Drug Delivery Strategies for Antivirals against Hepatitis B Virus

20 Pages·2017·2 MB·English

viruses
Review
Drug Delivery Strategies for Antivirals against
Hepatitis B Virus
LataviaSingh,SunainaIndermun,MershenGovender,PradeepKumar ID,LisaC.duToit,
YahyaE.ChoonaraandVinessPillay* ID
WitsAdvancedDrugDeliveryPlatformResearchUnit,DepartmentofPharmacyandPharmacology,Schoolof
TherapeuticSciences,FacultyofHealthSciences,UniversityoftheWitwatersrand,7YorkRoad,Parktown,
Johannesburg2193,SouthAfrica;[email protected](L.S.);[email protected](S.I.);
[email protected](M.G.);[email protected](P.K.);[email protected](L.C.d.T);
[email protected](Y.E.C.)
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:21April2018;Accepted:8May2018;Published:17May2018
Abstract: Chronic hepatitis B virus (HBV) infection poses a significant health challenge due to
associatedmorbidityandmortalityfromcirrhosisandhepatocellularcancerthateventuallyresults
inthebreakdownofliverfunctionality. Nanotechnologyhasthepotentialtoplayapivotalrolein
reducingviralloadlevelsanddrug-resistantHBVthroughdrugtargeting,thusreducingtherateof
evolutionofthedisease. Apartfromtissuetargeting,intracellulardeliveryofawiderangeofdrugs
isnecessarytoexertatherapeuticactionintheaffectedorganelles. Thisreviewencompassesthe
strategiesandtechniquesthathavebeenutilizedtotargettheHBV-infectednucleiinliverhepatocytes,
withasignificantlookatthenewinsightsandmostrecentadvancesindrugcarriersandtheirrolein
anti-HBVtherapy.
Keywords: Hepatitis B virus; intracellular drug delivery; liver targeting; hepatocyte;
asialoglycoproteinreceptor;nanoparticles;cell-penetratingpeptides
1. Introduction
AsignificantthreattopatientssufferingfromchronichepatitisBvirus(HBV)infectionliesinthe
emergenceofcirrhosis,oftenleadingtohepatocellularcancer,portalhypertension,orliverfailure.
TengenotypesofHBVhavebeenidentified,labeledAthroughJ,withgenotypesA,B,andCbeing
mostprevalent[1,2]. Anti-HBVtherapy,overthelastfewyears,promisedanenhancedeffectiveness
againstHBV.Drugssuchasinterferon-α2B,PEGylatedinterferon-α2A,lamivudine,adefovir,tenofovir,
entecavir,andtelbivudinehaveallbeenrecognizedandacknowledgedfortheirtreatmentanddirect
antiviralactivityagainstchronicHBVinfection[2,3]. Theprincipalaimsthatneedtobeaddressed
to attain a favorable therapeutic approach in an HBV infection should include a reduction in the
viralloadtoundetectablelevels,adecreaseintherateofprogressionofthedisease,andareduced
rate of evolution of drug-resistant HBV. The mechanics of targeted drug delivery are foremost in
considerationforparenteraladministrationinadditiontoshieldingtherapeuticsfromdegradation
anduntimelyelimination. Thesemechanicsaredrugdeliveryvehiclesconsistingofsolublecarriers
suchassyntheticpolymers,particulatecarrierssuchasmicro-andnanoparticles,andtarget-specific
recognitionmoietiessuchasmonoclonalantibodies.
Classes of drug targeting fall into two groups, namely active and passive targeting. Active
targeting utilizes certain interactions at the target site, such as those of ligand–receptor and
antigen–antibodybinding[4],orinothercases,signalssuchastemperatureormagneticfieldsthatcan
beappliedexternally. Passivetargetinginvolvesadjustingcarriersystems’physicochemicalproperties
Viruses2018,10,267;doi:10.3390/v10050267 www.mdpi.com/journal/viruses
Viruses2018,10,267 2of20
to that of the physiological and histological features of the target site. At the target site of action,
inthiscasebeingtheliver,adrugistransportedbyitscarrier. Forthisseeminglystraightforwardand
simpleapproachtowork,multiplefundamentalrequirements(nonspecificinteractions,accesstothe
targetsite,drugrelease,andsuitability)mustbesatisfied. Thisarticlethereforereviewsthecurrently
developeddrugdeliverystrategiesforthetreatmentofchronicHBVinfectionandprovidesabroad
discussionontheireffectivenesstodecreaseHBVviralcountsinthehumanbody. Emphasisisalso
providedonthedrugscurrentlybeingutilized,inadditiontothenoveldrugdeliveryplatformsthat
havebeenproducedwiththeaimofprovidingarationalebetweenthevariousdrugsbeingutilizedin
combinationwiththeadvanceddrugdeliverysystemsemployed.
2. Site-SpecificLiverTargeting
Hepato-orlivertargetingdoesnotnecessarilyreflecthepatocytetargeting,asothercellsinthe
liversuchastheKupffercellspossesstheabilitytophagocytose.Therefore,carriersneedtobemodified
chemicallytoachievetheabilityofencompassinglivercelltypespecificity[5]. Theasialoglycoprotein
receptor(ASGP-R)hasbeenamongstthemostdistinguishedandinvestigatedofallreceptorsandwas
firstcorroboratedbyAshwellandMorell[6]. TheASGP-Rhasprovidedalocationtoaccommodate
cell–cellintercommunicationthatismembrane-bound. Itallowsfortargetingspecificityofvarious
therapeuticagents,andhasbeentheresearchfocusforHBVuptake[7]. Severalattemptshavebeen
madetolabelcarriers(polymers,humanserumalbumin,andrecombinanthighdensitylipoproteins)
withASGP-R-specificligands(galactose,lactose,acetylgalactosamine,asialofetuin)todesignspecific
carriersfordrugandgenedeliverytohepatocytes[8].
