazine Paw edema severity N/A [146]

N/A No P(CE-MAA-MEG) (M) Mesalazine Colitis severity Colitis severity [102]

Disease model available No Eudragit S/PLGA (M) Curcumin Colitis severity N/A [147]

N/A No Eudragit P-4135 F (M) Tacrolimus Colitis severity N/A [148]

Disease model available No Eudragit S/PLGAb_(M) _Curcumin _{Tissue sections}

harvested, colitis severity

Visual accumulation in colon

[58]

N/A Yes Eudragit FS 30 D (C);

Eudragit L100-55 (C)

FITC-BSA; luciferase DNA plasmid; CpG vaccine Tissue sections harvested Cellular uptake in harvested tissue [57]

Disease model available No Eudragit FS 30 D/PLGAb

(M)

Cyclosporine; DiRc Tissue sections harvested; colitis severity

Visual observation of harvested tissue sections

[55]

Disease model available No Eudragit S (C) Budesonide; DiRc;

coumarin-6

NIR spectroscopy; tissue sections harvested; disease severity

NIR spectroscopy and colon concentrations

[56]

Disease model available No Eudragit S (M) Curcumin Colitis severity; plasma

samples; fecal matter

Fecal concentrations [48]

Mouse and rabbit Disease model available (mouse) and N/A (rabbit)

No Eudragit P-4135F (M) Enoxaparin Plasma samples Not possible because of

negligible systemic absorption of enoxaparin

[149]

Rabbit N/A No Eudragit S/Ethyl cellulose

(C)

Metronidazole Plasma samples Plasma samples and

colon arrival time from literature

[80]

GI tract comparable to human Yes Eudragit S/Ethyl cellulose (C)

Theophylline Radiography, plasma

samples

Radiography and colon arrival time from literature [62] a Not available. b Poly(lactic-co-glycolic acid. c

1,10-dioctadecyl-3,3,30,30-tetramethylindotricarbocyanine iodide.

1382 www .drugdisco verytoday .com Reviews KEYNOTE REVIEW

(11)

identified(iib,iiib,andiv)(Table4).Oneoptionistoretrievethe

drugdeliverysystemitself(iv)andtomeasurethedrugcontentin

thesystem,inwhichincompleterecoveryindicatesdrugrelease.

Intheotherapproaches(iibandiiib),itisimportantthatthedrug

isnotextractedfromthedrugdeliverysystemandthattheluminal

contentisincludedintheassayeitherwithorwithoutthetissue.

Drug release is then indicated by the presence of drug in the

extract.

Allmethodsdescribedabovemeasuredrugcontent.Nexttothis

approach,itispossibleto detectthe drugdeliverysystem (e.g.,

microspheresor tablets) in harvested tissue samplesvisually or

withalightmicroscope.Incaseofdissolvingorerodingsystems,

thesemethodsallowfortheconformationofdrugreleasefromthe

disappearanceofthedrugdeliverysystem[50–54].Whenthedrug

deliverysystemisstillpresent,drugreleasecannotberuledout.

Moreinformationisobtainedwhenreleasecanbevisualizedby

fluorescencemicroscopyorNIRfluorescencemicroscopy,whena

markerisincludedinthedeliverysystem.Forfluorescence

micros-copy,fluoresceinorcoumarin-6havebeenusedasmarkersandfor

NIR fluorescence imaging, 1,10-dioctadecyl-3,3,30,30

-tetramethy-lindotricarbocyanine iodide (DiR),hasbeen used[45,51,55,56].

Furthermore,cellularuptakeofthe marker/drugcan beusedto

verifyrelease[45,46,57,58].

When tissue sections areharvested, multipleanimals are

re-quired.The consequenceofthisisthatindividualanimalswith

variableGItransittimesarecompared,whichmakes

interpreta-tionofthedatalessreliablewhenthedataarepooled.Moreover,

the exact locationofdrug releasecannoteasilybedetermined,

especiallyformultiparticulateformulations,suchasmicrospheres,

becausetheyspreadthroughouttheGItract.Additionally,many

animals have to be sacrificed to obtain data at sufficient time

points. Furthermore, possible degradation or metabolization

and/ortransferofthedrugtotheplasmahavetobetakeninto

account.Theadvantageofharvestingtissuesamplesisthat itis

possibletoassesswhetherthedrughasreachedthecolon.Ifthe

drug doesnotreach the colon, nodrug wouldbemeasured or

visually detected in the colonictissue and/or luminalcontent.

