An ultra-long-acting dimeric bictegravir prodrug defined by a short pharmacokinetic tail

Domain: chemistry
Published: 2026-01-21
Editorial outcome: minor_revision

Abstract
Ultra-long-acting(ULA)antiretroviralparenteralformulations,withlowinjec-tionvolumes,highresistancebarriers,andshortpharmacokinetic(PK)tails,cantransformHIV-1therapeutics.Here,weconvertedbictegravir(BIC),apotentdailyoralantiretroviraldrug,intomonomericandhomodimericesterprodrugs.Thehomodimericprodrugnanosuspension,NMXBIC,showssustainedplasmaBIClevels>16timestheprotein-adjusted95%inhibitoryconcentration(PA-IC95)forsixmonthsafterasingleinjectioninSpragueDawleyrats.TheresultsparalleledashortPKtailwiththepotentialforlatedoseforgiveness.Themonomericprodrugnanosuspension,NM2BIC,showsloweryear-longplasmaBICconcentrationsabovePA-IC95afterasingleinjection.Afterrepeatedinjections,NMXBICandNM2BICarewelltoleratedinNewZealandWhiterabbits.NMXBIC’sphysicochemicalpropertiesandhighBICloading/unitmassoftheprodrugcontributetoitsuniqueULAPKprofile.TheseresultssupportitsdevelopmentasaULAformulationforHIV-1treatmentandprevention.

Introduction
Despitenotableadvancementsinantiretroviraltherapy(ART),thefight against HIV-1 remains reliant on strict regimen adherence1. While dailyoral ART is highlyeffective,suboptimal commitment leads to virological failure, transmission, and drug resistance2. Long-acting(LA)antiretroviral(ARV)formulations,suchasCabenuva,whichcombinescabotegravir(CAB)andrilpivirine(RPV),are a transformative alternative3. The Cabenuva LA regimen is as effective as daily oral ART when administered monthly or bimonthly.Italsoimprovesadherenceandtreatmentoutcomesin patientswithahistoryofchallengestoARTadherence4–7.However, transitioning from daily to LA ART has introduced healthcare challenges.Theseincludesustainingfrequentclinicvisits,laboratorytesting,andpatientconfidentiality8.

OtherinjectableARTobstaclesincludevariablepharmacokinetic(PK) profiles and a prolonged subtherapeutic plasma drug concentrationphaseknownasthePKtail9,10.Forinstance,23%ofmenand 63%ofwomen,whengiveninjectableCAB,experienceaprolongedtail phaseforuptoayearafterdiscontinuation9.SuchanextendedPKtail increasestheriskofdrugresistanceandbreakthroughinfectionduringtreatmentcessation9,11,12.Indevelopment,ultraLA(ULA)formulations focus now on optimizing dosing intervals, reducing injection volumes,andlimitingPKtaildurations.SynchronizingULAARTdosing with routine clinic visits could improve treatment outcomes and regimencontinuation.Thesubcutaneous(SC)injectionoflenacapavir (LEN,SUNLENCA)hasalreadymadeprogress.Fortreatmentsettings, SUNLENCAisgivenevery6monthsincombinationwithdailyoralART in people living with HIV (PLWH) who are infected with multidrug-resistantHIVstrains13.ThereisnootherULAsmall-moleculeagentthat canbecombinedwithSUNLENCAaspartofasynchronous,every-6-month treatment regimen. This study reports a ULA homodimeric bictegravir(BIC)prodrugnanosuspension,NMXBIC,withapotential 6-monthdosingregimenandtheabilitytofillaniche14.Notably,BICis ahighlypotentARVdrugwithahighresistancebarriertoviralmutation and is recommended as a first line of combination therapy15. Compared to biologics, the process of manufacture and long-term storage of ultra-long-acting formulations of small-molecule active pharmaceuticalingredients,suchasBIC,iscost-effective,whichcould potentiallyimprovepatientaccessandlowertherapycosts.

A nanocrystalline prodrug approach produced NMXBIC from a libraryofsynthesizedBICesterprodrugs.Thenewlibraryconsistedof monomeric BIC- myristate (MBIC), -stearate (M2BIC), -behenate (M2BIC),andadimericBIC-octadecanedioate(MXBIC).Whenadministered to BALB/cJ mice and Sprague Dawley (SD) rats as single intramuscular (IM) injections, nanosuspensions of homodimeric MXBIC (NMXBIC) and monomeric M2BIC (NM2BIC) facilitated controlledinjectionsitedrugrelease.Notably,singleIMinjectionsof45 and90mg/kgBICequivalentdosesofNMXBICtoSDratsprovideda “plateau”phaseofplasmaBICexposure,withBIClevels>16timesthe protein-adjusted95%inhibitoryconcentration(PA-IC95)for6months, followedbyashortPKtail.Bycomparison,asingleIMdoseofNM2BIC producedplasmaBIClevelsatorabovethePA-IC95for1year.

Alternative approaches to extended dosing frequencies and reducedPKtailsincludesubcutaneousARV-loadedsolidandinsituformingimplants16–20.Whileeachoneofthemcouldbroadenpatient options, their uptake could be impacted by the need for surgical procedures for removable and non-fillable implants, limited drug loadinganddosedumpingforbiodegradableimplants,delayeddrug absorption,PKvariability,andadverseeventsfromorganicsolvent-based formulations21–24. Notably, histopathological examination of lymphoid,mucosal,andguttissueshowednoadversereactionsinSD ratstreatedwithNMXBIC.SCandIMinjectionsofNMXBICadministered to New Zealand White (NZW) rabbits producedwell-tolerable injectionsitereactions(ISRs).Moreimportantly,thestrategydescribed in this work could potentially address prolonged PK tails of injectableULAformulations.Thesedatasetsdemonstratethepotentialfora6-monthaqueousnanosuspensionofBICwithashortPKtail.

Results
SynthesisandcharacterizationofmonomericanddimericBIC esterprodrugs
Water-insoluble monomeric (MBIC, M2BIC, and M3BIC) and homodimeric(MXBIC)prodrugsofBICweresynthesizedbyesterificationof the parent drug. The derivatization process employed naturally occurring fatty acids (Fig. 1A, B) with chemical yields of >70%. The single-step chemical synthesis enables a scalable process. Their inherentsolidformsfacilitatetheirformulationintoaqueousinjectable nanosuspensions. The chemical structure and composition of eachoftheprodrugsmadewereaffirmedbynuclearmagneticresonance (NMR), fourier-transform infrared (FT-IR) spectroscopy, and electrosprayionizationmassspectrometry(ESI-MS)(Fig.1CandSupplementaryFigs.1–8).Therecordedmolecularionpeaks(M+H)+at 660.33,716.39,772.45,and1177.48g/molintheESI-MSspectraidentifiedtheexpectedmolecularweightsforMBIC,M2BIC,M3BIC,and MXBIC, respectively (Supplementary Figs. 5–8). The unique X-ray powderdiffraction(XRD)patternsforBIC,M2BIC,andMXBICsuggest a distinct arrangement of atoms within the crystal lattices (SupplementaryFig.9A).Forthermogravimetricanalyses(TGAs),amassloss oflessthan0.1%acrossatemperaturerangeof30to300°Csuggests that the final prodrug powders were anhydrous (Supplementary Fig.10).ChemicalmodificationsmadetoBICwerereversibleforallthe synthesized prodrugs. Hydrolysis studies demonstrated that the solubilizedformsofM2BICandMXBICwere>95and45%hydrolyzed within 24h after exposure to dog, monkey, mouse, rat, and human plasma, respectively (Fig. 1E, F). Additionally, M2BIC exhibited pH-dependenthydrolyticstabilityacross arangeofpHbuffers.In contrast, MXBIC underwent uniform hydrolysis in different pH buffers, with up to half of the prodrug undergoing hydrolysis within 24h (Supplementary Fig. 11A, B). The role of chemical hydrolysis in the activationoftheBICprodrugswasfurthersupportedbytheconversion observed when prodrug solutions were incubated in heat-inactivatedplasma.Efficientconversionoftheprodrugsintoparent BICisalsoreflectedbyantiretroviralactivityevaluationagainstHIV-1 reversetranscriptase(RT)activityinHIV-1ADA-challenged monocyte-derived macrophages (MDMs) (Fig. 1D). The 50% inhibitory concentration(IC50)ofBIC,M2BIC,andMXBICsolutionswere3.75,7.11, and 4.33nM, respectively (Fig. 1D). MDMs exposed to M2BIC and MXBICsolutionsalsodemonstratedcomparablecellvitalityasmeasured by the mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay (Supplementary Fig. 12). MTT determinedcytotoxicitybyevaluatingcellularmetabolicactivity.The lowaqueoussolubilityofM2BICandMXBICfacilitateschemicalstabilityoftheprodrugswithinaqueousnanosuspensions.Theaqueous solubilitiesofBIC,M2BIC,andMXBICwere62.7±8.9,0.45±0.13,and 1.96±0.67µg/ml,respectively(Fig.1G).ComparedtoM2BIC,BICand MXBIC exhibited lower octanol solubilities (2499±127 and 2640±378µg/mlforBICandMXBIC,respectively,vs23.0±1.9mg/ml forM2BIC)(Fig.1H).

FormulationandcharacterizationofaqueousBICprodrug nanosuspensions
A scalable manufacturing process using microfluidization by high-pressurehomogenizationwasdevelopedtotransformBICprodrugs into surfactant-stabilized aqueous nanosuspensions25. The initial M2BIC and MXBIC formulations were produced in poloxamer 407 (P407)asthestabilizingsurfactantwithencapsulationefficienciesof >60%. The particle sizes of nanoformulated M2BIC (NM2BIC) and MXBIC(NMXBIC)were300nm(polydispersityindex,PDI0.29)and 375nm (PDI 0.43), respectively (Fig. 2A, B). These formulations remained stable over a 3-month observation period at room temperature, maintaining consistent prodrug concentration and physicochemical properties, including color, viscosity, syringeability, particle size, and homogeneity (Fig. 2A, B). An optimal formulation with polysorbate 20 (Tween 20) and polyethylene glycol-3350 (PEG3350) stabilizers was designed, improving the drug concentrationwithintheformulationfrom120mg/mLfortheP407-containing formulations to 300–330mg/mL (w/v) for the Tween 20/PEG3350-containing formulation. The optimized NM2BIC and NMXBIC nanosuspensions were produced at encapsulation efficiencies of >75%, particle sizes of 400–550nm, and narrow PDIs (Supplementary Fig. 13). These formulations were stable in color, viscosity, particle sizes,andhomogeneityoveranobservationperiodof142days(SupplementaryFig.13).XRDanalyses confirmedthecrystalline formof M2BIC and MXBIC within the formulation with distinct diffraction patterns from nanoformulated BIC (NBIC) (Supplementary Fig. 9B). Unlike unformulated M2BIC and MXBIC solutions, NM2BIC and NMXBICnanosuspensionsmaintainedcellvitalityandcellularsafety, underscoring the therapeutic potential (Fig. 2C). As expected for surfactant-stabilizedsoliddrugnanoparticles,nanoformulatedforms of BIC (NBIC) and its prodrugs (NM2BIC and NMXBIC) exhibited modestincreasesinIC50valuescomparedtotheirsolubilizedforms.In particular,theIC50valuesforNBIC,NM2BIC,andNMXBICinMDMs were9.04,56.0,and27.9nM,respectively(Fig.2D).