Organ-specifictargeteddrugdeliveryhasbeenachievedusingthelinearaminopolysaccharide
chitosananditsderivatives. Systemsthattargettheliveremploypassivetrappingofthedrug-loaded
delivery device by the reticuloendothelial system (RES) or active targeting established through
recognitionbetweenhepaticreceptorsandtheligand-bearingdrug-loadeddeliverydevice. Inone
suchstudy,lactosaminatedN-succinyl-chitosanwassynthesizedanditspotentialasaliver-specific
drugcarrierassessed[9]. Itwasfoundthatthiscarrieraccumulatedanddistributedthroughoutthe
liverasaresultofinteractionwiththeASGP-R.
Lactose conjugated to polyion complex micelles consisting of polyethylene glycol grafted
to chitosan also indicated promising potential as a liver-specific nanocarrier to deliver the drug
diammoniumglycyrrhizinate[10].AstudyundertakenbyLinandcoworkers[11]modifiedthesurface
of chitosan nanoparticles by conjugating it with glycyrrhizin. The conjugation occurred through a
processofoxidationoftheglycyrrhizinwithsodiumperiodate[11].Invitrostudiesshowedlocalization
of the glycyrrhizin-conjugated chitosan nanoparticles in hepatocytes, and the intracellular uptake
amountedto4.9timeshigherthanthatofhepaticnonparenchymalcells.Thenanoparticledoseaswellas
theincubationtimeweresignificantmarkersofthecellularuptakeprocess.Livertargetingwasachieved
byaspecificinteractionbetweentheglycyrrhizinandthehepatocytes: aligand–receptorinteraction.
High-densitylipoproteins(HDL)presentanothereffectivehepatictargetingcarrier. HDLtakes
upphospholipidsandcholesterolfromthebody’speripheraltissuesanddeliversthemtotheliver
hepatocytes via the apolipoprotein (apo) A-l, the major lipoprotein component in HDL. A study
undertakenbyBiessenandcoworkers[12]designeddivalentandtrivalentclusterglycosidesattached
totheantiviralnucleoside9-(2-phosphonylmethoxyethyl)adenine(PMEA).Thisconjugationpromoted
ASGP-Raffinity[12]. Datafromthisworkindicatedthatmorethan90%ofthetherapeuticagentwas
taken up by liver parenchyma cells, thereby reducing its localization in other tissues significantly.
Dextranconjugationisalsoseeninafavorablelight. Synergistically, modificationofdextranwith
galactose and mannose results in its selectivity for hepatocytes and Kupffer cells, respectively [5].
Liver-specific treatment and hepatocellular carcinoma (HCC)-targeting of doxorubicin is possible
whenthepolymersusedarefunctionalizedwithgalactosamine[13]. Figure1depictsandfollowsthe
movementofatherapeuticdrug-loadeddeliverysystemtoliverhepatocytes.
Viruses2018,10,267 3of20
Viruses 2018, 10, x 3 of 20

Figure 1. Liver targeting by nanoparticle (NP) therapeutics: (a) nanosized particles of less than 200
Figure1.Livertargetingbynanoparticle(NP)therapeutics:(a)nanosizedparticlesoflessthan200nm
nm with specific functionalities aid in the evasion of premature Kupffer cell clearance; (b) nanosized
with specific functionalities aid in the evasion of premature Kupffer cell clearance; (b) nanosized
particles extravasate into the space of Disse through sinusoidal fenestrations in basal lamina absence;
particlesextravasateintothespaceofDissethroughsinusoidalfenestrationsinbasallaminaabsence;
(c) a high local concentration of NP therapeutics diffusing across the loosely organized extracellular
(c)ahighlocalconcentrationofNPtherapeuticsdiffusingacrossthelooselyorganizedextracellular
matrix in the space of Disse; (d) nonspecific endocytic uptake; and (e) receptor-mediated uptake by
matrixinthespaceofDisse;(d)nonspecificendocyticuptake;and(e)receptor-mediateduptakebythe
the hepatocyte (reproduced with permission from [14], © Elsevier B.V. Ltd., 2010).
hepatocyte(reproducedwithpermissionfrom[14],©ElsevierB.V.Ltd.,2010).
Various drug carriers often fail in delivering drugs to extravascular sites. A strategic plan would
be to use low-molecular-weight prodrugs that have the ability to distribute themselves throughout
Various drug carriers often fail in delivering drugs to extravascular sites. A strategic plan
the body, but are cleaved intracellularly to the active form of the drug by an organ-specific enzyme.
would be to use low-molecular-weight prodrugs that have the ability to distribute themselves
HepDirect prodrugs are a series of phosphate and phosphonate prodrugs that pursue a cytochrome
throughoutthebody,butarecleavedintracellularlytotheactiveformofthedrugbyanorgan-specific
P450-catalyzed oxidative cleavage reaction inside the hepatocytes. These prodrugs are cyclic 1,3-
enzyme. HepDirect prodrugs are a series of phosphate and phosphonate prodrugs that pursue a
propanyl esters that contain a ring substrate which presents sensitivity to oxidative cleavage by the
cytocchyrtoocmheroPm4e5 P04-c5a0t, awlyitzhe sdpeocxifiidciatyti vtoe CcYlePa3vAa4g [e1r5e].a ctioninsidethehepatocytes. Theseprodrugsare
cyclic1,3-Sptruodpieasn wylerees taelrsso tphearftocromnetda itno atersitn tghes upobtsetnratitael owf hbiicleh apcirdess eton tdseslievnesri dtirvuigtys stopeocxifiidcaaltliyv teoc tlheea vage
bythleivceyrt, oscinhcreo tmheesPe 4b5il0e, awciidths asrpee tcriafincsiptyorttoedC aYcPro3sAs 4th[e1 5p]la.sma membrane in the portal domain [16].