However,ifnodrugismeasuredorvisuallydetectedinthecolonic

tissue,itcannotbeexcludedwithcertaintythatthedrughasnot

reachedthecolon.Morereliableconclusionscanbedrawnwhen

plasmasamplesaretaken atthe sametime pointsasthe tissue

samples,ontheconditionthatthedrugisimmediatelyabsorbed

intothesystemiccirculationafterrelease.Thepresenceofdrugin

plasmaindicatesdrugreleaseandthepresenceintissuesections

mightindicatethesiteofrelease.

Non-invasive

imaging

techniques

Anattractivealternative toharvestingtissuesamples,alsointhe

lightofthe3Rs(reduction,refinement,andreplacement)foranimal

experiments[59],istheuseofnon-invasiveimagingtechniques.

Mostfrequentlyusedareradiographyandfluoroscopy,followedby

g

-scintigraphyandNIRfluorescenceimaging[60–71].

Ionizingradiation,X-rays,areusedinradiographyand

fluoros-copytocapturetheimageswhilein

g

-scintigraphythemarkerin

thedosageformemitsionizing

g

-radiation[72–74].Tovisualize

thedosageforminradiographyorfluoroscopy,a contrastagent

(e.g., barium sulfate) has to be integrated in the drug delivery

system[73,74].Forradiography,fluoroscopy,and

g

-scintigraphy,

thecumulativeionizingradiationexposurehastobeconsideredin

thestudydesigntoensurehumanetreatmentoftheanimals.This

is especially the case when animals are notsacrificed after the

study,becauseradiationcancauselong-termeffects[75].WithNIR

fluorescenceimaging,noionizingradiationisusedandinsteada

fluorescentagent(e.g.,DiR)isusedasamarkercompound[60,74].

ThedownsideofNIRfluorescenceimagingisthatitsuffersfroma

lowresolutionbecauseofattenuation,scattering,anddispersion

oftheemittedlightwhenitpassesthroughtissues[60,74].With

all imaging techniques, the animals have to be restrained or

broughtunderanesthesiatopreventblurredimages,whichcauses

TABLE3

Examplesofcolonicdiseasemodelsinrabbits

Disease Diseaseinduction Refs

Coloncancer VX2carcinomatransplantation [150,151] Azoxymethanesubcutaneously [152,153] Irritablebowel

syndrome

Moistheat,stress,andlow-doselaxatives [154] Colorectaldistensionwithinducedvisceral

hypersensitivity

[155,156] Inflammatory

boweldisease

Dilutedaceticacidintrarectally [157] Haptenintrarectally(e.g.,

2,4,6-trinitrobenzenesulfonicacidor dinitrochlorobenzene)

[158_–161]

InoculationwithEimeriamagnaoocytes intragastrically

[162] Degradedcarrageenanorally [163] Lipopolysaccharideintrarectallyafter1%

formalinenema

[164] InoculationwithCrohn’stissuehomogenates intraileally

[165] Immunecomplexintravenouslyin

combinationwithdiluteformalinintrarectally [166] InoculationwithBacteroidesvulgatus

intra-appendiceal

[167] Dextransodiumsulfateorally [168]

TABLE4

Overviewofmethodsusedtoanalyzeharvestedtissuesamples

Method Drugextracted Drugreleaseindicatedby Applicablefordrugs

thatarenotabsorbed Tissue Luminalcontent Deliverysystem

i X Presenceofdrug

iia X X X Incompleterecoverya

iib X X Presenceofdrug X

iiia X X Incompleterecovery

iiib X Presenceofdrug X

iv X Incompleterecovery X

a

Thetotaldrugcontentisnotretrieved.

Reviews

KEYNO

TE

(12)

discomfort to the animal [75]. Radiography, fluoroscopy, and

g

-scintigraphyare alsoused in clinicalstudies.The advantages

anddisadvantagesofthesemethodsweredescribedinmoredetail

inpartIofthisseries[1].

Basedontheevaluationofpublishedanimalstudiesinwhich

non-invasiveimagingtechniquesareapplied,wesuggestaspects

thatcouldbeimproved.Generally,onlyimagesweretakenfrom

one angle, generating a 2D imageof the abdomen[60–71,73].