Macrophagesfacilitatedrugabsorptionandsecondarydepots
Macrophagesfacilitatetheabsorptionofnanoformulateddrugsfrom the injection site. We previously demonstrated that macrophages infiltrate and uptake prodrug nanosuspensions at the injection site depotfollowingIMinjection26.Deliveryofsoliddrugnanoparticlesto macrophagesenhancestheirhalf-lifebyfacilitatingintracellularand tissuedrugstorage27.Wethereforeexamineddruguptake,retention, and antiretroviral activities in human MDMs after treating the cells with25µMofeachnanoformulation.AsshowninFig.3AandSupplementaryFig.14A,BICprodrugnanosuspensionsfacilitatedenhanced intracellular drug delivery compared to NBIC. At 24h, intracellular prodruglevelswere61.9,101,200,and35.7nM/millioncellsforNMBIC (nanoformulatedMBIC),NM2BIC,NM3BIC(nanoformulatedM3BIC), and NMXBIC,respectively(Fig. 3AandSupplementaryFig. 14A).To assesstheprodrug’sintracellularconversionintotheparentdrug,BIC levelsinthesamesampleswerequantified.IntracellularBIClevelsfor NMBIC,NM2BIC,NM3BIC,NMXBIC,andNBIC-treatedMDMsat24h were4.62,5.50,9.47,7.95,11.8nM/millioncells(Fig.3AandSupplementaryFig.14A).Alltheprodrugnanosuspensions,exceptNMBIC, facilitatedsustaineddrugretentionforupto30daysfollowingasingle treatment(Fig.3BandSupplementaryFig.14B).Onday30,intracellularprodruglevelsforNM2BIC,NM3BIC,andNMXBIC-treatedMDM were 4.95, 42.4, and 12.9nM/million cells, respectively (Fig. 3B and SupplementaryFig.14B).Nodetectabledrugwasobservedat24hfor NBIC,suggestingrapiddrugreleaseintoculturemedia.Intracellular retentionofBICprodrugsparalleledimprovedantiretroviralactivities. NM2BICandNMXBICprotectedagainstinfectionforupto30days afterMDMsweretreatedfor8hwith25µMofNM2BICorNMXBIC, followed by an HIV-1 challenge administered at 10-day intervals (Fig.3CandSupplementaryFig.15).TheresultsdemonstratethatBIC prodrugs improve intracellular drug delivery for sustained antiretroviralactivities.

PKandbiodistributionofnanoformulatedmonomericprodrugs
The PK profiles of monomeric prodrug nanosuspensions (NMBIC, NM2BIC, and NM3BIC) were compared againstnanoformulated BIC (NBIC)inmaleBALB/cJmiceaftersingleIMinjectiosat45mg/kgbody weightBIC-equivalentdoseintotherightcaudalmuscle.Alltheformulationshadidenticalexcipients(P407stabilizer)andcomparable particle sizes, homogeneity, and drug concentrations. On day 1, IM injectionofNBICandNMBICdemonstratedthehighestpeakplasma BICconcentrationscomparedtoNM2BICandNM3BIC(Fig.4A).Cmax valuesforNBICandNMBICwere17,865±2266and13,235±1355ng/ml, respectively, compared to 5135±2038 and 787±75.35ng/ml for NM2BICandNM3BIC,respectively(Fig.4C).One-wayANOVAfollowed byTurkey’spost-hocanalysisdemonstratedthattheCmax valuesfor NM2BICaresignificantlylowerthanforNMBICandNBICtreatments (p<0.0001). Rapid and uncontrolled drug absorption for NBICand NMBIC resulted in higher initial plasma BIC levels that rapidly fell below 4x PA-IC95within 21 (305ng/ml) and 68 days (207ng/ml), respectively(Fig.4A).Onday180,atsacrifice,BIClevelswerebelow thelimitofquantitationforNBICtreatmentandslightlyabovethelimit ofquantitationforNMBICtreatment(Fig.4A).LowerBIClevelswere recordedforNM3BICtreatment,whereBIClevelsfellbelowthePA-IC95within2weeks,suggestinginefficientprodrugconversionintoBIC (Fig.4A).Notably,NM2BICshowedimprovedPKprofilesoverNBIC, NMBIC,andNM3BICbysustainingplasmaBIClevelsatorabovePA-IC95untilday295(163ng/ml)(Fig.4A).Onday365,themeanplasma BICconcentrationforNM2BICtreatmentwas129ng/ml(Fig.4A).The meanresidencetime(MRT)forNM2BICwas165±9.12dayscompared to 5.8±0.56, 17.4±0.45,and 79.1±9.34 days for NBIC, NMBIC, and NM3BIC,respectively(Fig.4C).NM3BICexhibitedalowerplasmaBIC decay rate (λz=0.016±0.003 1/day) compared to NBICand NMBIC (λz=0.276±0.06and0.039±0.0031/day),resultinginanextended MRT of 79.1±9.34 days (Fig. 4C). The calculated half-life (T1/2) was 110±8.48daysforNM2BIC,comparedto2.60±0.51,17.9±0.94and 45.3±6.5daysforNBIC,NMBICandNM3BIC(Fig.4C).

NM2BICandcontrolNBICformulationswerefurtherevaluatedin SDratsadministeredasingleIMinjectionof45mg/kgBICequivalent dose.Nanosuspensionswithsimilarparticleparameterswereprepared forthisPKstudyusingTween20andPEG3350stabilizingsurfactants. SimilartoobservationsmadeinBALB/cJmice,NM2BICcontrolledthe initialburstreleaseofBICmoreeffectivelythanNBIC.Acomparisonof themeanCmaxvaluesforNM2BICandNBICusingatwo-tailedt-test with Welch’s correction revealed statistically significant differences between the two treatments (3374±805ng/ml for NM2BIC vs. 23,150±3145ng/mlforNBIC,p<0.0001)(Fig.4B).MeanplasmaBIC concentrationsforNBICweresustainedabove4xPA-IC95and1xPA-IC95 for 84 days (700ng/ml) and 168 days (183ng/ml), respectively (Fig. 4B). However, plasma BIC levels among individual animals administeredNBICwerehighlyvariable.Bycomparison,plasmaBIC concentrationsforNM2BIC-treatedanimalsweresustainedatorabove 4xPA-IC95foratleast4months(668ng/mlonday140)andabovethe PA-IC95for1year(162ng/mlonday365).Themedian(minimum-maximum) plasma BIC concentration from day 7 to 140 was 1409 (668–3263) ng/ml (Fig. 4B). NM2BIC demonstrated sustained tissue druglevels.TheaveragetissueBICconcentrationsat365daysafter NM2BIC treatment were 57.6, 30.9, 23.0, 16.8, 16.0, 8.20, 2.90, and 356ng/g tissue in the lymph nodes, kidney, lung, liver, gut, spleen, brain,andinjectionsite,respectively(Fig.4D).Prodruglevelswerelow in all tissues except the muscle injection site (77074ng/g of tissue) (Fig.4E,F).Thesedataconfirmedthatthemuscleinjectionsitewasa primarydrugdepot.Bycomparison,BICwasnotdetectedinanytissues following NBIC treatment. No differences in animal weights, organ-to-body weight ratio, and serum chemistry were recorded among NM2BIC-treated and age-matched control animals (SupplementaryFigs.16–18). Histopathologicalexamination of hematoxylin andeosin(H&E)-stainedtissuesectionsfromNM2BIC-treatedanimals andage-matchedcontrols,conductedbyaboard-certifiedpathologist, demonstrated normal liver, spleen, lung, kidney, and muscle tissue histology(SupplementaryFig.19).

NMXBICPK,tissuebiodistribution,andviralrestriction
NMXBICPKwasfirsttestedinBALB/cJmice,whichdiffersfromNBICor the monomeric prodrugs nanosuspensions. Plasma BIC levels for NMXBIC-treatedmiceexhibitedabiphasicdrugreleaseprofilecomparedtolinearplasmaBICdecaycurvesforthemonomericandparent drugformulations(Fig.5A).FollowingasingleIMinjectionof45mg/kg BIC equivalent dose, the initial peak plasma BIC level for NMXBIC treatmentwasrecordedwithin24hpost-injection,withaCmaxvalueof 2328±197ng/ml (Fig. 5A). One-way ANOVA followed by multiple comparisonswithTukey’stestshowedstatisticallysignificantdifferences between the Cmax values of NBIC (17,865±2265ng/ml) and NMXBIC(p<0.0001).However,therewerenostatisticallysignificant differencesbetweenthemeanCmaxvaluesforNM2BICandNMXBIC treatments(p>0.05),suggestingcontrolleddrugabsorptionforboth prodrugformulations(Fig.5A).Byday7,meanplasmaBIClevelsfrom NMXBICdeclinedto970ng/ml.However,beyondday7,plasmaBIC levelsstabilized,fluctuatingbetweenthreeandsixtimesthePA-IC95 overa6-monthperiod(Fig.5A).Notably,arapiddeclineinplasmaBIC levels was observed 6 months post-NMXBIC dosing, with a 29-fold decreasefromday180today257(from28.0ng/mL).Byday365,BIC levels dropped below 1ng/ml. These data sets demonstrate the potentialforNMXBICtosustainefficaciousBIClevelsfor6months withashortPKtail(Fig.5A).