STthued liiveserw-spereecifailcs dorpuge rcfholromraemdbtuocitle wstasth uesepdo atnend tcioavlaolfenbtillye lainckideds ttoo 7d aellpivhea,r 1d2r aulpghsas, p-deichifyidcarollxyy-tothe
liver,3s ibnectea-(tohmeseegab-ialmeiancoiadlskoaxrye)-t5r-abnetsap-ochrtoeldan-a2c4r-oosisc athcied ptloa sfmoram mchelmorbarmabnueciiln-btilhee apciodr tcaolndjuogmataeisn, [16].
and studies have revealed the success of bile acid molecules in behaving as carriers for drug
Theliver-specificdrugchlorambucilwasusedandcovalentlylinkedto7alpha,12alpha,-dihydroxy-3
molecules and their specificity for the liver. Researchers designed a nanoparticle-based model
beta-(omega-aminoalkoxy)-5-beta-cholan-24-oicacidtoformchlorambucil-bileacidconjugates,andstudies
delivery system that simulated nonviral gene delivery particles with respect to their surface
haverevealedthesuccessofbileacidmoleculesinbehavingascarriersfordrugmoleculesandtheir
properties in an attempt to identify design constraints to aid next-generation gene delivery to the
specificityfortheliver.Researchersdesignedananoparticle-basedmodeldeliverysystemthatsimulated
liver [17,18]. Four nanoparticles were formulated from polystyrene beads, namely Gal-50 and Gal-
nonv1ir4a0l, gwehniechd aerleiv gearlyacptoasrytliaclteeds;w anitdh MreesOp-e5c0t atondt hMeeirOs-u14rf0a, cwehpicrho pareer tmieesthinoxayn-taetrtmeminpattetdo. Tidheen 5t0if ayndde sign
const1ra40in dtsentootaeisd thnee xmt-egaenn edriaamtioenterg eonf ethdee nliavneorypatrotitchlees liinv enra[n1o7m,1e8t]e.rFs.o Guralnaactnoospe aisr tiinclceosrpwoerraetefdo rams ua lated
fromtpaorglyestitnygre lnigeabneda, dasn,dn apmroevliysiGona lo-5f 0searnudmG satal-b1i4li0ty, wish aitcthaianreedg waliathc tPosEyGlaytleadti;oann odf Mthee On-a5n0opanardtiMcleesO. -140,
whichReasrueltsm sehtohwoxedy -tthearmt ai nsalitgehdt .anTiohneic5, 0gaalnacdto1s4e0-PdEGenyolatteesd tnhaenmopeaarnticdlei asmhoeutledr hoafvteh ae snizaen oofp uaprt itcol es in
nanom eters.Galactoseisincorporatedasatargetingligand,andprovisionofserumstabilityisattainedwith
PEGylationofthenanoparticles.Resultsshowedthataslightanionic,galactose-PEGylatednanoparticle
shouldhaveasizeofuptoaround50nmand140nmindiameterinordertoselectivelytargethepatocytes
Viruses2018,10,267 4of20
and Kupffer cells, respectively. Overall, these studies and findings support the concept that targeted
deliveryofantiviralstotheliverincreasestheefficacyofthesetherapeuticagentsinthetreatmentofviral
liverinfectionsandreducestheirtoxicityprofilesinotherbodilytissuesandorgansasaresultofsystemic
exposure[5].
3. ConventionalAnti-HBVTherapiesandDrugs
3.1. CytokinesandNucleot(s)ideAnalogues
Theinnateimmunesystemrepresentsthefirstlinedefenseofahostcelltoviralinfection[19].One
suchexampleistheIFN-αcytokine,whichhasdisplayedsupportivefindingstowardHBVreduction
and suppression of HBV viral copying. IFN-α provides for the restitution of T-helper lymphocyte
responsesasaresultofitsantiviralactivitiesandimmunoregulatoryprocesses,thusitremainsthe
mainstaycytokinepresentlyutilizedintheinterventionofchronicHBV.Solely,itallowsforanti-hepatitis
Be-antigen(HBeAg)seroconversionin25%ofchronicHBVdiseasecases[20,21].Researchersexamined
theimpressionthatIFNleftonpatientswithanti-HBeAgpositivechronicHBVdiseaseintermsof
diseaseprogressionbypursuingreexaminationafteranaverageofsixyears[22]. Conclusiveresults
werea2.5-folddeclineintheprogressionofHBV.Continuousevaluationofitseffectswasvalidatedin
otherstudiesdeclaringthatIFNtreatmentstimulatesanenlargedsurvivalrate,adiminishedpossible
growthofHCC,andconsequently,animprovementofliverhistology. Futurefollow-upsshoweda
sustainedclinicalsubsidenceandhepatitisBsurfaceantigen(HBsAG)seroconversion[23,24]. Research
haspointedoutthatlambda-λhasthepotentialtobeusefulfortherapeuticpurposesinthealleviation
ofchronicHBVorhepatitisCvirus(HCV)[25]. IFN-λissimilartoIFN-αandIFN-βinitsantiviral
behaviortoinhibitHBVwhenitconveysitseffectsbymeansofadistinguishablereceptorcompositein
amodifiedmurinehepatocytecellline. ItalsoloweredHCVreplicationinHuh7cells. Datainastudy
undertakenbyZoulim[26]assessedtheantiviralimpactofIFN-α2aorIFN-α2bwithattachedPEG
demonstratingcloseto30%HbeAgseroconversionand3–5%HbsAgseroconversionrates,respectively,
withHBsAgseeninaround7%ofpatientsafterasix-monthfollow-up. AcombinationofNUCtherapy
(entecavirortenofovir)withrecombinanthumanIL-7(CYT107)orwithbothCYT107andHBVvaccine
(GenHevacB®,Pasteur,Merieux,Lyon,France)isnowbeingstudied[27].