Reasonsfortakingonlyoneimageanglecouldbe,forinstance,

reductionofradiationexposure,animalwelfare,orpractical

con-siderations.Imagesfromonlyoneanglemightleadto

misinter-pretation of disintegration and/or erosionof the drug delivery

systemoroftheexactlocationofthedrugdeliverysysteminthe

GI tract because the depth cannot be determined. When the

locationofthedifferent segmentsoftheGItract isdetermined

with,forinstance,abariumsulfatemealstudyinradiographyor

fluoroscopystudies,interpretationoftheimagesismore

straight-forward [61,62,66,67,73,76]. Interpretation of images is more

problematic inanimal studiesthaninclinical trialsbecause, in

humans,thelocationofthelargeintestineisfixedinthebodyand

theoutlineofthelargeintestinebecomesvisiblewhenthemarker

isreleased[1].Inaddition,theprojectionofthe2Dimages(e.g.,

dorsoventral)isoftennotgivenandtheprintedimagequalityand

sizeissometimesnotoptimal[60–62,64–70],whichcomplicates

interpretationoftheseimagesbythereaders.Furthermore,image

exposureandanimalpositioningcouldbeimproved,whichwould

simplify interpretation of the images [60–64,66–69,71]. When

these factors are notoptimal and when only imagesfrom one

angle areused, one should bereluctant withstatements about

colontargeting,becausestructuresintheabdomenoverlapand

theexact3Dpositioncannotbedeterminedwithcertainty.

Imagingtechniques,whenperformedcorrectly,provideuseful

informationaboutthepositionofadrugdeliverysystemintheGI

tract. However, these techniques do not provide information

about the drug release from a system and, therefore, do not

automatically give information about the ileocolonic targeting

ability.Thisshortcomingcanbeovercomebycombiningimaging

techniques with measuring plasma concentrations, especially

whenthedrugandanimagingmarkerarecombinedinthesame

drugdeliverysystem[60,62,67,71].However,thisisonlyvalidfor

drugsthatareabsorbedovertheentireGItract.

Plasma

samples

Drug and/ordrug metaboliteconcentrationsinplasmasamples

havebeenfrequentlyusedasaproofforileocolonicdrugdelivery

inanimalstudies(Table2)[52,77–81].However,plasma

concen-trationsgenerallydonotprovideenoughinformationtoconfirm

ileocolonicdrugdelivery.Anoptionistocomparethe

pharmaco-kineticdataobtainedfromtheplasmacurvetothecolonarrival

time,todeterminewhethertheobservedlagtimeofthesystemis

sufficiently long to warrant targeting to the colon [62,77–80].

Kennedyetal.developedamethodtomeasurethecolonicarrival

time,also calledmouth-to-cecal transittime,in humans,using

sulfasalazine [82],whichhasbeenvalidated byothers[83].This

methodisbasedonthefactthatsulfasalazineispoorlyabsorbedby

theGItractbutisconvertedbybacteriainthecoloninto

sulfa-pyridine,whichissubsequentlyabsorbed[84,85].Several

research-ershaveusedthismethodtocircumventinterindividualvariation

incolonarrivaltimes[52,86].Itisalsopossibletocircumventthe

intraindividualvariationincolonarrivaltimebycombining

sul-fasalazinewith areleasemarker(e.g.,theophylline) inthedrug

deliverysystem[1,87].Inthismethod,theophyllineisusedasa

markerfordrugrelease,sinceitisabsorbedovertheentireGItract.

Ifthereisnodifferenceinplasmaarrivaltimebetween

theophyl-lineandsulfapyridine,itindicatesthattheformulationreleasedits

contentsintothecolon.Adownsideofthismethodisthatitonly

givesananswertothequestionwhetherthedrugdeliverysystem

releasesitscontentsintothecolon,butdoesnotprovide

informa-tionastotheexactlocationinthecolon.Todeterminetheexact

location of drug release, imaging techniques can be used in

combination with the theophylline-sulfasalazine method.

Fur-thermore, the pharmacokinetics and/or pharmacodynamics of

theinvestigateddrugmightbeinfluencedbyeithersulfasalazine

orthereleasemarker(incasethedrugitselfcannotbeusedasa

releasemarker). In addition, sulfasalazine is degraded into not

onlysulfapyridine,butalsomesalazine,whichisa

pharmacologi-callyactive compound used in the treatment of inflammatory

boweldisease [82,88].This should betakenintoaccount ifthe

compoundsarecombinedintoonedrugdeliverysystem.