Inaparallelstudy,NMXBIC’sPKprofilewasvalidatedinSDrats administeredsingleIMinjectionsof45and90mg/kgBICequivalent dose. NMXBIC used in these studies consisted of Tween 20 and PEG3350asthestabilizingsurfactants.Thedifferencesintheshapeof thePKprofilecurvesforNMXBICandthemonomericesterprodrugs werealsoobservedinSDrats.However,inratswithsufficientmuscle mass for IM injections, NMXBIC produced a horizontal and consistentlyhigherBICconcentrationfromday1to6monthscomparedto linearplasmaBICdecaycurvesforthemonomericandparentdrug nanoformulations(Fig.5B).Notably,therewerenosharpfluctuations in plasma BIC concentrations in the first 6 months at both dose strengths(Fig.5B).Forthe45mg/kgBICequivalentdoseofNMXBIC, themedian(minimum-maximum)plasmaBICconcentrationfromday 1today180was3154(696–6712)ng/ml(Fig.5C).MeanplasmaBIC concentrationsondays28,56,84,142,and180were3951,2688,2798, 2541,and3659ng/ml.DespitethehighplasmaBICconcentrationsin SDrats,theterminaldeclineinplasmaBIClevelsmirroredtheshortPK tailinmice,withdrugconcentrationsdecliningto682ng/mlbyday 225(Fig.5B).DoublingthedoseinSDratsdidnotproduceproportionalplasmaBICexposure(Fig.5B).Themedian(minimum-maximum) plasma BIC concentration from day 1 to 180 was 3558 (1377–9088)ng/ml(Fig.5C).MeanplasmaBICconcentrationsondays 28,56,84,142,and180were4637,3656,3586,3152,and4034ng/ml. Similarly,rapidplasmaBICdecaywasobservedafterday180(Fig.5B). Onday365,themeanplasmaBICconcentrationwas19ng/ml(Fig.5B). Tissuedruglevelsdifferedbetweenthelowandhigh-dosegroups, withthehigherdoseyieldinghigherBICandprodruglevels(Fig.5D,E). Forthehigh-dosegroup,BICconcentrationsonday90were668,569, 553,531,and465ng/gtissueinlymphnodes,rectum,lung,kidney,and liver,respectively(Fig.5D).Bycomparison,meanBIClevelsonday90 forthelow-dosegroupwere422,307,391,356,and257ng/gtissuein lymphnodes,rectum,lung,kidney,andliver,respectively(Fig.5D). TissueMXBIClevelsinthelow-dosegroupwere11.0,1.00,andbelow the limit of quantitation in the spleen, lung, and liver, respectively (Fig.5E).Bycomparison,thehigh-doseresultedinnotablyincreased MXBICtissueconcentrations,particularlyinthespleen,lung,andliver, withcorrespondinglevelsof88.0,62.0,and59.0ng/g,respectively,on day90(Fig.5E).Notably,bothBICandMXBICweredetectedatthe injectionsiteonday90forbothdosegroups,supportingtheformation of primary drug depots at the injection site (Fig. 5F). MXBIC concentrationsattheinjectionsitewere850,558and1,110,100ng/g tissue for 45 and 90mg/kg BIC equivalent dosegroup, respectively (Fig. 5F). BIC levels at the injection site muscle were 153,577 and 204,222ng/gmtissuefor45and90mg/kgBICequivalentdosegroup, respectively (Fig. 5F). Similar to the observed short PK tail after 6monthspost-dosing,adecreaseintissueBICandMXBIClevelswere alsorecordedonday225forthe45mg/kgBICequivalentdose,suggestingefficientclearanceofBICandMXBICfromtissuereservoirsas well.ThecorrespondingBIClevelsonday225were80.0,77.0,66.0, 37.0,and34.0ng/gtissueinthelung,lymphnode,kidney,liver,and rectum,respectively(Fig.5G).NoMXBICwasdetectedintissueson day225.

ToevaluatetheeffectofinjectionvolumeandformulationconcentrationonNMXBICabsorptionandPKprofile,alessconcentrated formulation (NMXBIClow concentration) was made and injected intramuscularly to SD rats at a dose of 45mg/kg BIC equivalent. NMXBIClow concentration also demonstrated a comparable PK profile curve that was observed with the high concentrated formulation (Supplementary Fig. 20A). However, notable differences were observed inthe Cmax and plasmaBIC exposure. Median(minimum-maximum) plasma BIC concentrations from day 1 to day 180 for NMXBIClow concentration wererecordedat1882(470–3288)ng/mlwith an average Cmax of 2705±425ng/ml (Supplementary Fig. 20B). Consistentwithourpreviousobservations,plasmaBIClevelsrapidly declinedafter6months,affirmingtheshortPKtailofNMXBIC(SupplementaryFig.20A).TheobservedlowBICexposurefromthedilute formulation could potentially be linked to the effect of drug-depot volumeon drug absorption. Histopathologicalexamination of H&E-stainedtissuestreatedwithNMXBIConday365foundnomorphological difference in all tissues compared with age-matched controls (SupplementaryFig.21).Therewerenonotabledifferencesinanimal bodyweights,organ-to-body-weightratios,orcomprehensiveserum chemistry between the groups treated with NMXBIC and the age-matchedcontrolgroups(SupplementaryFigs.22and 23). Toselect dosesforantiretroviralsuppression,NMXBICPKprofileswerefurther determinedinBALB/cJandNOD/scid-IL-2Rγcnull(NSG)miceatsingle intramusculardosesof50,100,and200mg/kgBICequivalent.The data demonstrated dose-dependent BIC exposure (supplementary Fig.24).InNSGreconstitutedwithhumanperipheralbloodlymphocytes (hu-PBL),NMXBICsuppressedHIV-1inadose-dependentmanner. These results were coordinated with reductions in HIV-1p24 antigenexpressioninspleentissues(supplementaryFig.24).

Injectionsiteformulationtolerability
The injection site tolerability of NMXBIC and NM2BIC formulations was comprehensively evaluated in NZW rabbits, a well-established modelfordepotinjectionstudiesduetotheirrobustinflammatoryand immune responses, as well as their suitability for testing human-equivalentdoses28.AnimalsreceivedIMorSCinjectionsofNM2BICor NMXBICorformulationvehicleonday1,followedbyasecondinjectionatadifferentsiteonday26.Onday29,theanimalsweresacrificed, and tissues from the injection sites were collected for histological analysis.Notably,1mlIMinjectionsofNMXBIC(150mg)orNM2BIC (300mg)onday1andday26werewelltoleratedanddidnotresultin macroscopicalterationsattheinjectionsites.Histologicalexamination of H&E-stained injection site tissue sections on day 3 post-second injection revealedminimalmyofibernecrosisinoneofsixandminimal to mild mixed cellular inflammatory reactions in six of six animals treated with NMXBIC (Table 1). Day 29 post-first injection analyses revealedmoderategranulomatousinflammationandpseudocystformationinoneofsixanimalstreatedwithNMXBIC(Table1).Similarly, forNM2BIC,H&E-stainedhistologicalanalysisonday3post-second injection revealedminimalmyofibernecrosisinoneofsixanimalsand mildmixedcellularinflammationinthreeofsixtreatedanimals.Byday 29 post-first injection, moderate granulomatous inflammation and pseudocystformationwerenotedintwoofsixanimalstreatedwithNM2BIC(Table2).

Two milliliters SC injections of NMXBIC (300mg) or NM2BIC (600mg)orformulationvehiclewerealsowelltoleratedbutcaused thickening at the injection sites due to pseudocyst and pyogranulomatous inflammation. Minimalpyogranulomatous inflammation,mildmixedcellularinflammation,andpseudocystpresencewere observed on day 3 post-second injection for NMXBIC treatment (SupplementaryTable1).Histopathologicalevaluationonday29post-first injection of NMXBIC indicatedmildfibrosis,minimaltomoderate mixed cellular inflammation, and pseudocyst development (SupplementaryTable1).SimilartoNMXBIC,microscopicevaluationofday3 after SCadministration of NM2BICrevealed minimalto mildmixed cellular inflammation, isolated adipose necrosis, and pseudocyst formation (Supplementary Table 2). Histological analysis of tissue sections from the injection site on day 29 post-first injection predominantlyrevealedminimaltomildfibrosiswithinthesubcutaneous layer,variousdegreesofcellularinflammation,minimalmyofiberand adiposenecrosis,andpseudocystformation(SupplementaryTable2). Overall,theinjectionsitefindingsforNMXBICandNM2BICrepresent thereportedexpectedforeignbodyresponsetoinsolubledrugdepots attheinjectionsite29,30.

Methods
Studyapproval
TheUniversityofNebraskaMedicalCenterInstitutionalAnimalCare and Use Committee (IACUC) approved all the PK studies involving laboratoryanimalsbythestandardsincorporatedintheGuideforthe CareandUseofLaboratoryAnimals(UNMCIACUCprotocolnumbers: 22-075-02-FCand22-045-06-EP).Humanperipheralbloodmonocyte isolationandpurificationfromHIV-1/2andHBVseronegativeparticipantswereperformedaccordingtoanexemptprotocolbytheUNMC Institutional Review Board (IRB) with informed consent. The local injectionsitestudywasconductedusingaprotocolapprovedbythe IACUCatLabcorpEarlyDevelopmentLaboratoriesInc.(Madison,WI, USA-53704)(studynumber:848296).

Reagents
Reagentswereobtainedfromcommercialsourcesanduseddirectly; exceptionsarenoted.BICwaspurchasedfromBOCSciences(Shirley, NY,USA).Myristoylchloride,stearoylchloride,behenicacid,octadecanedioicacid,pyridine,dimethylformamide(DMF),N,N-diisopropylethylamine(DIEA),polysorbate20(Tween20),polyethyleneglycol-3350(PEG3350),PluronicF127(poloxamer407;P407),ciprofloxacin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO),1-octanol,paraformaldehyde(PFA),and 3,3′-diaminobenzidine(DAB),werepurchasedfromSigma-Aldrich(St. Louis,MO,USA).Diethylether,acetonitrile(ACN),methanol,optima-grade water, ethyl acetate, hexanes, dichloromethane (DCM), Dulbecco’s Modified Eagle’s Medium, phosphate-buffered saline (PBS), gentamicin,L-glutamine,potassiumphosphatemonobasic(KH2PO4), bovineserumalbumin(BSA),andTritonX-100werepurchasedfrom ThermoFisherScientific/Gibco(Waltham,MA,USA).Cellculturegrade water(endotoxin-free)waspurchasedfromCytiva(Logan,UT,USA). Heat-inactivatedpooledhumanserumwaspurchasedfromInnovative Biologics(Herndon,VA,USA).

SynthesisandcharacterizationofBICprodrugs
WesynthesizedthreelipophilicmonomericprodrugsofBICthrough single-step esterification reactions, each using linear biocompatible fattyacidchloridesofvaryingcarbonchainlengths(C-14,C-18,andC-22),coinedasMBIC,M2BIC,andM3BIC,respectively.Oneequivalent ofBICwasfirstdriedfromanhydrouspyridineandthensolubilizedin anhydrousdimethylformamide(DMF)solventunderanargonatmosphere.Thesolutionwasthencooledto4°C,followedbytheaddition of2equivalentsofdiisopropylethylamine(DIEA)base.Subsequently, 1.5 equivalents of the acyl chlorides were added, and the reactions werecontinuedfor18hat45°C.Uponcompletionofthereactions,the solventswereevaporatedonarotaryevaporator,followedbydilution of the reaction mixture in dichloromethane (DCM) and successive extractionwith0.1NHCl,NaHCO3,andbrinesolution.TheDCMlayer was dried over Na2SO4 and then concentrated using a rotary evaporator.Thedesiredproductswereisolatedbyflashchromatography eluting with a 4:1 mixtureofethyl acetate and hexane. The desired compoundfractionswerecollected,driedonarotaryevaporator,and thenprecipitatedfromhexanetoobtainoff-whitepowders.Theprodrugpowderswerefurtherdriedundervacuum,producingaverage chemicalyieldsof70–75%.