Nucleot(s)ideanaloguescanbeperceivedasprodrugs,astheirroleasinhibitorsofpolymerasesis
generatedbythephosphorylationprocessoftheirnucleosidetriphosphatesordiphosphates[28].Even
thoughnucleot(s)ideanaloguessuppressHBVreplicationandpreventliverdiseaseprogression,theydonot
affectHBVRNAtranscriptionfromthecovalently-closedcircularDNAmoleculeofHBVwithintheinfected
hepatocytes. TheythushavealimitedimpactonHBVsurfaceantigenserumlevels[29]. Lamivudine
(LMV)isacytidineanaloguethatwhencomparedtoIFN-α,doesnothaveatargetedimmunoregulatory
outcome,butdoesshowtheresponserestorationofT-helperlymphocytessuitedtoHBVfortheinitial
monthsofitstherapy.Theothernucleotideanaloguescouldalsofeaturethesamefunction[30].LMVis
agenerallywell-enduredantiviralanditstreatmentconducesafallinHBVDNAlevelby3to5log10
copies/mLfollowing12monthsoftreatmentequatedtostartinglinemeasurements,anaccompanied
promptremovalofHBeAg,andstandardized/decreasedlevelsofserumalanineaminotransferase(ALT).
ProlongedLMVtherapymayresultinadropintheriskofdevelopinglivercirrhosisandHCC[31].Patients
intheUSAharboringuntreatedchronicHBVinfectionexhibitedwell-disposedresultsofHBVeffects
involvingchemicalprocessesinthebodywithreferencetoitshistologicalcharacteristics.HBeAgresponses
weregenerallykeptupafterLMVadministration[32];however,itwasshowninanotherexamination
thatHBeAgresponseswerenotsignificantlykeptupafterLMVadministrationwasceased[33]. LMV
therapyissuggestedinpeoplewhodonotshowanyseroconversion.Theproblemofdrugresistanceis
seenimmenselywithLMVanditsresistancemoderatesahigherHBVviralload[26].
Oneofthenucleotideanalogues,adefovirdipivoxil(ADV),proveditsabilityindecreasingthe
HBV DNA levels in addition to improving serum hepatic enzyme levels. It is also an option for
treatmentofLMV-resistantpatientswithcasesofchronicHBVinfection[34,35]. Patientswithchronic
Viruses2018,10,267 5of20
HBVinfectionwererandomlyassignedtoreceivespecificdosagesofADVeverydayfortheduration
of48weeksofastudy[36]. Resultssuggestedahistologicliverprogression, arecededamountof
HBVDNAandALT,andanincreasedrateofHBeAgseroconversion. Astudydemonstratedthat
solidlipidnanoparticles(SLNs)arenoveldrugdeliverysystemsforADVforanti-HBVactivity[37].
SLNs provide nonbiotoxicity, drug targeting, and sustained drug release as well as harboring the
incorporatedcompoundinaprotectivefashion,anditisalsochemicallydegradable. Afluorescence
marker(octadecylamine-fluoresceinisothiocyanate)showedtheuptakeofSLNsbyHepG2.2.15cells.
Compared to the free ADV, a significant drop in levels of HBsAg, HBeAg, and HBV DNA levels
invitrowasobserved. ADVwasreviewedinitstreatmentofchronicHBV,anditwasreportedthat
ADVwasfoundtoenhanceitstherapeuticefficiencyafterthreeyearsofitscontinuedtreatment[34].
Entecavir(ETV)isanotherpowerfulinhibitorofHBVreplicationandwasapprovedin2005for
itsuseagainstactiveHBVandliverdiseaseincludingaffirmationofacontinuousriseinserumALT.
Italsohasanamicableside-effectprofileandalesserchanceofdrug-resistanceprogression[26,38].
The long-term productiveness of ETV monotherapy in patients with detectable HBV DNA levels
wasexploredbyZoutendijkandcoworkers[39]. Conclusivefindingsbythisstudysuggestedthat
ETVtreatmentcouldbeextendedafter48weeks,particularlyinpatientswithlessHBVDNA,asit
precipitatesavirologicalreactioninmostofthesepatients.
Tenofovir disoproxil fumarate (TDF) also has a potent antiviral activity against HBV, but has
beeninvestigatedmoreinpatientswhoareHIV-positiveandhavebeencoinfectedwithHBV.Many
studieshaveimpliedthatTDFnotablyreducedHBVviralloadandtheprobabilitythatitismore
effectiveintherapycomparedtoADV[26]. ETVandTDFwerereviewedintheclinicalsetting,where
itwasshownthatETVproducedanaverageof86%virologicresponsesinpatientsuntilfouryearsof
treatmentandTDFproducedanaverageof82%virologicresponsesuntiloneyearandninemonthsof
treatment. Reportsonthetolerabilityrevealedcommendablesafetyprofilesandlesseroccurrencesof
drugresistanceforbothETVandTDF[40]. Accordingtoresearchers,fiveyearsoftreatmentwithTDF
iseffectiveineliminatingHBV,possiblyleadingtothereversionoflivercirrhosis[36].
Inastudycomprisingoftreatment-naïvechronicHBVpatients,theefficacyandsafetyofAL-3778
(then called NVR 3-778) was assessed, given alone and in combination with PEG-IFN for 28 days.
Dose-relatedHBVDNAreductionsandearlyHBeAgreductionswereshownwiththeseeffectsand
increasedwhenNVR3-778wasgivenwithPEG-IFN[27].
Thenucleot(s)idetelbivudineisknowntobeaffiliatedwiththelargestHBeAgseroconversion
rates,withtheadditionaleffectofimmunomodulation. ThismakesitsimilarincharactertoPEGylated
IFN[41]. SerumHBsAglevels,after36monthsoftelbivudinetreatment,wereevaluatedin162cases
thatwerepositiveforHBeAganddisplayeddecreasedHBVDNAserumlevelsinapersistentmanner.
TherapidfallinserumHBsAglevelsinthefirst12monthsoftelbivudinetherapypinpointspatients
who are more inclined to acquiring the compete removal of HBsAg [42]. Table 1 summarizes the
impressionthatcytokinesandnucleot(s)ideanalogueshaveonHBV.
Table1. Summaryofanti-hepatitisBvirus(HBV)cytokineandnucleot(s)ideanaloguetherapeutic
agentsasatreatmentplan.