Breath

and

urine

samples

Inaddition to usingplasmasamples todetermine theorocecal

transit time, it is possible to use breath samples [89,90]. This

methodutilizes13C-urea(astableisotope),whichismetabolized

bybacteriainto13CO2 thatis subsequentlyexhaled. Inhuman

volunteers,thismethodincombinationwithmeasuring13C-urea

and15_N-urea_(an_internal_standard)_in_urine_has_been_used_to_verify

colonicdrugdelivery[1,91–93].Thisprinciplecouldalsobeused

inlaboratoryanimals.Collectionofurineinanimalsispossibleby

usingametaboliccageorbycatheterization[94].However,theuse

ofmetaboliccagesgeneratesastressfulenvironmentforanimals

becauseofindividualhousingandthewiremeshfloors,whichcan

influence the therapeuticeffect ofa drug or exacerbatedisease

symptoms[94,95].The latterstressorcanbepreventedby using

hydrophobicsandforurinecollection[96],butindividualhousing

wouldremainanissue.Inaddition,catherizationisproblematic

because,amongotherissues,thecathetercanberemovedbythe

animal,andinsertingthecatheterisastressoronitsown[97].The

collectionofbreathsamplesisalsoproblematicbecauseofstress

causedbyhandlingorindividualhousinginabreath-testsystem

[94,98].The non-invasivecharacter ofthe methodmakes itan

ideal method to verify colonic drug delivery in humans [1];

however,becauseoftheimplicationsofurineandbreath

collec-tioninlaboratoryanimals,wedonotrecommendthismethodfor

animalstudies.

Therapeutic

effect

The last method, abundantlyused to evaluateileocolonicdrug

deliverysystemsin laboratoryanimals,isto determinea

thera-peuticeffect(Table2)[99–103].However,thetherapeuticeffectas

suchdoesnotdirectlyanswerthequestionwhetherthedrugisin

facttargetedto,andreleasedinto,theileocolonicregion.

Thera-peuticproteins or peptides givenorally in an ileocolonicdrug

deliverysystemareanexceptionbecausetheywillbedegradedin

theupperGItract.Therefore,they canonlyelicit atherapeutic

effect if they arereleased in the colon [104]. Inother cases, a

(13)

therapeuticeffectshouldnotbeusedassuchtoverifyileocolonic

drugtargeting,butshouldbeusedincombinationwithimaging

techniques and plasma sampling to verifysuccessful targeting.

Whenthetheophylline–sulfasalazinemethodisusedorurine is

collected with a metabolic cage, the influence on therapeutic

effectsofthedrugunderinvestigationshouldfirstbetakeninto

accountbeforeconductingtheexperiments.

Overall,thedeterminationofthetherapeuticeffectisvaluable,

becauseit cananswer the questions whetherthe drugdelivery

systemcanimproveclinicalsymptomsandwhetheritissuperior

tonontargeteddrugdeliverysystems.

Concluding

remarks

For testing novel drugs forileocolonic delivery and/or for

pH-dependent ileocolonic drug delivery systems containing novel

excipients,animalshaveto beused.Toobtain ileocolonicdrug

delivery,asharpdistinctpHpeakintheterminalileumiscrucial,

thusasimilarpHtohumansisnotessential.However,ifthepH

profile ofthe GItract in animals is similar tothat of humans,

establishedileocolonicdrugdeliverysystemscanbeusedtotest

novel drugs. When novel pH-dependentexcipients have to be

tested,apHprofilesimilartothatinhumansisaprerequisiteto

obtainileocolonicdrugdeliveryinhumans.Inthisrespect,the

rabbitisthemostappropriateanimalspeciescomparedwithother

frequentlyusedlaboratoryanimals,becausetheirGIpHvaluesare

mostsimilartothoseofthehumanGItract.However,notonlythe

pHvaluesoftheGItract,butalsothedesireddiseasemodeland

thesizeofthedeliverysystemhavetobetakenintoaccount.Ifthe

rabbitcannotbeused,thenthepig,rat,anddogmightbesuitable

alternatives, onthe condition that the pHvaluesof individual

animalsareverifiedfirst.Toproperlydrawconclusionsfromthe

obtainedefficacydata,ileocolonicdrugdeliverymustbeverified.

Thedifferentmethodsusedforthisverificationallhavespecific

advantagesand limitations,thustheoptimalmethodshouldbe

determinedforeachstudy.Non-invasive imagingtechniquesin

combinationwithplasmasamplingcanbeusedifthetherapeutic

effectofanoveldrugisinvestigated.Whenanovelexcipientinthe

drugdeliverysystemitselfissubjectofinvestigation,the

theoph-ylline-sulfasalazine methodis an elegant way to verifycolonic

drugdelivery.