Additionally,onedimericBICprodrug,MXBIC,wassynthesized by activating octadecanedioic acid using thionyl chloride (SOCl2), followed by conjugation to BIC. Briefly, octadecanedioic acid (0.5 equivalent)wasdriedfrombenzeneandreactedwithtenequivalents ofSOCl2for4hat75°C.Thereactionmixturewasthendriedbyco-evaporatingwith20mlofbenzeneonarotaryevaporator.Thedried samplewasresuspendedin20mlofanhydrousDMFunderanargon atmosphere and cooled to 4°C. Four equivalents of DIEA and one equivalentofBICwereaddedtothereactionmixtureandwarmedto 45°C.After16h,thereactionmixturewasconcentratedusingarotary evaporatorandsubjectedtoacid-baseextractionasdescribedforthe monomericprodrugs.TheDCMlayerwasdriedoverNa2SO4 andthen concentratedusingarotaryevaporator.Theconcentratedsamplewas then purified by flash column chromatography, eluting with a 4:1 mixtureofethylacetateandether.Thedesiredcompoundfractions werecollectedanddriedonarotaryevaporatortoproducecolorless foams.ThedriedcompoundwasthenlyophilizedfromTHFandwater, producingoff-whitepowderswithaveragechemicalyieldsof>70%.

All chemical reactions were conducted under an argon atmosphereunlessotherwisespecified.ThefinalproductswerecharacterizedbyprotonandcarbonNMR(1Hand13CNMR)spectroscopyona BrukerAdvanced-IIIHD(Billerica,MA,USA)operatingat400MHz,a magneticfieldstrengthof11.7T.Distinctfunctionalgroupswithinthe synthesized compounds were corroborated by FT-IR spectroscopy, performed on a PerkinElmer universal attenuated total reflectance Spectrum Two (Waltham, MA, USA). The molecular masses of the purifiedcompoundswereconfirmedonanExploris480massspectrometer. Comparative crystallographic analyses of the synthesized compoundswereconductedviapowderX-raydiffraction(XRD)within a2ørangeof2–50°,utilizingaPANalyticalEmpyreandiffractometer (PANalytical Inc., Westborough, MA, USA) equipped with Cu-Ka radiation(1.5418Å)atsettingsof40kVand45mA.

AqueousandoctanolsolubilityofBICprodrugs
Anexcessamountofeachcompoundwascontinuouslymixedineither wateror1-octanolatroomtemperaturefor24h.Aftersaturation,the samplemixturewascentrifugedat20,000×gfor10mintoseparate theinsolubledrugfromthe supernatant.The1-octanolsupernatant solutionsweredirectlyanalyzedfordrugcontentbyLC-MS/MS.Aqueoussupernatantsolutionswerelyophilizedpriortoreconstitutionin methanol,anddrugquantificationwasperformedbyLC-MS/MS.

SolubilizedprodrugshydrolysisinplasmaanddifferentpH buffersolutions
ToinvestigatetheroleofenzymaticandchemicalhydrolysisinBIC prodrugsactivationintotheparentdrug,1µMsolutionofM2BICand MXBICinmethanol(1%v/v)wasincubatedinplasmaobtainedfrom various species (dog, monkey, mice, rat, and human) or in buffer solutionsofdifferentpHvalues(2.0,6.0,8.0,and10.6).BICandprodrug levels in each sample were determined at pre-specified time points.Briefly,theplasmaorpHsolutionwaspreincubatedat37°Cfor 5min.Then,10µLofa100µMsolutionofM2BICorMXBICinmethanol wasincubated in 990µl ofplasmaor pH solution at37°C for 24h, yieldingafinalprodrugconcentrationof1µM.Atpredeterminedtime intervals,a25µLsamplewasdrawnfromthemixtureandmixedwith 475µLof80%methanolinwatercontaining0.1%formicacid(Optima-grade).Thesamplemixturewasvortexedfor3minandcentrifugedat 16,000×gfor10min.Theresultingsupernatantswerecollectedand analyzed for drug content by UPLC-MS/MS using the quantitation methodoutlinedinthesupplementarysection.Controlsampleswere prepared following the same protocol; however,M2BIC and MXBIC solutions wereadded to plasmacontaining methanol. The pHsolutions investigated in this experiment included 7.5mM ammonium acetate(pH6.0,adjustedwithaceticacid),7.5mMammoniumbicarbonate (pH 8.0, adjusted with acetic acid), as well as pH-adjusted solutions involving 0.1% formic acid (pH 2.0) and 0.1% ammonium hydroxide(pH10.6).

Nanoformulationpreparationandcharacterization
BICandeachprodrugwereformulatedasaqueousnanosuspensions stabilizedbynon-ionicsurfactantsusedinotherparenteralproducts. Briefly,thewater-insolubleprodrugpowderswereeitherdispersedin solutionsofsurfactantsinPBS(11.9mMpotassiumphosphatemonobasic, 137mM sodium chloride, 2.7mM potassium chloride) or endotoxin-free water (pH 7.0), followed by microfluidization of the presuspension on an Avestin EmulsiFlex-C3 high-pressure homogenizer(Ottawa,ON,Canada)at20,000 ± 1000PSIuntilthedesired nanoparticle size was achieved. Each formulation was assessed for stability(hydrodynamicparticlediameter(size)),polydispersityindices (PDIs), and zeta potential, appearance, and drug concentration (prodrugstabilitywithintheformulation)atroomtemperature.Size, PDI,andzetapotentialweredeterminedbydynamiclightscattering (DLS) using a Malvern Zetasizer Nano-ZS (Worcestershire, UK). The stabilitiesofthenanosuspensionsweremonitoredatroomtemperature. Compound (BIC or prodrugs) concentration in each nanosuspension was determined by diluting the formulations in methanol (1000–10,000-fold dilution), followed by drug quantitation by UPLC–ultraviolet/visible (UV/vis) spectrometry. The encapsulation efficiencywascalculatedusingtheequation:encapsulationefficiency (%)=(weight of drug in formulation/initial weight of drug added)× 100.Endotoxinconcentrationsintheformulationsweredetermined byusingaCharlesRiverEndosafenexgen-PTSsystem(CharlesRiver, USA).Allthepreclinicalformulationswerereadilysyringeableusinga 28Gneedle.

The initial formulation screening for NBIC, NM2BIC, NMXBIC, NMBIC, and NM3BIC was produced in endotoxin-free water, using poloxamerP407asthesurfactant.ForSDrats,optimalformulations werepreparedinpolysorbate20(Tween20)andpolyethyleneglycol-3350(PEG3350)solutionsinPBS.AlltheformulationsusedinSDrats PKstudieshadparticlesizesof300–550nmandPDIsof0.3–0.4.

PKandBDstudiesofmonomericprodrugsnanosuspensions
For mice PK studies, male BALB/cJ mice (weighing 26–30g, aged 6–8weeks,obtainedfromJacksonLabs,BarHarbor,ME,USA)were administeredasingleIMinjectionofeitherNBIC,NMBIC,NM2BIC,or NM3BIC at a dose of 45mg/kg BIC equivalent. NBIC, NMBIC, and NM3BICgroupshadfivemicepergroup,andtheNM2BICgrouphad threemice.Bloodsampleswerecollectedondays1,7,14,21,28,and thenevery2weekspost-day28.Onday180post-dosing,NBIC,NMBIC, andNM3BIC-treatedmicewerehumanelyeuthanized,whileNM2BIC micewerehumanelyeuthanizedonday365.Uponeuthanization,tissues(liver,lung,spleen,lymphnodes,kidney,injectionsitemuscle, gut,andrectum)werecollectedfordrugandprodrugquantitationby UPLC-MS/MSandhistopathologicalassessment.

For PK and BD evaluation in Sprague Dawley rats, male rats (weighing 270–300g, aged 8 weeks, purchased from SASCO, Wilmington,MA,USA)wereadministeredasingleIMinjectionofeither NBIC or NM2BIC at a dose of 45mg/kg BIC equivalent in the right caudalmuscle(n=5,SDratsineachgroup).Bloodsampleswerecollectedfordrugquantificationondays1,3,7,14,21,28,andthenevery 2weekspost-day28.Atotalof3animalsintheNBICgroupwerelost during blood sampling on days 98, 126, and 194, respectively. One animalintheNM2BICgroupwaslostduringbloodsamplingonday 168.Theratswerehumanelyeuthanizedonday365fortissue(liver, lung, spleen, lymph nodes, kidney, injection site muscle, gut, and rectum)collection.

PKandBDstudiesofthedimericprodrugnanosuspension
ForNMXBICmicePKstudies,maleBALB/cJmice(n=3),matchedin weightandagetothoseusedinmonomericprodrugsPKstudies,were administeredasingleIMinjectionofNMXBICatadoseof45mg/kgBIC equivalentonday0.Bloodsampleswerecollectedondays1,7,14,21, and28,andthenevery2weeksuntilday365,whentheanimalswere humanely euthanized. Male SD rats, matched in weight and age to those used in monomeric prodrugs PK studies, were divided into threegroups.Group1(n=5SDrats)wasinjectedwithasingledoseof 45mg/kg BIC equivalent NMXBIC on day 0. Group 2 (n=8 SD rats) was injected with a single dose of 45mg/kg BIC equivalent NMXBIC formulation, while Group 3 (n=8 SD rats) was injectedwithahigherdoseof90mg/kgBICequivalentNMXBIConday 0. All the injections were administered by IM into the right caudal muscle.Forgroup1,bloodwascollectedfromallfiveanimalsondays 1,3,7,14,21,28,andevery2weeksuntilday365,whenanimalswere humanelyeuthanized.Forgroups2and3,bloodsamplingwasalternatedbetweenfouranimals.Fouranimalsfromgroups2and3each weresacrificedonday90,whiletheotherfouranimalsfromgroups2 and3weresacrificedondays225and365,respectively.Tissueswere collected for drug and prodrug quantitation by UPLC-MS/MS and histopathologicalassessment.