TherapeuticAgent EffectivenessasAnti-HBVTherapy Reference
SuppressedHBVviralcopying,restoredT-helperlymphocyte
responses/immunoregulatory,allowsforanti-HbeAgseroconversion,
IFN-α/-PEG/-λ prolongeddeclineintheprogressionofHBV,prolongeddiminished [20–24]
growthofHCC,sustainedclinicalsubsidenceandHBsAG
seroconversion,subduedHCVreplication
T-helperlymphocyteresponserestored,HBVDNAleveldecline,rapid
HBeAgremoval,decreasedlevelsofserumALT,minimalriskof
LMV [26,30–33]
developinglivercirrhosisandHCC,improvedliverhistology,sustained
HBeAgresponsesaftertreatment
Viruses2018,10,267 6of20
Table1.Cont.
TherapeuticAgent EffectivenessasAnti-HBVTherapy Reference
DecreasedHBVDNAlevels,improvedserumliverhistology,treatment
ADV ofLMVresistance,decreasedALT,increasedHBeAgseroconversion, [34–37]
decreasedHBsAgandHBeAglevels,prolongedtherapeuticefficacy
PotentHBVinhibition,minimaldrug-resistanceprogression,effective
ETV [26,38,39]
long-term,precipitatesvirologicalresponses,favorableside-effectprofile
PotentHBVinhibition,decreasedHBVviralload,moreeffectivetherapy
TDF comparedtoADV,sustainedvirologicresponses,favorablesafetyprofile, [26,36,40]
prolongedlivercirrhosisreversal
HighestHBeAgseroconversionrate,immunomodulation,rapiddecrease
Telbivudine [41,42]
inHBsAGlevels,possiblecompleteremovalofHBsAg
IFN-α/-PEG/-λ:interferon-α/PEGylated-interferon/lambda-interferon;anti-HbeAg:anti-hepatitisBe-antigen;
HCC:hepatocellularcarcinoma;LMV:lamivudine;ADV:adefovirdipivoxil;HBsAG:hepatitisBsurfaceantigen;
HCV:hepatitisCvirus;LMV:lamivudine;ALT:alanineaminotransferase;ADV:adefovirdipivoxil;ETV:entecavir;
TDF:tenofovirdisoproxilfumarate.
3.2. ThiazolideAnti-Infectives
Thiazolideshaverecentlydisplayedpromiseasantiviraltherapeuticagentsthatmayamplifyexisting
orforthcomingtreatmentsagainsthepatitis. Nitazoxanide(NTZ)isathiazolideanti-infectivethathas
activityagainstarangeofvirusesincellculturemodels,anaerobicbacteria,protozoa,andhelminths.NTZ
antiviral exertion was first stumbled upon when patients with AIDS (coinfected with HBV or HCV)
werebeingtreatedforcryptosporidialdiarrhea,asitwasthefirstthiazolidethatwasprimarilyusedfor
thetreatmentoftheprotozoanCryptosporidiumparvum. InstudiesofpatientswithchronicHBV,NTZ
administrationevokedtheseroconversionofHBeAgandHBsAgwiththeoveralleliminationofHBVDNA
levels.InotherstudiesofpatientswithchronicHCV,theadministrationofNTZtogetherwithPEGylated
IFN-α2a,withorwithouttheadditionofribavirin,manifestedtheefficiencyandacceptablesafetyofthe
thiazolide[43–45].TogetherwiththemetaboliteofNTZ,tizoxanide,andotherthiazolides,reportshave
indicatedtheeffectiveinhibitionofbothHBVandHCVreplicationinstandardantiviralassays. NTZ
showedactivityagainstbothgenotypes1aand1bofHCVandthefrequentLMV-andADV-resistantHBV
mutantsaswellascertainHCVmutants.ItinducedareductionintheHep2.2.15cellsproducingHBV
proteins;however,itdidnothaveaneffectonHBVRNAtranscription[46].NTZiscurrentlyundergoing
preclinicaltrials[47].ResearchersperformedaQSARexaminationonavarietyofthiazolidesandthey
assessedtheirinterferenceonHBVreplicationandactivity[48].Briefly,thebroad-spectrumNTZpresented
efficacy against viruses, amongst other things; the novel 2-hydroxybenzoyl-N-(5-chlorothiazol-2-yl)
amidedisplayedrobustandselectiveHBVreplicationhindranceandincomparisontosomeanalogous
salicyloylanilides, favorable activity against HBV was shown with numerous 4(cid:48)- and 5(cid:48)-substituted
thiazolides.Ithasbeensuggestedthattheactionofremovalwithsubstitutionofthenitrogroupfoundon
NTZwithagroupthatisnotreduciblewillallowfornovelthiazolidestobeformedthatwouldsustaina
widespectrumofviralactivityonly,whichwouldplayavaluableroleinthereductionofHBV[49].
3.3. SmallInterferingRibonucleicAcids
Smallinterferingribonucleicacids(siRNAs)withanefficientdeliverysystemcanovercomebarriers
andcausetheinhibitionofgeneexpressionofspecificproteins.Themechanismbywhichthisisachieved
isknownasRNAinterference(RNAi). Itisanaturalprocedurethatprotectsthegenomebytargeting
aparticularmessengerRNA(mRNA)fordegradation,thusinhibitingproteinsynthesis,andtherefore,
viralgenetranscription,expression,andultimatelyreplication.Thereareseveralnotablestudiesthathave
beenconductedconcerningsiRNA.MerelytoachievesiRNAhepatocytetargeting,inonesuchstudy,a
vehiclewasdeveloped:siRNADynamicPolyConjugates[50].Thistechnologyinvolvesamembrane-active
polymerthatconfersitsactivityonlyuntilitreachestheendosomeacidicenvironmentandacquiresthe
abilitytodropoffitssiRNAcargopreciselytohepatocytesafterasimpleintravenousinjection. Novel
Viruses2018,10,267 7of20
hepatotropicnontoxiclipid-basedvectorsystemsweregeneratedtodeliverchemicallyunmodifiedsiRNAs
totheliver[51].TriggeredPEGylatedsiRNAnanoparticleswerethereforeformulated.DuetothePEG
coupling,theresultingpH-sensitiveoximelinkagecausesthereleaseofnucleicacidsfromtheendosomes.