Declaration

of

Competing

Interest

Theauthorsdeclarethefollowingfinancialinterests/personal

rela-tionships which maybeconsideredas potentialcompetinginterests:

HWFisoneoftheinventorsofapatent(WO2007/013794)

describ-ingamethodforcolontargeting,whichisheldbyhisemployer.The

otherauthorsdeclarethattheyhavenoknowncompetingfinancial

interests or personal relationships that could have appeared to

influence the workreported inthis paper. The literature search

was partiallyfundedbyJanssenPharmaceutica.Janssen

Pharma-ceuticahadneitherroleinstudydesign,inthecollection,analysis,

andinterpretationofdata,norinthewritingofthereportandinthe

decisiontosubmitthepaperforpublication.

Acknowledgments

ThisliteraturesearchwaspartiallyfundedbyJanssen

Pharmaceutica.JanssenPharmaceuticahadneitheraroleinthe

studydesign,inthecollection,analysis,andinterpretationofdata,

norinthewritingofthereportandinthedecisiontosubmitthe

paperforpublication.

References

1Broesder,A.etal.(2020)pH-dependentileocolonicdrugdelivery,partI:invitro

andclinicalevaluationofnovelsystems.DrugDiscov.Today.http://dx.doi.org/

10.1016/j.drudis.2020.06.011PublishedonlineJuneXX,2020

2Bespalov,A.etal.eds(2020)GoodResearchPracticeinNon-ClinicalPharmacologyand Biomedicine,Springer

3ICH(2020)SafetyGuidelines.ICH

4ICH(2000)GuidelineS7ASafetyPharmacologyStudiesforHumanPharmaceuticals. ICH

5Hatton,G.B.etal.(2015)Animalfarm:considerationsinanimalgastrointestinal physiologyandrelevancetodrugdeliveryinhumans.J.Pharm.Sci.104,2747– 2776

6Sjo¨gren,E.etal.(2014)Invivomethodsfordrugabsorption–comparative physiologies,modelselection,correlationswithinvitromethods(IVIVC),and applicationsforformulation/API/excipientcharacterizationincludingfood effects.Eur.J.Pharm.Sci.57,99–151

7Arndt,M.etal.(2013)Dissolutionmediasimulatingtheproximalcanine gastrointestinaltractinthefastedstate.Eur.J.Pharm.Biopharm.84,633–641

8Akimoto,M.etal.(2000)GastricpHprofilesofbeagledogsandtheiruseasan alternativetohumantesting.Eur.J.Pharm.Biopharm.49,99–102

9Lui,C.Y.etal.(1986)ComparisonofgastrointestinalpHindogsandhumans: implicationsontheuseoftheBeagledogasamodelfororalabsorptioninhumans. J.Pharm.Sci.75,271–274

10Sagawa,K.etal.(2009)FedandfastedgastricpHandgastricresidencetimein consciousbeagledogs.J.Pharm.Sci.98,2494–2500

11Mahar,K.M.etal.(2012)GastricpHandgastricresidencetimeinfastedandfed consciousBeagledogsusingtheBravo1pHsystem.J.Pharm.Sci.101,2439–2448

12Steinberg,W.H.etal.(1965)HeidelbergcapsuleI.Invitroevaluationofanew instrumentformeasuringintragastricpH.J.Pharm.Sci.54,772–1726

13Dickman,R.andFass,R.(2006)AmbulatoryesophagealpHmonitoring:new directions.Dig.Dis.24,313–318

14Chotiprashidi,P.etal.(2005)ASGETechnologyStatusEvaluationReport:wireless esophagealpHmonitoringsystem.Gastrointest.Endosc.62,485–487

15Becker,D.etal.(2014)NovelorallyswallowableIntelliCap1devicetoquantify regionaldrugabsorptioninhumanGItractusingdiltiazemasmodeldrug.AAPS PharmSciTech15,1490–1497

16Smith,H.W.(1965)Observationsonthefloraofthealimentarytractofanimals andfactorsaffectingitscomposition.J.Pathol.Bacteriol.89,95–122

17Smith,H.W.andJones,J.E.T.(1963)Observationsonthealimentarytractandits bacterialflorainhealthyanddiseasedpigs.J.Pathol.Bacteriol.86,387–412

18Merchant,H.A.etal.(2015)Gastrointestinalcharacterisationanddrugsolubility determinationinanimals.J.Pharm.Pharmacol.67,630–639

19Gupta,P.K.andRobinson,J.R.(1988)Gastricemptyingofliquidsinthefasteddog. Int.J.Pharm.43,45–52

20Kalantzi,L.etal.(2006)Canineintestinalcontentsvs.simulatedmediaforthe assessmentofsolubilityoftwoweakbasesinthehumansmallintestinalcontents. Pharm.Res.23,1373–1381