For mouse and rat studies in both monomeric and dimeric PK studies, a 28G×½″ needle was used for injections, and the total injectionvolumedidnotexceed1µL/gweightforSDratsor1.6µL/g weightforBALB/cJmice.Duringthebloodsamplecollection,animals wereanesthetizedusinginhaledisoflurane.Bloodsampleswerecollected in heparinized tubes. Blood sampling was collected via submandibularpunctureforBALB/cJmiceandviaretro-orbitalortail-vein bleeding from SD rats. Plasma was isolated from blood by centrifugation at 2000×g for 8min and analyzed for drug content by UPLC-MS/MSusingmethodsspecifiedinthesupplementarysection. Alltherodentswereeuthanizedusingisoflurane,followedbycervical dislocation. The tissues were homogenized and analyzed for drug contentbyUPLC-MS/MS.Non-compartmentalPKforplasmaBICinall specieswasperformedbyPhoenixWinNonlin-8.3.3.33software(Certara,Princeton,NJ,USA).SafetyevaluationinSDratsandBALB/cJmice was assessed by monitoring animal body weights, organ to body weightratiosaftersacrifice,serumchemistry,andtissuehistopathology.Fortissuehistopathologyassessments,5-µMparaffin-embedded tissue sections were stained with H&E and imaged according to a previouslypublishedprotocol45.Anindependentcertifiedpathologist assessedtheimagedtissuesectionsinablindedway.AVetScanVS-2 instrument(AbaxisVeterinaryDiagnostics)wasusedtoassessserum chemistry profiles, and the data were compared with age-matched controlSDratsorBALB/cJmice.

AlltheanimalexperimentswereapprovedbytheUNMCIACUC, andanimalswereregularlymonitoredbyanimalcarepersonneland veterinarystafffromtheComparativeMedicinedepartmentatUNMC. BALB/cJmiceandSDratsusedinthePKstudieswerekeptincontrolled conditions, including a 12-h light/dark cycle, temperatures ranging from20to24°C,andhumiditylevelsbetween30and70%.Theanimalsweregivenasterilized7012Tekladdiet(Harlan,Madison,WI)and hadaccesstoacidifiedwateradlibitum.

InjectionsitereactionsinNZWrabbits
Twenty-fourfemaleNZWrabbitsweregroupedintofour(Groups1–4), eachcomprisingsixanimals.Eachanimalingroup1receivedatotalof foursubcutaneousinjectionsinfourdistinctsitesonday1andday26 (oneinjectionofNM2BICandoneinjectionofplaceboeachday).On day1,600mgofNM2BIC(2mlinvolume)andplacebo(2ml)were administeredsubcutaneouslyintheupperrightandupperleftscapularregions,respectively.Onday26,asimilardoseofNM2BICand placebo was given subcutaneously in the lower right and lower left scapularregions,respectively.

Eachanimalingroup2receivedatotaloffourIMinjectionsinfour distinctmusclesitesonday1andday26(oneIMinjectionofNM2BIC and oneIMinjection ofplacebo each day).On day1,they received 300mgofNM2BIC(1mlinvolume)andaplacebo(1mlinvolume)in therightthighandleftparalumbarmuscles,respectively.Onday26,a secondinjectionofasimilardoseofNM2BICandplacebowasadministeredintheleftthighandrightparalumbarmuscle,respectively. Group3mirroredtheadministrationpatternsofGroups1,substituting 600mg of NM2BIC (2ml) for 300mg of NMXBIC (2ml). Similarly,Group4mirroredtheadministrationpatternsofGroup2, substitution300mgofNM2BIC(1ml)for150mgofNMXBIC(1ml). Thevehiclecontrolforeverygroupconsistedof88.6%PBS(pH7),5.7% PEG3350,and5.7%Tween20.

All the injection sites were shaved and cleaned with a surgical scrubbeforeadministration.Dosesitesweremarkedandmaintained throughout the study. Dose sites were observed daily, including immediatelypost-doseonDays1and26andjustbeforenecropsyon Day 29, and scored/graded using a Draize technique. Abnormal observationsorindicationsoftheordinarywererecorded.Ifedemaor erythema at the injection site was observed, measurements of the affected area were recorded separately (in cm) for each finding accordingly. Photographs of abnormal dose site observations (discharge,ulceration,etc.),ifnoted,weretaken.Animalswereanesthetizedwithsodiumpentobarbitalandexsanguinatedonday29post-first injection. Injection sites from each animal were collected and preservedin10%neutralbufferedformalin:InjectionSites(foursites/animal),includingtheoverlyingskin,stapledtocardboardtomaintain the orientation of the dose site and any gross lesions (if present). Collectedtissueswereembeddedinparaffin,cutintofiveµMsections, and stained with H&E for histopathological assessment. Histology slides were examined by Experimental Pathology Laboratories Inc. (Sterling,VA,USA-20166).

Histologyslidesweresubjectedtoqualitativeassessmentbased ontheDraizescale,whichincludeddiagnosisandgradingofthe overall inflammatory response and cellular infiltrate, local tissue reaction and/or damage, including tissue degeneration/necrosis, gradingofedema,hemorrhage,fibrosis,orformationofgranulation tissue.Theseverityofthenon-neoplastictissuelesionswasgradedon ascaleof1–5,representedbyminimal,mild,moderate,marked,and severe. Non-gradable lesions, such as cysts, were noted as “P” for present.Adetaileddescriptionoftheseveritygradingscaleisincluded inthesupplementarysections(SupplementaryTable3).

Isolationandcultivationofhumanmonocytes
MonocytesfromHIV-1/2andhepatitisBseronegativedonorswere acquired through leukapheresis and purified using a counter-current centrifugal elutriation process. Following isolation, the monocyteswereculturedaccordingtoprotocolsdescribedinour prior publications27,46. After differentiation, MDM were used to evaluate drug-particle uptake, retention, and antiretroviral activity.

ProdrugnanoformulationsuptakeandretentioninMDMs
MDMuptakeandretentionofBICprodrugnanoformulationsfollowed the methodology detailed in our previously published work27,46. In brief,foruptakestudies,MDMswereexposedtoeither25µMofBIC nanoformulation (NBIC) or prodrug nanoformulation for 24-h. In contrast,forretentionstudies,MDMswereinitiallytreatedwith25µM ofNBICorprodrugnanoformulationsfor8handsubsequentlyculturedinadrug-freemediumforadurationof30days.Todetermine theintracellularBICandprodruglevelsinbothuptakeandretention studiessamples,MDMsampleswerecollectedatspecifictimeintervals,lysedinmethanol,andquantifiedusingUV-VisUPLC,aselaboratedinthesupplementarysections.

BICprodrugnanoformulationsinHIV-1ADA-challengedMDMs
Assessments of the antiretroviral effectiveness of BIC prodrug nanoformulations were performed according to the methodology establishedinourpreviouslypublishedresearch27,46.Inbrief,MDMs were initially exposed to 25µM of either NBIC or prodrugs nanoformulationfor8handsubsequentlyculturedinadrug-freemedium for30days.Atspecifictimeintervals,MDMswereexposedtoHIV-1ADA atamultiplicityofinfection(MOI)of0.1infectiousvirionsper cell for 16h, following the protocols outlined in our earlier publications27,46. To perform the HIV-1p24 antigen immunohistochemistry,theMDMswerefixedwith4%PFAateachtimepoint. Furthermore,post-challengecellculturesupernatantswerecollected to determine the reverse transcriptase (RT) activity percentage in HIV-1-infectedcells.

BICprodrugshalf-maximalinhibitoryconcentration(IC50)
A previously published protocol was used to determine the IC50 in MDMs27,46. In brief, MDMs were exposed to varying concentrations, ranging from 0.01 to 1000nM of either BIC or prodrug solution, dissolvedin0.1%DMSO(v/v)ornanoformulations.Onehourafterthe drugornanoformulationtreatment,MDMswerechallengedwithHIV-1ADA atanMOIof0.1for16h.Afterthat,cellswereculturedindrug-containing media. On day 10, cell supernatants were collected and analyzedforHIV-1RTactivity.

NMXBICHIV-1suppression
NOD/scid-IL-2Rγcnull(NSG)mice(6weeksofage;JacksonLabs,Bar Harbor,ME)reconstitutedwithhumanperipheralbloodlymphocytes (hu-PBL)wereusedfortheinvivoefficacystudy.Atstudyinitiation, micewerereconstitutedwith25×106hu-PBLperanimalbyintraperitoneal (IP) injection. Groups of 4–6 animals received a single IM injectionofNMXBIConday1atdosesof50,100,or200mgBICeq./kg. Control groups included HIV-1-infected, untreated, and uninfected animals.hu-PBLmicewerechallengedwithHIV-1ADA byIPinjectionon days10and15.HIV-1challengewasmadeataMOIof104infectiousviral particles/mL.Duringthebloodsamplecollection,animalswereanesthetized using inhaled isoflurane. Blood samples were collected in heparinizedtubesviasubmandibularpuncture.Cellandplasmasamples were collected from blood at equivalent time points. Blood mononuclear cells were analyzed for human T-cell populations, including CD45⁺, CD3⁺, CD4⁺, CD8⁺, and CD4:CD8 ratios, by flow cytometry. At study termination (day 25), mice were humanely euthanized using isoflurane, followed by cervical dislocation, and blood and tissues were collected for viral load quantification and immunohistochemical(IHC)analyses.Viralloadwasquantifiedusing theRocheAmplicorandTaqman-48system[HIV-1kitV2.0according tothemanufacturer’sinstructions(Indianapolis,IN)].ForIHCanalysis, spleentissueswerefixedovernightin4%paraformaldehyde,embeddedinparaffin,sectionedat5µm,andmountedonglassslides.Sections were stained for HIV-1p24 antigen, developed with DAB, and counterstainedwithMayer’shematoxylin.Slideswerevisualizedusing a Nikon Eclipse E800 microscope (Melville, NY) equipped with a Nuance EX multispectral imaging system (PerkinElmer, Hopkinton, MA).BrownDAB-positivecellsindicatedp24viralantigen,whichwas comparedacrossuntreatedHIV-1-infectedandNMXBIC-treatedcontrolandexperimentalgroups.

ThermalandCrystallographicanalyses
TGAandXRDofparentandprodrugsweregeneratedbyapreviously describedmethod27.Briefly,TGAwasperformedfrom30to300°Cat therateof10°C/mininanitrogenenvironment(flowrate20mL/min) inaTGA550(TAInstruments,NewCastle,DE,USA)withathermal analysis operating system (TA DSC 25) and TRIOS software V5.1.1 (Waters/TAInstruments,NewCastle,DE,USA).XRDanalysesofparent and prodrugs solid powder and lyophilized nanosuspensions were carriedoutinthe2θrangeof2–70°atarateof1°/susingPANalytical Empyreandiffractometer (PANalytical Inc., Westborough, MA) with Cu-Kαradiation(1.5418Å)at40kV,45mAsetting.

Cytotoxicityassays
Cellular vitality of MDMs treated with either free drug solutions or their nanoformulated forms wasassessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)assay,asdetailed in our prior publications27. MDMs were exposed to varying concentrations(rangingfrom1.95to1000µM)offreedrugsolutions(BIC, M2BIC,orMXBIC)ornanoformulations(NBIC,NM2BIC,orNMXBIC) foradurationof24h.Controlgroupsconsistedofvehicle-treatedand untreated cells. Following the 24-h drug exposure, the cells were thoroughlywashedandthenincubatedwithMTTsolutioninculture mediafor45min.Subsequently,theMTTsolutionwasremoved,and 200µlofDMSOwasaddedtoeachwell.Absorbancewasmeasuredat 490nmusingaMolecularDevicesSpectraMaxM3platereader,and data analysis was performed with SoftMax Pro 6.2 software from Sunnyvale,CA.