Suppression of markers of HBV replication on account of triggered PEGylated siRNA nanoparticles
increasebythree-foldrelativetocontrols.ResultsfromanotherstudyshowthatsiRNAdeliveryfollows
asurfacecharge-andsize-dependentmanner[52]. Inanotherstudy,theaimwastooptimallydesign
siRNAstargetingHBV,andthiswasauthenticatedviaquantitativestructure–activityrelationship(QSAR)
analysismethods[53].CocktailsofsiRNAscouldalsobeproblem-solving,ascombinationsofsiRNAs
wouldcleavemultiplesitesonthemRNAtarget,makingitdifficultforrestorationthereof[54].Researchers
demonstratedastudythatshowedtheeffectsofanHBV-specific21-bpsiRNAthatisdirectedtothe
HBsAgregion,asitewherethreeprominentviralmRNAsprojectoveroneanother,onHBVreplicationin
acellculturesystemandamousemodel[55].Resultspointedoutthemarkedinhibitionofviralantigens,
theirtranscripts,andDNA,andthusHBVreplicationasawhole.
AstudydemonstratedanevaluationofsyntheticsiRNAcombinationstargetingvarioussitesofHBV
transcriptsonitsreplicationandantigenexpressioninvitro[56].ResultsshowedthatthesiRNAstargeting
thepolymeraseandprecoreregionspecificallyinhibitedvirusreplicationandantigenexpressionina
dose-dependentmanner.ThiswasdoneefficientlycomparedtotheuseofsinglesiRNAsatthesamefinal
concentration.Inaddition,noapoptoticchangewasobservedinthecellsafterthecombinationsiRNA
treatment.AspecializeddeliverysystemknownasaSNALP(stablenucleic-acid-lipidparticle)containing
chemicallymodifiedsiRNAinitsliposomalformwasused[57].Thesewereadministeredintravenously
intomousehostsofreplicatingHBV.HBVDNAreductionsspecificallylastedfordaysandweekswith
thisparticulardosing.Taking3mg/kg/dayintravenousinjectionsthreetimesadayreducedserumHBV
DNAby>1.0log10.AmousemodelcarryingreplicatingHBVwasinjectedintravenouslywithsynthetic
siRNA/apoA-I/1,2-dioleoyl-3-trimethylammonium-propanecomplexes[58].Thenanoparticlesdisplayed
liverspecificitywithlowdosesoflessthanorequalto2mg/kg,stillrenderingeffectivenessinonlya
singletreatmentwithpersistenceofthetherapeuticeffectforeightdays.Additionally,administrationof
thesenanoparticlessignificantlydiminishedviralproteinexpressionbyreceptor-mediatedendocytosis.
ResearchersinanotherstudycoinjectedacompoundedN-acetylgalactosamine-melittin-likepeptidewitha
siRNAcompoundedtocholesterolthatwasdirectedtocoagulationfactor7,andvalidatedthereductionof
HBVRNA,DNA,andproteinswithalengthyeffectivecontinuity[59].Datafromthisstudyvalidatedthe
useofRNAi-basedtherapeuticsinthetreatmentofchronicHBVinfections.Anotherrecentstudyaddedto
theillustrationofRNAidecreasingHBVbyproclaimingviralclearanceofHBVfromtheliveroftransgenic
micebyrecombinantadenovirusesexpressingHBV-directedshorthairpinRNAs[60].
ARC-520,designedtoreducetheexpressionandreleaseofnewviralparticlesandtheviralprotein
loadbythemechanismofRNAi,althoughithadshowedefficacyinachimpanzeechronicallyinfected
withHBV[61],theclinicaltrialswerediscontinuedduetosafetyconcernsregardingthetoxicityofthe
deliveryvehicle.
3.4. Heteroarylpyrimidines
Heteroarylpyrimidines (HAPs) were identified as powerful inhibitors of the HBV capsid
maturationstep.Theinteractionsiteofthecoreprotein–HAPandtheexactpointinthereplicationcycle
wheretheHAPallowsitsprincipalfunctionisunknown,howeverthemodeofactionistheattachment
toanddegradationofthecoreprotein. Cell-basedHBVreplicationassaysportrayedagreaterpotency
ofHAPsthanLMVinthetherapeutictreatmentofHBV[5]. ResearcherstestedtheeffectthataHAP
(methyl4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate)
hadonHBVcapsidproteincongregation[62]. ResultsdepicttheHAPcommandingthedetachment
ofHBVcapsidsandconsequentlybeingcapableofrelayingdiversifiedeffectsemanatingfromthe
incongruous assembling of the HBV capsid proteins. Therefore, actuating and decontrolling the
assemblageofaviruscouldleadtoprevailingantiviral-basedtreatments. ArangeofHAPsbuiltona
crystallizedarrangementofacapsid–HAPconjugatewasformulatedtoattempttobetterunderstand
Viruses2018,10,267 8of20
theHBVcapsidassemblyandreplicationinHepG2.2.15cellsinvitro[63]. Thekineticsofassembly
invitrocorrespondedsufficientlywiththeprohibitionofHBVinthecellculture. Resultsalsoalluded
to contention between suitable and unsuitable assembly because of the interrelation of assembly
kineticsandvirusrestriction. BAY41-4109isaHAPthatwasevaluatedforitsantiviralactivityin
transgenicrodentscarryingHBVatvaryingdosages(3–30mg/kg,b.i.d/t.i.dfor28days).Inrelationto
LMV,BAY41-4109wasjustascompetentinloweringHBVDNAinadose-dependentfashion. Results
also suggested lowered HBcAg in excised hepatic sections in disparity with LMV-treated rodents,
indicatingthatBAY41-4109bearsnoresemblancetothemechanismofactionofLMVasanantiviral.