21Graysel,D.M.andMiller,E.G.(1928)ThepHofthecontentsofthegastrointestinal tractindogs,inrelationtodietandrickets.J.Biol.Chem.76,423–436

22Kohl,K.D.etal.(2013)EffectsofanatomyanddietongastrointestinalpHin rodents.J.Exp.Zool.AEcol.Genet.Physiol.319,225–229

23McConnell,E.L.etal.(2008)MeasurementsofratandmousegastrointestinalpH, fluidandlymphoidtissue,andimplicationsforin-vivoexperiments.J.Pharm. Pharmacol.60,63–70

24Cummings,J.H.andMacfarlane,G.T.(1991)Thecontrolandconsequencesof bacterialfermentationinthehumancolon.J.Appl.Bacteriol.70,443–459

25Merchant,H.A.etal.(2011)AssessmentofgastrointestinalpH,fluidandlymphoid tissueintheguineapig,rabbitandpig,andimplicationsfortheiruseindrug development.Eur.J.Pharm.Sci.42,3–10

26Ward,F.W.etal.(1986)Nitratereduction,gastro-intestinalpHandN-nitrosation ingnotobioticandconventionalrats.FoodChem.Toxicol.24,17–22

27Haiba,M.H.(1954)ThepHofthealimentarytractinnormalandGiardia-infected culturemice.Parasitology44,387–391

28Haiba,M.H.andWilliamson,J.(1952)ThepHofthesmallintestineofnormal, starvedandGiardia-Infectednorwayrats.Trans.R.Soc.Trop.Med.Hyg.46,85–93

Reviews

KEYNO

TE

(14)

29Ward,F.W.andCoates,M.E.(1987)GastrointestinalpHmeasurementinrats: influenceofthemicrobialflora,dietandfasting.Lab.Anim.21,216–222

30Eaimtrakarn,S.etal.(2001)Retentionandtransitofintestinalmucoadhesivefilms inratsmallintestine.Int.J.Pharm.224,61–67

31Mori,C.andKondo,H.(2006)Effectofgastricacidityregulationonthe gastrointestinaltransittimeandsecretionofgastricfluidsinbeagledogs.J.Drug Deliv.Sci.Technol.16,467–472

32Yamada,I.andHaga,K.(1990)MeasurementofgastricpHduringdigestionofa solidmealindogs.Chem.Pharm.Bull.(Tokyo)38,1755–1756

33Gidenne,T.andLebas,F.(2006)Feedingbehaviourinrabbits.InFeedingin DomesticVertebrates:FromStructuretoBehaviour(Bels,V.,ed.),pp.179–194,CABI

34Chen,E.P.etal.(2008)GastricpHandgastricresidencetimeinfastedandfed consciouscynomolgusmonkeysusingtheBravo1pHsystem.Pharm.Res.25, 123–134

35Polentarutti,B.etal.(2010)ModificationofgastricpHinthefasteddog.J.Pharm. Pharmacol.62,462–469

36Ewe,K.etal.(1999)InflammationdoesnotdecreaseintraluminalpHinchronic inflammatoryboweldisease.Dig.Dis.Sci.44,1434–1439

37Pye,G.etal.(1990)GastrointestinalintraluminalpHinnormalsubjectsandthose withcolorectaladenomaorcarcinoma.Gut31,1355–1357

38Ovesen,L.etal.(1986)IntraluminalpHinthestomach,duodenum,andproximal jejunuminnormalsubjectsandpatientswithexocrinepancreaticinsufficiency. Gastroenterology90,958–962

39Farmer,A.D.etal.(2014)CecalpHisabiomarkerofexcessivecolonic fermentation.WorldJ.Gastroenterol.20,5000–5007

40Press,A.G.etal.(1998)GastrointestinalpHprofilesinpatientswithinflammatory boweldisease.Aliment.Pharmacol.Ther.12,673–678

41Xu,Q.etal.(2013)Preparation,invitroandinvivoevaluationofbudesonideloaded core/shellnanofibersasoralcolonicdrugdeliverysystem.J.Nanosci.Nanotechnol. 13,149–156

42Hirayama,M.etal.(2011)Concentrationdependenceof5-aminosalicylicacid pharmacologicalactionsinintestinalmucosaafteroraladministrationofa pH-dependentformulation.Mol.Pharm.8,1083–1089

43Dong,K.etal.(2014)Assessmentofthesafety,targeting,anddistribution characteristicsofanovelpH-sensitivehydrogel.ColloidsSurf.BBiointerfaces.123, 965–973