UPLC-UV/VisquantificationofBICandprodrugs
DruglevelswerequantifiedonaWatersACQUITYUPLCH-Classsystemcoupledwithanultraviolet/visible(UV/Vis)detectorandEmpower 3software.AnalyticalseparationofBIC,MBIC,M2BIC,andM3BICwas achievedonaPhenomenexKinetex5μmC18column(150×4.6mm) (Torrance,CA,USA).MXBICwasseparatedonaWatersXBridge3.5μm C8column(150×3.0mm)(Milford,MA,USA).Themobilephasefor BICanalysisconsistedof50mMKH2PO4 atapHof3.2(PhaseA)and 100%acetonitrile(ACN)(PhaseB).Forallprodruganalyses,themobile phaseswere100%water(PhaseA)and100%ACN(PhaseB).Isocratic elutionforBICutilized65%PhaseAand35%PhaseBover10minata flow rate of 1ml/min. MBIC, M2BIC, and M3BIC were eluted isocraticallyatratiosof10%A/90%B,5%A/95%B,and2%A/98%Bataflow rateof1ml/min,respectively,whileMXBICemployeda20%A/80%B mixata flowrate of0.5ml/min. Detectionwavelengths were setat 254nmforBICand230nmforallprodrugs.

QuantitationofBICandtheprodrugsbyUPLC-MS/MS
BIC,M2BIC,andMXBICconcentrationsinrodentplasmaandtissues were quantified by UPLC-MS/MS using a Waters ACQUITY Premier System UPLC (Milford, MA, USA) coupled with a Xevo TQ-XS mass spectrometerandWatersMassLynxV4.2software(Milford,MA,USA). Formouseplasmasamples,25μLoftheplasmasamplewasaddedinto 1mL ACNspiked with 10μL of internalstandard (IS).Samples were vortexedfor3minandcentrifugedat17,000×gfor10minat4°C.The supernatantsweredriedusingaSpeedVacandreconstitutedin100μl of0.1%(v/v)formicacid(FA)containing80%methanolinH2O.10μlof reconstituted sample was injected for BIC and prodrug analyses in UPLC-MS/MS.ForSDratsplasmasamples,50µLofplasmasamplewas addedinto950µlof0.1%(v/v)FAcontaining80%methanolinH2O spikedwith10µLofISsolution.Thiswasfollowed byvortexing for 3minandcentrifugationat17,000×gfor10minat4°C.Supernatants werecollected,and3µlofsupernatantwasinjectedintoUPLC-MS/MS for analysis. d3-Dolutegravir (d3DTG) and stearoylated darunavir (SDRV),atafinalconcentrationof5and10ng/ml,respectively,were used as ISs for BIC and prodrugs analyses, respectively. Standard curveswerepreparedinblankmatrixintherangeof0.2–500ng/mL forrespectiveanalytes.

For rodent tissue drug quantitation, 3–200mg of sample was homogenized in 4–29 volumes of 0.1% (v/v) FA containing 80% methanol in H2O. BIC and prodrugs were extracted from 100µl of tissuehomogenatebyadding890µlof0.1%(v/v)FAcontaining80% methanolinH2Ospikedwith10µlofISsolution.Thiswasfollowedby 3min vortexing and centrifugation at 17,000×g for 10min. The supernatantwascollected,and3µlofsupernatantwasinjectedinto UPLC-MS/MS for BIC and prodrug quantitation. d3-Dolutegravir (d3DTG)andstearoylateddarunavir(SDRV),atafinalconcentration of5and10ng/mL,respectively,wereusedasISsforBICandprodrugs analyses, respectively. Standards were prepared similarly using respectiveblanktissuehomogenates.

For simultaneous quantitation of BIC and prodrugs, chromatographic separation of 3µL supernatant was achieved on a Waters ACQUITY UPLC BEH RP18 column (1.7μm, 2.1×50mm) over 7min usinggradientsofmobilephaseA(0.1%FAinH2O)andmobilephaseB (0.1% FA in ACN) at a flow rate of 0.7ml/min, with a column temperatureof45°C.Duringthefirst0.5min,mobilephaseBwasheldat 20%,followedbyalineargradientfrom20%Ato80%Bbetween0.5 and1.5min.Subsequently,alineargradientfrom80%Bto95%Bwas appliedfrom1.5to5min.MobilephaseBwasheldat95%from5to 6minandthenreturnedtoitsoriginalconditionof20%Bat6.5min, whereitwasheldfor0.5minforequilibration.

IntheanalysiswhereonlyBICwasquantified,chromatographic separation was carried out on a Waters ACQUITYUPLC BEH Shield RP18column(1.7μm,2.1×100mm)witha10-mingradientofmobile phase A and mobile phase B at a flow rate of 0.25mL/min. The compositionofmobilephaseAwas7.5mMammoniumformatebuffer atapHof3.2,andmobilephaseBwasACN.Duringtheinitial3.5min, the mobile phase consisted of 35% B, which was subsequently increasedto95%Bwithin0.5minandhelduntilthe8thminute.Mobile phaseBwasthenresettoitsoriginalcompositionover0.5minand retainedforanadditional1.5min.Intheanalysiswhereonlyprodrug (M2BIC or MXBIC) was quantified, chromatographic separation was achieved using a Waters ACQUITY UPLC BEH Shield RP18 column (1.7μm, 2.1×30mm) withan8-mingradientofmobilephaseAandBat aflowrateof0.25ml/min.Theinitialmobilephasecompositionwas 85%and80%Bforthefirst5minforM2BICandMXBIC,respectively. Themobilephasecompositionwasincreasedto95%Bwithin0.5min andmaintaineduntilthe6.5thminute.Subsequently,themobilephase compositionrevertedtoitsoriginalstateover0.5minandwasheldat theoriginalconditionforanadditional1mintoensureequilibration.