BAY41-4109pharmacokineticsrevealedswiftabsorptionanda30%bioavailability[64]. BAY41-4109
wasalsostudiedforitscausativeeffectontheintracellularEGFP–corefusionproteinsintoHepG2
cells. ItisconclusivethatBAY41-4109isapowerfulinhibitorofHBV,havingnumerouseffectsonthe
arrangementoftheHBVcapsidtoachievethisantiviralresult[65]. However,studiesbyWangand
coworkers[66]reportedGLS4tobemorepotentthanBAY41-4109invitro,demonstratinganEC50
of12nMinstablytransfectedHepG2.2.15cells. AphaseIstudyisongoing,butnoclinicalresults
have been reported to date [47]. GLS4, a potent inhibitor of the replication of both wild-type and
ADV-resistantHBVmutantstrains,iscurrentlyundergoingaphaseIstudyinChina[47].Isothiafludine
(NZ-4), a leucamide A derivative, has been investigated for the inhibition of HBV replication in
HepG2.2.15cells. NZ-4wasmechanisticallyshowntoincreasereplicationofdeficientcapsids,devoid
ofpgRNA[67]. Table2reflectstheefficiencyofthiazolides,siRNAs,andHAPsasHBVeradicators.
Table2.Summaryofanti-HBVthiazolide,siRNA,andHAPtherapeuticagentsasatreatmentplan.
TherapeuticAgent EffectivenessasAnti-HBVTherapy Reference
PotentandselectiveHBVreplicationinhibition,HBeAgandHBsAgseroconversion,
Thiazolides decreasedHBVDNAlevels,treatmentinLMV-andADV-resistance,decreased [43–46,48,49]
Hep2.2.15cellHBVproteins,noHBVRNAtranscriptioneffect
Geneexpressioninhibited,viralantigeninhibition,HBVtranscriptinhibition,decreased
siRNAs serumHBVDNAandRNAlevels,suppressedHBVreplicationmarkers,mRNA [50,51,54,60]
cleavage,polymeraseandprecoreregiontargeting,effectivecontinuanceoftreatment
PotentHBVcapsidmaturinginhibition,HBVcoreproteindegradation,increased
HAPs potencycomparedtoLMV,potentantigeninhibitioneffect,misassemblyofHBVcapsid [5,62–65,68]
proteins,dose-dependentdecreaseinHBVDNAlevels,decreasedHBcAg
SiRNAs: small interfering ribonucleic acids; RNA: ribonucleic acid; mRNA: messenger RNA; HAPs:
heteroarylpyrimidines;HBcAg:hepatitisBcoreantigen.
3.5. SulfamoylBenzamideCapsidAssemblyModulators
A relatively new class of HBV nucleocapsid assembly modulators is the sulfamoyl benzamides
(SBAs).WhencomparedwithBAY41-4109andAT-61,SBAswerefoundtoinhibitviralreplicationina
mannersimilartothatofthephenylpropenamidederivatives,thoughtheinteractionofSBAswithcore
proteinsorcapsidsisstilltobeextensivelyreportedon[67]. AstudybyOhtsukiandcoworkers[69]
demonstratedareductionofHBVDNAsimilartothatofentecavirwhenahumanizeduPA/SCIDmouse
modelreceivedamonotherapyofNVR3-778forsixweeks.Asynergisticeffectwasalsoobservedwhen
NVR3-778wasdosedincombinationwithPEG-interferon,reducingHBVDNAlevelstobelowthelimit
ofquantitation.Recently,sulfamoylbenzamidecapsidassemblymodulatorsinthetreatmentofHBVhave
beenprovenefficaciousinPhaseIBstudies,validatingNVR3-778asaclassofanti-HBVcompounds[67].
4. NovelDrugDeliveryStrategiesforAnti-HBVTherapeutics
4.1. NanoparticleSystems
4.1.1. InorganicNanoparticles
Astudyconcernedwithimaginginlivercancerplacedemphasisonaproteoglycanknownas
glypican-3(GPC-3), whichisinvolvedwithenablingcellgrowthandisfoundtobeoverexpressed
Viruses2018,10,267 9of20
inHCC.Superparamagneticironoxidenovelmultifunctionalnanoparticlesweredeveloped,where
particleswereconjugatedtostreptavidinandAlexaFluor647. Itwasfoundviaconfocalfluorescence
microscopy(CFM)thatbiotin-conjugatedGPC-3monoclonalantibodywasconfinedonlytothecellular
surfaceofHepG2cellsexpressingGPC-3. TheGPC-3nanoparticlesystemprovedthatitcanbeutilized
inimagingforHCCvisualizationaswellashavingcapabilitytobeusedasavehiclefordeliveryof
therapeuticstargetingtumors[70]. Researchersalsousedmagneticnanoparticlesurfacesinanother
studytopreparemultifunctionalHCC-targetingagentsbyblendingbis-N-hydroxysuccinimideester
andOSu-activatedfluorescentdyeCy3. Amonoantennaryandtriantennarygalactosylligandswere
eachfixedontothefluorescentmagneticnanoparticlesandtheiruptakeintoHepG2andHeLacellswere
evaluatedbyCFM.Resultsshowthatthissystemisagoodligandtransporterandthatitsmultivalent
ligandassemblyimprovesonthecellinteractionwithHepG2receptors. ThegalactosylCy3magnetic
nanoparticleswerealsofoundtobenoncytotoxic[8]. SiO nanoparticleswerealsousedforattachment
2
toHBV-likeparticlesinordertotransitimmuneresponse-regulatingagentsfortargetedtreatment[71].