44Kietzmann,D.etal.(2010)ColonicdeliveryofcarboxyfluoresceinbypH-sensitive microspheresinexperimentalcolitis.Eur.J.Pharm.Biopharm.76,290–295

45Kim,H.Y.etal.(2020)Ternarynanocompositecarriersbasedonorganicclay-lipid vesiclesasaneffectivecolon-targeteddrugdeliverysystem:preparationandin vitro/invivocharacterization.J.Nanobiotechnol.18,17

46Gan,L.etal.(2013)NovelpH-sensitivelipid-polymercompositemicrospheresof 10-hydroxycamptothecinexhibitingcolon-specificbiodistributionandreduced systemicabsorption.J.Pharm.Sci.102,1752–1759

47Makhlof,A.etal.(2009)pH-sensitivenanospheresforcolon-specificdrug deliveryinexperimentallyinducedcolitisratmodel.Eur.J.Pharm.Biopharm.72, 1–8

48Kesharwani,S.S.etal.(2018)Site-directednon-covalentpolymer-drugcomplexes forinflammatoryboweldisease(IBD):formulationdevelopment,characterization andpharmacologicalevaluation.J.Control.Release290,165–179

49Morishita,I.etal.(1993)Enteralinsulindeliverybymicrospheresin3different formulationsusingEudragitL100andS100.Int.J.Pharm.91,29–37

50Deol,P.K.andKaur,I.P.(2013)Improvingthetherapeuticefficiencyofginger extractfortreatmentofcoloncancerusingasuitablydesignedmultiparticulate system.J.DrugTarget.21,855–865

51Jeong,Y.-I.etal.(2001)ApplicationofEudragitP-4135Fforthedeliveryofellagic acidtotheratlowersmallintestine.JPharmPharmacol.53,1079–1085

52Hu,Z.etal.(1999)Characterizationofnorfloxacinereleasefromtabletcoatedwith anewpH-sensitivepolymer,P-4135F.J.DrugTarget.7,223–232

53Geary,R.S.andSchlameus,W.H.(1993)Vancomycinandinsulinusedasmodels fororaldeliveryofpeptides.J.Control.Release23,65–74

54Lo,Y.-L.etal.(2013)pH-andthermo-sensitivepluronic/poly(acrylicacid)insitu hydrogelsforsustainedreleaseofananticancerdrug.J.DrugTarget.21,54–66

55Naeem,M.etal.(2018)Colon-targeteddeliveryofcyclosporineAusing dual-functionalEudragit1FS30D/PLGAnanoparticlesamelioratesmurine experimentalcolitis.Int.J.Nanomed.13,1225–1240

56Naeem,M.etal.(2018)pH-triggeredsurfacecharge-reversalnanoparticlesalleviate experimentalmurinecolitisviaselectiveaccumulationininflamedcolonregions. Nanomedicine14,823–834

57Zhu,Q.etal.(2012)Largeintestine-targeted,nanoparticle-releasingoralvaccineto controlgenitorectalviralinfection.Nat.Med.18,1291–1296

58Beloqui,A.etal.(2014)pH-sensitivenanoparticlesforcolonicdeliveryof curcuminininflammatoryboweldisease.Int.J.Pharm.473,203–212

59Russell,W.M.S.andBurch,R.L.(1959)ThePrinciplesofHumaneExperimental Technique.Methuen&Co.Ltd

60Hou,L.etal.(2016)Smartnanocompositehydrogelsbasedonazocrosslinked grapheneoxidefororalcolon-specificdrugdelivery.Nanotechnology27,315105

61Sharma,P.andPathak,K.(2013)Inulin-basedtabletincapsuledeviceforvariable multipulsedeliveryofaceclofenac:optimizationandinvivoroentgenography. AAPSPharmSciTech14,736–747

62Patel,M.M.andAmin,A.F.(2011)Designandoptimizationofcolon-targeted systemoftheophyllineforchronotherapyofnocturnalasthma.J.Pharm.Sci.100, 1760–1772

63Hong,S.etal.(2012)Colon-targetedcell-permeableNFkBinhibitorypeptideis orallyactiveagainstexperimentalcolitis.Mol.Pharm.9,1310–1319

64Omar,S.etal.(2007)Colon-specificdrugdeliveryformebeverinehydrochloride.J. DrugTarget.15,691–700

65Avachat,A.M.andShinde,A.S.(2016)Feasibilitystudiesofconcomitant administrationofoptimizedformulationofprobiotic-loadedVancomycin hydrochloridepelletsforcolondelivery.DrugDev.Ind.Pharm.42,80–90