Themassspectrometer condition and Multiple reaction monitoring (MRM)transitionsaredetailedinTable3.Spectrawereanalyzedand quantifiedbyWatersMassLynxV4.2software(Milford,MA,USA).All quantitation were determined using analyte peak area to internal standardpeakarearatios,usingtheMRMtransitionsofBIC,M2BIC, MXBIC, DTG-d3, and SDRV: 450.07>289.04, 716.39>450.14, 1177.3828>450.1328,423.1>280.06,814.46>658.54,respectively.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a nice study that reports the chemical production and experimental assessment of two new prodrugs of bictegravir with improved pharmacokinetics, as longer half-life and shorter tails. In animal models, NMXBIC provided plasma inhibitory concentrations enough to be administered twice a year. Furthermore, its short PK tail would allow late dose forgiveness, a concern with the currently available ultra-long acting cabotegravir. Given that bictegravir has a good safety profile and greater resistance barrier than cabotegravir, NMXBIC would be more appealing than cabotegravir for both HIV prevention and treatment. Thus, further clinical development of NMXBIC is warranted. The research team has long experience in the development of ultra-long acting drugs and has pioneered several formulations in the HIV and hepatitis fields.\nThe Discussion is quite simple and would benefit from expanding the scenarios for HIV Treatment and prevention with the adevent of a 6-month potent antiviral such as NMXBIC. Specific comments for the Discussion section:\n1. Drug resistance. From the clinical standpoint, two major differences exist between cabotegravir and bictegravir. The first is resistance barrier by HIV. Whereas HIV integrase mutations Q148R and N155H may compromise the activity of cabotegravir, they have not demonstrated to reduce bictegravir efficacy in vivo.\n2. Drug interactions. A second difference between cabotegravir and bictegravir refers to its metabolism. Cabotegravir is largely catabolized in the liver by UGT1A1 and in less extent by CYP3A4. Rifampin may induce its metabolism. Overall the potential for drug interactions is lower with bictegravir.\n3. Drug volume. The new bictegravir prodrug NMXBIC needs low volume nanosuspensions and may overcome one of the major side effects of current parenteral long-acting cabotegravir formulations, which is local pain at injection sites. They may extent for a few days, with common development of induration and nodules. The data presented by the author’s testing 1 ml injection site reactions of NMXBIC in the New Zealand rabbit model support its favorable safety profile.\n4. PK tail. The long PK tail of long-acting cabotegravir accounts for prolonged subtherapeutic drug concentrations when timely dosing is forgotten, favoring development of drug resistance. In contrast, the new bictegravir prodrug NMXBIC depicts a shorter PK tail and may allow dosing forgiveness with lesser risk.\n5. Combination options. The opportunity to combine the new bictegravir prodrug NMXBIC with lenacapavir, which is prescribed subcutaneously every 6 months as HIV treatment, opens a further clinical scenario for NMXBIC. Since HIV treatment with long-acting cabotegravir-rilpivirine every 2-months has revolutionized HIV therapeutics, the advent of a new treatment option every 6 months will definitively transform the HIV field and improve patient’s management.\n6. Pre-exposure prophylaxis. The new NMXBIC formulation given twice a year should be more appealing than currently approved options, making easier PrEP indication in some developing regions with high HIV endemicity, as in Sub-Saharan Africa, where 25 out of the 40 million HIV-positive persons live.", "reviewer_2": "This manuscript by Nayan et al describes the design, synthesis, and in vitro and in vivo evaluation of novel monomeric and dimeric bictegravir (BIC) ester prodrugs formulated as injectable nanosuspensions. The primary focus is on the dimeric prodrug (NMXBIC), which achieves ultra-long-acting (ULA) antiretroviral drug exposure with a short pharmacokinetic (PK) tail, addressing a key limitation of current long-acting HIV therapies. The study employs comprehensive in vitro and in vivo methods, including pharmacokinetics, biodistribution, efficacy, and safety assessments in rodent and rabbit models. The development of a dimeric BIC prodrug (NMXBIC) with a short PK tail is a significant advancement, potentially overcoming the issue of prolonged subtherapeutic drug exposure seen with existing long-acting injectables. The authors thoroughly characterize the synthesized prodrugs using NMR, FT-IR, mass spectrometry, and XRD, and assess their physicochemical properties, hydrolysis profiles, and antiretroviral activity in vitro.\nNotwithstanding the promising data of the prodrugs shown by the authors, a few key experiments are missing to ensure that the conclusions drawn by the authors are valid:\n- Statistical tests are completely absent from the manuscript. These need to be conducted, without them any comparative statements should be removed throughout.\n- While the manuscript demonstrates sustained BIC plasma levels and in vitro antiviral activity, there is no in vivo efficacy data (e.g., viral load reduction in infected animal models). Such data would strengthen the translational impact and provide direct evidence of therapeutic benefit\n- The manuscript references other long-acting BIC formulations but does not provide direct head-to-head experimental comparisons in their models. Including such data is necessary to contextualize the advantages of the here developed prodrugs more clearly.\n- While the study infers hydrolysis mechanisms from plasma/tissue BIC levels, this is insufficient evidence for the metabolic fate and prodrug release. Identification of hydrolysis intermediates (e.g., monomeric BIC-lipid conjugates for MXBIC) should be conducted. Enzyme-specific hydrolysis assays (e.g., carboxylesterase knockdown models) would further delineate esterase specificity of prodrug release and indicate tissue selectivity of the prodrugs. Biliary/fecal excretion profiles should be conducted in vivo to rule out intact prodrug elimination and therefore incomplete prodrug conversion in vivo.\n- While prodrug stability was assessed room temperature, it should be investigated at accelerated conditions (e.g., 50°C/75% RH) to further delineate stability. It would also be important to assess stability under clinical-grade sterilization (e.g., autoclaving, filtration) to assess the clinical translatability of the developed prodrugs and formulations.\n- While injection site reactions were minimal to moderate in rabbits, the long-term implications of repeated dosing, especially in humans, warrant further discussion in the manuscript.\nMinor comments:\n- The brackets indicating the extended tail seem to be missing in Figure 1B for MXBIC.\n- Error bars should be shown as SD instead of SEM in several of the line plots to more clearly indicate experimental variability, e.g. Fig 1E, 1F, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B.\n- Standard deviations of PK parameters should be indicated in the Tables in Fig 4C and 5C."}, "author_response": "Re: NCOMMS-25-08967, “An Ultra-Long-Acting Dimeric Bictegravir Prodrug Defined by a Short\nPharmacokinetic Tail”\nDear Reviewers,\nOn behalf of the authors, I thank you for the thorough and reasoned review of our manuscript titled, “An\nUltra-Long-Acting Dimeric Bictegravir Prodrug Defined by a Short Pharmacokinetic Tail.” We are\npleased to respond to each of the queries raised point-by-point. The required amendments made in the\ntext are highlighted in yellow.\nReviewer 1\n“This is a nice study that reports the chemical production and experimental assessment of two new\nprodrugs of bictegravir with improved pharmacokinetics, as longer half-life and shorter tails. In animal\nmodels, NMXBIC provided plasma inhibitory concentrations sufficient to be administered twice a year.\nFurthermore, its short PK tail would allow late-dose forgiveness, which is a concern with the currently\navailable ultra-long-acting cabotegravir. Given that bictegravir (BIC) has a good safety profile and a\ngreater resistance barrier than cabotegravir, it would be more appealing than cabotegravir for both HIV\nprevention and treatment. Thus, further clinical development of NMXBIC is required. The research team\nhas long experience in the development of ultra-long-acting drugs and has pioneered several\nformulations in the HIV and hepatitis fields.”\nResponse: Thank you for your thoughtful comment. We certainly agree.\nPoint 1. “The discussion is quite simple and would benefit from expanding the scenarios for HIV treatment\nand prevention with the advent of a 6-month potent antiviral such as NMXBIC. Specific comments for the\ndiscussion section…”\nResponse: Thank you. We have addressed each comment in the revised manuscript (Please refer to lines\n402-411 on page 18 of the main manuscript text).\nPoint 2. Drug resistance. “From a clinical standpoint, there are two major differences between\ncabotegravir (CAB) and bictegravir (BIC). The first is the barrier to resistance caused by HIV-1 infection.\nWhereas HIV integrase mutations Q148R and N155H may compromise the activity of cabotegravir,\nthey have not demonstrated to reduce bictegravir efficacy in vivo.”\nResponse: We agree! CAB has been reported to have a lower genetic barrier to resistance compared\nto BIC and DTG [1]. Specific integrase mutations linked to CAB resistance cluster at positions 74,\n97, 138, 140, 147, 148, 155, and 263; combinations of these mutations (e.g., Q148H/K/R with G140A/S\nand/or E138A/K, or N155H with secondary changes) are typically required for high-level CAB resistance\n[2]. In contrast, BIC retains activity against a broader range of single, double, and triple INSTI mutants\ncompared with dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL) [1] Clinically, fixed-dose\nBIC/FTC/TAF has shown durable efficacy and safety in virologically suppressed individuals, including\nthose with pre-existing NRTI resistance (e.g., archived M184V/I), with no treatment-emergent resistance\nobserved across randomized trials and pooled analyses with multi-year follow-up [3, 4]. These strengths\nhave been referenced in the discussion section (please refer to lines 409-411 on page 18 of the main\ntext).\nPoint 3. Drug interactions. “The second difference between CAB and BIC is their metabolism. CAB is\nlargely catabolized in the liver by UGT1A1, and to a lesser extent by CYP3A4. Rifampin may induce its\nmetabolism. Overall the potential for drug interactions is lower with BIC.”\nResponse: We agree with the reviewer’s comment on the differences in metabolic pathways of CAB and\nBIC, which affect their susceptibility to drug-drug interactions such as CYP3A4 inducer. Several studies\nhave shown that BIC is metabolized by cytochrome P450 (CYP)3A and uridine diphosphate-\nglucuronosyltransferase UGT1A1. Co-administration of BIC with strong CYP3A and UGT1A1 inducers\nsuch as atazanavir, nilotinib, and rifampicin is contraindicated [5]. Strong UGT1A1 inducers likewise\ndecrease CAB concentrations [6]. Thus, although the metabolic mechanisms for BIC (dual CYP3A +\nUGT1A1) and CAB (predominantly UGT1A1) slightly differ, co-administration of rifampin with these\nagents significantly lowers their concentration and dose adjustment is required.\nPoint 4. Drug volume. “The new BIC prodrug, NMXBIC, requires low-volume nanosuspensions and may\novercome one of the major side effects of current parenteral long-acting cabotegravir formulations, which\nare local pain at injection sites. They may extend for a few days, with common development of induration\nand nodules. The data presented by the author’s testing 1 ml injection site reactions of NMXBIC in the\nNew Zealand rabbit model support its favorable safety profile.”\nResponse: Agree! The discussion section has been amended to reflect this added advantage of NMXBIC\nformulations (please refer to lines 402-411 on page 18 of the main manuscript text).\nPoint 5. PK tail. “The long PK tail of long-acting CAB accounts for prolonged sub-therapeutic drug\nconcentrations when timely dosing is forgotten, favoring the development of drug resistance. In contrast,\nthe new bictegravir prodrug NMXBIC depicts a shorter PK tail and may allow dosing forgiveness with\nlesser risk.”\nResponse: We agree! The prolonged PK tail of ULA formulations is a concern in chronic viral infections\nsuch as HIV, where subtherapeutic exposure may facilitate resistance selection. Contemporary LA\ndesign efforts therefore aim to limit tail duration or otherwise mitigate tail-related risk. The novelty of\nNMXBIC is its ability to maintain biannual therapeutic concentrations while exhibiting a shorter terminal\ntail. For context, LA CAB has reported tail durations of approximately 43.7 weeks in males and 67.3\nweeks in females, which can complicate missed-dose management and regimen switches [7]. By\ncontrast, the shorter tail observed with NMXBIC may offer added flexibility for adherence lapses and\ntherapeutic transitions, without compromising LA coverage. We have revised the discussion section to\nreflect this strength (please refer to lines 402-411 on page 18 of the main manuscript text).\nPoint 6. Combination options. “The opportunity to combine the new BIC prodrug NMXBIC with\nlenacapavir, which is prescribed subcutaneously every 6 months for HIV treatment, opens a further\nclinical scenario for NMXBIC. Since HIV treatment with long-acting CAB-RPV every 2-months has\nrevolutionized HIV therapeutics, the advent of a new treatment option every 6 months will definitively\ntransform the HIV field and improve patient’s management.”\nResponse: Agree! The discussion section has been revised to reflect this potential benefit (please refer to\nlines 412-416 on page 18 of the main manuscript text)).\nPoint 7. Pre-exposure prophylaxis. “The new NMXBIC formulation given twice a year should be more\nappealing than currently approved options, making easier PrEP indication in some developing regions\nwith high HIV endemicity, as in Sub-Saharan Africa, where 25 out of the 40 million HIV-positive persons\nlive.”\nResponse: Thank you for outlining the potential strengths of an ultra-long acting bictegravir formulation.\nWe have incorporated these points in the discussion section (please refer to lines 402-411 on page 18\nof the main manuscript text).