4.1.2. PolymericNanoparticles
Poly(vinylbenzyl-O-β-D-galactopyranosyl-D-gluconamide)(PVLA)isknowntobesite-specifically
taken up into hepatocytes via ASGPR. Therefore, researchers synthesized a copolymer, poly(N-p-
vinylbenzyl-[O-β-D-galactopyranosyl-(1→4)-D-gluconamide]-co-N-p-vinylbenzyl-6-[2-(4-dimethylamino)
benzaldehydehydrazono]nicotinate) (P(VLA-co-VNI)), and this was tagged with (99m)Tc for liver
imaging. Results revealed promising potential for more of its application in the evaluation of liver
cell function [72]. Researchers have also achieved site specificity to hepatocytes by synthesizing a
galactosylatedPEG-graft-PEIderivativeviathemodificationofabiscarbamatecross-linkedPEIbyproduct
withPEGandlactobionicacid.Thecomplexheldagalactosemoietyforhepatocytetargeting.Itcould
alsoefficientlyincorporateplasmidDNAintonanoparticles.Datadisplayeditssuccessfultargetingtothe
hepatocytes[73].Inadifferentstudy,nanoprecipitationandsolventevaporationtechniqueswereusedto
preparecationicpoly(lactide)(PLA)-basednanoparticlestogetherwithPEIandchitosanassurfacecoating
components.mPEG-PLA-PEInanoparticlesshowedthebestabilitytoimpedeHBVsurfaceantigenand
thusallowfortransfectionofsiRNA[52].Researchersusedadouble-emulsificationtechniquetoprepare
HBsAgpassivelyadsorbedontothesurfaceofcationicPLGAnanoparticlesforsite-specificdeliveryof
IFN-αtohepatocytes.HbsAg-coated(99m)Tc-labeledPLGAnanoparticlesresultspresentednotableliver
recovery,incontrasttonormalPLGAnanoparticles[74].Theparticlesizeandhydrophobicityeffectsof
porousPLAandPLGAnanoparticleswereassessedoncell-mediatedandmucosalimmuneresponses.
ThenanoparticlesaccommodatedasetamountofHBsAg,andadministrationoccurredviapulmonary
delivery.Datarevealedthathydrophobicparticlesthatwerelargerinsize,greaterthan500nm,derived
apotentincreaseinsecretoryIgA,IFN-γ,andinterleukin-2levels,asopposedtohydrophilicparticles
thatweresmallerthan500nm.Thelargersizedhydrophobicparticlesalsoshowedthattheycouldbe
moreeasilytakenupintoratalveolarmacrophages. Thestudydemonstratedthecompetenceofthe
nanoparticlestoinduceaugmentedimmuneresponses[75].
ResearchershavealsodevelopedpolymericnanoparticlesformulatedforHBVgenesilencing
makinguseofthecommonbiodegradablepolymerPLGAagain,butinthisstudy,thecationicpolymer
chitosan is embodied into its matrix. The idea is to advance plasmid DNA loading efficiency and
cellularinternalization. ConclusivedatabyZengandcoworkers[76]revealedbetterHBVsilencing
with the chitosan–PLGA system compared to plain plasmid DNA (pDNA) alone or simple PLGA
nanoparticles(Figure2). Furthermore,thechitosan–PLGAsystemshowedalackofadverseeffects.
Micelles were developed in one study for incorporating the lamivudine (LMV) prodrug LMV
stearate.LMVstearatewassynthesizedtoincreaseLMVlipophilicity,duetoitbeingaveryhydrophilic
drug.Stearicacid-graft-chitosanoligosaccharidemicelleswerepreparedandloadedwithLMVstearate.
Results showed that the micelles had constrained effects on HBV antigen expression and DNA
replication, and this was more noticeable when compared with LMV or its prodrug alone [77].
Acyclovirwasalsocomplexedtochitosan-g-stearatethroughtheuseofasuccinatelinkerforanti-HBV
Viruses2018,10,267 10of20
activity. The acyclovir–chitosan-g-stearate was able to self-assemble in aqueous solution, constructing
micelles.Accordingtodata,therewasasignificantescalationinitsinhibitoryeffectofHbsAgcomparedto
plainacycloviralone.Anall-roundsuccessofcellularinternalizationandanti-HBVeffectswasobservedwith
thecomplexationofacyclovirtochitosan-g-stearate[78].Authorsalsorevealedanassuringhepatic-targeted
siRNAdeliverysystemforgeneexpressionsilencingformulatedfromN-acetylgalactosamine-functionalized
mixed micellar nanoparticles. The N-acetylgalactosamine micellar nanoparticles were self-aggregated
inaqueoussolutionfromN-acetylgalactosamine-functionalizedPEG-b-poly(ε-caprolactone)andcationic
poly(ε-caprolactone)-b-poly(2-aminoethylethylenephosphate)(PCL-b-PPEEA).Thetargetingeffecttothe
hepatocyteswasdisplayedbynoteworthyenhancedfluorescentsiRNAfoundinprimaryhepatocytes,
implyingpositiveanti-HBVtherapyinliverdisease[73].Adrugcarrierwassynthesizedfromglycyrrhetinic
acid-modified sulfated chitosan, with glycyrrhetinic acid being the targeting ligand to HepG2 cells.
The micelles formed from this complex revealed swift and eminent ability for invivo liver targeting.
Moreover,theformedmicellesshowedspecificityforlivercancercells,incontrasttonormallivercells[79].
Figure 2. (1) PLGA–CHS nanoparticle binding efficiency and loading capacity to adsorb pDNA.
(A)PLGA–CHS–pDNAcomplexeswithincreasingamountsofPLGA–CHSNSwerepreparedandanalyzed
forpDNAimmobilizationability. Electrophoresiswascarriedoutusing1%agarosegelinTAEbuffer
containing0.5µg/mLethidiumbromideatpH8. (B)TheamountsoffreeDNAwererelatedtonaked
pDNA(100%mobile)runonthesamegel.ToquantifythepDNA-immobilizationability,thePLGA–CHS
NS/pDNA ratios (w/w) required for 100% immobilization are compared in this graph (solid bars =
percentageoffreeDNA;whitebars=100%immobilization). (2)Confocallasermicroscopicimagesof
HepG2.2.15cellsfollowing48htransfectionwith(A)plain-PLGA–pDNANSand(B)CHS–PLGA–pDNA
NS.Scalebar=75µm(reproducedwithpermissionfrom[76],©ElsevierB.V.Ltd.2011).