66Yassin,A.E.B.etal.(2010)Newtargeted-colondeliverysystem:invitroandinvivo evaluationusingX-rayimaging.J.DrugTarget.18,59–66

67Patel,M.M.andAmin,A.F.(2011)Formulationanddevelopmentofrelease modulatedcolontargetedsystemofmeloxicamforpotentialapplicationinthe prophylaxisofcolorectalcancer.DrugDeliv.18,281–293

68Moghimipour,E.etal.(2018)InvivoevaluationofpHandtime-dependent polymersascoatingagentforcolonicdeliveryusingcentralcompositedesign.J. DrugDeliv.Sci.Technol.43,50–56

69Amrutkar,J.R.andGattani,S.G.(2012)AnovelhydrogelplugofSterculiaurensfor pulsatiledelivery:invitroandinvivoevaluation.J.Microencapsul.29,72–82

70Mastiholimath,V.S.etal.(2007)TimeandpHdependentcolonspecific,pulsatile deliveryoftheophyllinefornocturnalasthma.Int.J.Pharm.328,49–56

71Patel,M.M.(2017)Formulationanddevelopmentofdi-dependent

microparticulatesystemforcolon-specificdrugdelivery.DrugDeliv.Transl.Res.7, 312–324

72Perkins,A.C.andFrier,M.(2004)Radionuclideimagingindrugdevelopment. Curr.Pharm.Des.10,2907–2921

73Owens,J.M.andBiery,D.N.(1998)RadiographicInterpretationfortheSmallAnimal Clinician(2ndedn),Williams&Wilkins

74Kiessling,F.etal.eds(2017)SmallAnimalImaging,Springer

75UniversityofBritischColumbiaAnimalCareCommittee(2014)UBCACC GuidelineonImagingofRodents.UBC

76Hardy,J.G.etal.(1987)Evaluationofanenteric-coateddelayed-release 5-aminosalicylicacidtabletinpatientswithinflammatoryboweldisease.Aliment. Pharmacol.Ther.1,273–280

77Takaya,T.etal.(1998)Importanceofdissolutionprocessonsystemicavailability ofdrugsdeliveredbycolondeliverysystem.J.Control.Release50,111–122

78Gao,C.etal.(2006)Invitroreleaseandinvivoabsorptioninbeagledogsof meloxicamfromEudragit1FS30D-coatedpellets.Int.J.Pharm.322,104–112

79Lamprecht,A.etal.(2004)pH-sensitivemicrospheredeliveryincreasesoral bioavailabilityofcalcitonin.J.Control.Release98,1–9

80Shah,N.etal.(2016)Designofexperimentapproachforformulatingmulti-unit colon-targeteddrugdeliverysystem:invitroandinvivostudies.DrugDev.Ind. Pharm.42,825–835

81Dong,K.etal.(2016)Assessmentofthedrugloading,invitroandinvivorelease behaviorofnovelpH-sensitivehydrogel.DrugDeliv.23,174–184

82Kennedy,M.etal.(1979)Apharmacologicalmethodofmeasuringmouthcecal transittimeinman.Br.J.Clin.Pharmacol.8,372–373

83Kellow,J.E.etal.(1986)Sulfapyridineappearanceinplasmaafter salicylazosulfapyridine.Gastroenterology91,396–400

84Dahan,A.andAmidon,G.L.(2009)SmallintestinaleffluxmediatedbyMRP2and BCRPshiftssulfasalazineintestinalpermeabilityfromhightolow,enablingits colonictargeting.Am.J.Physiol.LiverPhysiol.297,G371–G377

85Schroˆder,H.etal.(1973)Metabolismofsalicylazosulfapyridineinhealthysubjects andinpatientswithulcerativecolitis;Effectsofcolectomyandofphenobarbital. Clin.Pharmacol.Ther.14,802–809

86Takaya,T.etal.(1995)Developmentofacolondeliverycapsuleandthe pharmacologicalactivityofrecombinanthumangranulocytecolony-stimulating factor(rhG-CSF)inBeagledogs.J.Pharm.Pharmacol.47,474–478

87Ishibashi,T.etal.(1999)Evaluationofcolonicabsorbabilityofdrugsindogsusing anovelcolon-targeteddeliverycapsule(CTDC).J.Control.Release59,361–376

88Khan,A.K.A.etal.(1977)Anexperimenttodeterminetheactivetherapeutic moietyofsulphasalazine.Lancet310,892–895

89Uchida,M.andYoshida,K.(2009)Non-invasivemethodforevaluationof gastrocecaltransittimebyusingabreathtestinconsciousrats.J.Pharmacol.Sci. 110,227–230