\nReviewer 2\nPoint 1. “This manuscript by Nayan et al describes the design, synthesis, and in vitro and in vivo\nevaluation of novel monomeric and dimeric bictegravir (BIC) ester prodrugs formulated as injectable\nnanosuspensions. The primary focus is on the dimeric prodrug (NMXBIC), which achieves ultra-long-\nacting (ULA) antiretroviral drug exposure with a short pharmacokinetic (PK) tail, thus addressing a key\nlimitation of current long-acting HIV therapies. The study employed comprehensive in vitro and in vivo\nmethods, including pharmacokinetics, biodistribution, efficacy, and safety assessments in rodent and\nrabbit models. The development of a dimeric BIC prodrug (NMXBIC) with a short PK tail is a significant\nadvancement, potentially overcoming the issue of prolonged subtherapeutic drug exposure observed\nwith existing long-acting injectables. The synthesized prodrugs were thoroughly characterized using\nNMR, FT-IR, mass spectrometry, and XRD, and their physicochemical properties, hydrolysis profiles, and\nantiretroviral activities were assessed in vitro. Notwithstanding the promising data of the prodrugs shown\nby the authors, a few key experiments are missing to ensure that the conclusions drawn by the authors\nare valid:”\nResponse: Understood and appreciated. As suggested, these comments have been addressed in detail.\nThe missing few experiments were focused on data analysis and interpretations, and each were corrected\nincluding the statistical tests, comparisons between BIC formulations. A revised discussion was made\nand highlighted with yellow mark.\nPoint 2. “The statistical tests were completely absent from the manuscript. These need to be conducted,\nwithout them any comparative statements should be removed throughout.”\nResponse: All statistical tests were conducted. The completed test results are now contained in revised\nfigures (please refer to the legends of Figures 1, 3, 4, 5 in the main text and lines 198-199, 219-220, 249-\n253 and lines 740-743 of the main text).\nPoint 3. “While the manuscript demonstrates sustained BIC plasma levels and in vitro antiviral activity,\nthere is no in vivo efficacy data (e.g., viral load reduction in infected animal models). Such data would\nstrengthen the translational impact and provide direct evidence of therapeutic benefit”\nResponse: We agree and have added new data demonstrating efficacy of bictegravir monotherapy in a\nmouse model of HIV infection. Proof-of-concept viral restriction study in hu-PBL mice demonstrated that\nsingle IM doses of 50, 100, and 200 mg BIC. eq./kg of MXBIC monotherapy sustain suppression of\nplasma viral load. This is remarkable compared to our prior observations with cabotegravir injectable in\nthis mouse model (Biomaterials 151, 53-65 (2018). (Please refer to lines 314-320 and 417-425 of the\nmain text. Supplementary Figure 24 demonstrates proof of concept efficacy data in NSG PBL humanized\nmice)\nPoint 4. “The manuscript references other long-acting BIC formulations but does not provide direct head-\nto-head experimental comparisons in their models. Including such data is necessary to contextualize the\nadvantages of the here developed prodrugs more clearly.”\nResponse: We thank the reviewer for this important suggestion. However, direct head-to-head\ncomparison of proprietary drug delivery technologies described by others in our models is not feasible.\nNMXBIC and NM2BIC formulations are highly differentiated from those referenced. Our injectable\nprodrug formulations are aqueous nanosuspensions of chemically distinct compounds (prodrugs) versus\ndevice or polymeric based nanoparticles loaded with native bictegravir. We also note that other\ndifferences are linked to (i) primary depot compartments for each technology (intradermal vs.\nintramuscular), (ii) drug release mechanisms (polymer erosion/diffusion vs. particle and prodrug\ndissolution/hydrolysis), and (iii) lymphatic trafficking. However, we reiterate that animal studies to date\nsupport significantly longer half-life and dosing interval, attenuated peak-to-trough pharmacokinetics, and\nwell tolerated injections for our aqueous based bictegravir prodrug formulations versus those described\nin the literature (lines 402-405 of the main text were rewritten with appropriate references to clarify prior\nalternative long-acting BIC approaches).\nPoint 5. “While the study infers hydrolysis mechanisms from plasma/tissue BIC levels, this is insufficient\nevidence for the metabolic fate and prodrug release. Identification of hydrolysis intermediates (e.g.,\nmonomeric BIC-lipid conjugates for MXBIC) should be conducted. Enzyme-specific hydrolysis assays\n(e.g., carboxylesterase knockdown models) can further delineate the esterase specificity of prodrug\nrelease and indicate the tissue selectivity of the prodrugs. Biliary/fecal excretion profiles should be\nconducted in vivo to rule out intact prodrug elimination and therefore incomplete prodrug conversion in\nvivo.”\nResponse: We agree that metabolic studies of new chemical entities is scientifically warranted and\nrequired; however, radiochemical synthesis of the lipids and prodrugs, and multi-matrix bioanalytical\nmethod development required for these studies is beyond the scope and goal of our preclinical study.\nThere are no FDA approved long acting bictegravir formulations and our study sought to identify an ultra-\nlong acting injectable bictegravir prodrug formulation with a short PK tail. Our prodrugs are transient and\nare efficiently hydrolyzed into bictegravir upon dissolution from the particle. Extensive hydrolysis studies\non similar prodrug nanocrystals from our laboratory have demonstrated equal involvement of both\nenzymatic and chemical hydrolysis in our ultra-long-acting prodrug activation to release parent drugs with\nknown metabolic pathway and biocompatible linkages [8-10]. Identification of intermediates, esterase\nmapping, will be addressed in follow-up studies with clinical grade compounds and formulations (please\nrefer to lines 121-122, 443-444 and 447-450 of the main text).\nPoint 6. “While prodrug stability was assessed room temperature, it should be investigated at accelerated\nconditions (e.g., 50°C/75% RH) to further delineate stability. It would also be important to assess stability\nunder clinical-grade sterilization (e.g., autoclaving, filtration) to assess the clinical translatability of the\ndeveloped prodrugs and formulations.”\nResponse: We appreciate this comment and agree that stability under accelerated conditions and\ncompatibility with clinical-grade sterilization are essential elements of a future CMC program. Our present\nwork, however, is a preclinical study focused on prodrug screening and identification of novel ultralong\nacting BIC formulations for future clinical development. NM2BIC and NMXBIC have been licensed TO\nExavir Therapeutics Inc. for clinical development. It is also worth noting that our aqueous prodrug\nnanosuspensions are compatible with terminal sterilization by gamma irradiation. This was demonstrated\nfor another licensed program from our laboratory that is undergoing clinical development.\nPoint 7. “While injection site reactions were minimal to moderate in rabbits, the long-term implications of\nrepeated dosing, especially in humans, warrant further discussion in the manuscript.”\nResponse: We have added a comment in the revised discussion-text to highlight the implications of\nrepeated dosing (please refer to lines 398-400 of the main text).\nMinor comments\nPoint 8. “The brackets indicating the extended tail seem to be missing in Figure 1B for MXBIC.”\nResponse: Corrected, thank you.\nPoint 9. “Error bars should be shown as SD instead of SEM in several of the line plots to more clearly\nindicate the experimental variability, for example, Fig 1E, 1F, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B.”\nResponse: We agree, thank you. Figures 1, 2, 3, 4 and 5 data of the main text are expressed as mean SD.\nPoint 10. “Standard deviations of PK parameters should be indicated in the Tables in Fig 4C and 5C.”\nResponse: We agree, thank you. The PK parameters for Tables in Fig 4C and 5C are now expressed as\nmean SD.\nReferences\n1. Smith, S.J., et al., Efficacies of Cabotegravir and Bictegravir against drug-resistant HIV-1\nintegrase mutants. Retrovirology, 2018. 15(1): p. 37.\n2. Parikh, U.M., C.A. Koss, and J.W. Mellors, Long-Acting Injectable Cabotegravir for HIV\nPrevention: What Do We Know and Need to Know about the Risks and Consequences of\nCabotegravir Resistance? Curr HIV/AIDS Rep, 2022. 19(5): p. 384-393.\n3. Sax, P.E., et al., High efficacy of switching to bictegravir/emtricitabine/tenofovir alafenamide in\npeople with suppressed HIV and preexisting M184V/I. Aids, 2022. 36(11): p. 1511-1520.\n4. Sax, P.E., et al., Switching to Bictegravir, Emtricitabine, and Tenofovir Alafenamide in Virologically\nSuppressed Adults With Human Immunodeficiency Virus. Clin Infect Dis, 2021. 73(2): p. e485-\ne493.\n5. Stader, F., M. Battegay, and C. Marzolini, Physiologically-Based Pharmacokinetic Modeling to\nSupport the Clinical Management of Drug-Drug Interactions With Bictegravir. Clin Pharmacol\nTher, 2021. 110(5): p. 1231-1239.\n6. Wang, H., et al., Drug-drug interactions of Integrase Strand Transfer Inhibitors among older\npeople living with HIV: Interazioni farmacologiche degli inibitori delle integrase tra le persone\nanziane che vivono con HIV. J HIV Ageing, 2022. 7(2): p. 29-36.\n7. Landovitz, R.J., et al., Tail-phase safety, tolerability, and pharmacokinetics of long-acting\ninjectable cabotegravir in HIV-uninfected adults: a secondary analysis of the HPTN 077 trial.\nLancet HIV, 2020. 7(7): p. e472-e481.\n8. Deodhar, S., et al., Transformation of dolutegravir into an ultra-long-acting parenteral prodrug\nformulation. Nature Communications, 2022. 13(1): p. 3226.\n9. Kulkarni, T.A., et al., A year-long extended release nanoformulated cabotegravir prodrug. Nature\nmaterials, 2020. 19(8): p. 910-920.\n10. Gautam, N., et al., Lipophilic nanocrystal prodrug-release defines the extended pharmacokinetic\nprofiles of a year-long cabotegravir. Nature Communications, 2021. 12(1): p. 3453."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed adequately all points noticed on the original submission.\nMy guess is that the manuscript can be accepted in its current form.", "reviewer_2": "The authors have made substantial improvements to the manuscript. The addition of in vivo efficacy data and the inclusion of statistical analyses throughout the figures and results markedly improve the transparency, rigor, and interpretability of the work. These additions strengthen the manuscript and provide much-needed validation for the claims of antiviral activity and pharmacokinetic performance.\nHowever, several areas where the authors indicate that additional work will be required are not sufficiently caveated or acknowledged in the main text. In particular:\n1. Comparative claims — While the rebuttal appropriately explains that head-to-head testing against other long-acting bictegravir formulations was not feasible, the manuscript still contains statements that could be interpreted as implying superiority of the current formulation. These claims should be tempered or explicitly qualified to clarify that no direct comparative studies were performed.\n2. Mechanistic interpretation of prodrug release — The manuscript continues to describe prodrug conversion and release as fully efficient, without adequately acknowledging the complexity of in vivo metabolic pathways and the absence of direct data on intermediates or enzyme-specific hydrolysis. The Discussion should explicitly note that while hydrolysis is supported by indirect evidence, a full mechanistic and metabolic characterization remains future work.\n3. Stability and CMC development — The authors’ rebuttal mentions that further accelerated stability, sterilization compatibility, and CMC characterization are planned, but these are not discussed in the manuscript. To present a balanced view of translational readiness, the text should acknowledge that such studies are essential future steps to fully derisk the compound for clinical development.\nOverall, the manuscript is now much improved in data quality and presentation, but it would benefit from clearer self-containment and transparency in delineating what has been demonstrated versus what remains to be established in future studies."}, "author_response": "Re: “An Ultra-Long-Acting Dimeric Bictegravir Prodrug Defined by a Short Pharmacokinetic Tail”\nDear Reviewer,\nOn behalf of the authors, I thank you for the review of our manuscript titled, “An Ultra-Long-Acting Dimeric Bictegravir Prodrug Defined by a Short Pharmacokinetic Tail.” We are pleased to respond to the queries raised point-by-point. The required amendments made in the text are highlighted in yellow.\nReviewer 2\nPoint 1. “Comparative claims — While the rebuttal appropriately explains that head-to-head testing against other long-acting bictegravir formulations was not feasible, the manuscript still contains statements that could be interpreted as implying superiority of the current formulation. These claims should be tempered or explicitly qualified to clarify that no direct comparative studies were performed.”\nResponse: Reference to prior alternative preclinical bictegravir formulations described in the International Journal of Pharmaceutics, 2023. 642: p. 123108 and Antimicrob Agents Chemother, 2021. 65(4) has been deleted from the discussion.\nPoint 2. “Mechanistic interpretation of prodrug release — The manuscript continues to describe prodrug conversion and release as fully efficient, without adequately acknowledging the complexity of in vivo metabolic pathways and the absence of direct data on intermediates or enzyme-specific hydrolysis. The Discussion should explicitly note that while hydrolysis is supported by indirect evidence, a full mechanistic and metabolic characterization remains future work.”\nResponse: Clarity has been provided in the revised manuscript discussion\nPoint 3. “Stability and CMC development — The authors’ rebuttal mentions that further accelerated stability, sterilization compatibility, and CMC characterization are planned, but these are not discussed in the manuscript. To present a balanced view of translational readiness, the text should acknowledge that such studies are essential future steps to fully derisk the compound for clinical development.”\nResponse: Clarity has been provided in the revised manuscript discussion"}]

Decision letter