Antiviral potency of long-acting islatravir subdermal implant in SHIV-infected macaques

Abstract

Treatment nonadherence is a pressing issue in people living with HIV (PLWH), as they require lifelong therapy to maintain viral suppression. Poor adherence leads to antiretroviral (ARV) resistance, transmission to others, AIDS progression, and increased morbidity and mortality. Long-acting (LA) ARV therapy is a promising strategy to combat the clinical drawback of user-dependent dosing. Islatravir (ISL) is a promising candidate for HIV treatment given its long half-life and high potency. Here we show constant ISL release from a subdermal LA nanofluidic implant achieves viral load reduction in SHIV-infected macaques. Specifically, a mean delivery dosage of 0.21 ± 0.07 mg/kg/day yielded a mean viral load reduction of −2.30 ± 0.53 log₁₀ copies/mL at week 2, compared to baseline. The antiviral potency of the ISL delivered from the nanofluidic implant was higher than oral ISL dosed either daily or weekly. At week 3, viral resistance to ISL emerged in 2 out of 8 macaques, attributable to M184V mutation, supporting the need of combining ISL with other ARV for HIV treatment. The ISL implant produced moderate reactivity in the surrounding tissue, indicating tolerability. Overall, we present the ISL subdermal implant as a promising approach for LA ARV treatment in PLWH.

Graphical Abstract

1. Introduction

Human immunodeficiency virus (HIV) is considered a chronic yet manageable disease owing to advances in antiretroviral therapy (ART). Strict adherence to ART ensures continued viral suppression in people living with HIV (PLWH) to preserve the immune function, reduce the risk of opportunistic diseases and prevent transmission to people without HIV. Despite encouraging progress in some regions, most countries fell short of the 2020 “90-90-90” target issued by the Joint United Nations Program on HIV/AIDS (UNAIDS). The goal was to have 90% of PLWH be aware of their status, 90% of those diagnosed with HIV be provided with ART, and 90% of those treated to achieve continued viral suppression[1]. By the end of 2019, only ~59% of PLWH globally had viral suppression[1]. This could be partially attributed to the unprecedented stressors to the global healthcare system during the coronavirus disease 2019 pandemic, which further exacerbated challenges to the HIV care continuum[2, 3]. Given that the progress to end AIDS as a public health threat is off-track[4], transformative innovations to improve diagnosis, ART access and continued medication adherence is warranted.

Treatment nonadherence remains a prevailing medical challenge, albeit not isolated to HIV care[5, 6]. For PLWH, poor adherence could lead to emergence of ART resistance, increased morbidity and mortality, progression to AIDS, and viral transmission to others[7]. Factors contributing to ART nonadherence include pill fatigue, forgetfulness, discordance with social activities and lifestyle, as well as stigma[8–10]. To this end, long-acting (LA) approaches for prolonged ART delivery duration could alleviate user-dependent dosing. In line with this, advances in drug delivery technologies have yielded LA ART innovations, inclusive of once-weekly oral pills, intramuscular, subcutaneous or intravenous injectables as well as subdermal implants[11].

Cabenuva^®, consisting of cabotegravir (CAB) and rilpivirine (RPV) combination, is the first LA ART injectable clinically approved for HIV treatment[12]. However, Cabenuva^® requires two separate large volume injections, causing pain at the site of administration[13]. The monthly or bi-monthly injection regimen necessitates more frequent clinic visits than those prescribed with conventional oral tablets[11]. Further, irretrievability after administration poses challenges in the event of adverse side effects necessitating treatment discontinuation. Lenacapavir, a LA ART, was recently approved for heavily pre-treated individuals with multidrug resistant HIV, in combination with other approved ART (NCT04150068, NCT04143594)[11, 14]. Lenacapavir is administered orally during initiation phase followed by maintenance dosing twice yearly via subcutaneous injection.

Given its long intracellular half-life (~128.0 hrs), high potency at low doses, in vitro activity against multi-drug resistant strains, islatravir (ISL) is a candidate for LA HIV treatment[15–19]. ISL is clinically investigated as a once-weekly oral pill in combination with other ART such as lenacapavir (NCT05052996) or ulonivirine (MK-8507; NCT04564547). In individuals at risk of contracting HIV, a LA subdermal ISL-eluting implant has demonstrated preventive efficacy[20]. LA ART implants offer the convenience of constant delivery at a lower daily dose, avoidance of large injection volumes or long pharmacokinetic tail[21], as well as retrievability (for non-biodegradable types)[22]. Although LA implants offer advantages over injectables, thus far, few are in development for HIV treatment[23, 24]. Most LA ART implants are in preclinical studies[25–27] or early stages of clinical development for HIV pre-exposure prophylaxis[19, 20, 28].

Here we investigated the treatment efficacy of LA ISL monotherapy in simian HIV (SHIV)-infected macaques via a refillable subdermal nanofluidic implant. In a one-month study, we evaluated plasma ISL and intracellular ISL-triphosphate (ISL-TP) concentrations in peripheral blood mononuclear cells (PBMC) and tissues of relevance to HIV transmission. At the same time, we monitored the logarithmic viral load reduction in SHIV-infected NHP. Further, we histologically assessed the surrounding fibrotic capsule (FC) after one month of continuous ISL release in evaluation of implant tolerability. Overall, we present the potential of our nanofluidic technology to serve as a LA platform for HIV treatment.

2. Materials and Methods

2.1. Nanofluidic implant assembly

Refillable oval-shaped medical-grade 6AI4V titanium implants measuring 20 × 13 × 4.5 mm (length × width × height) were machined at the Houston Methodist Research Institute Machine Shop. The silicon membrane harbored nanochannels sized ~260 nm was glued to the implant using UV epoxy (OG116, Epoxy Technologies, Inc.) and cured with a UV lamp (UVL-18, UVL). Implants were assembled and primed with sterile 1X PBS as previously described[6] and were gamma sterilized at 30 kGy (VPTRad). Implants were maintained in sterile 1X PBS in a hermetically sealed container until implantation. ISL was transcutaneously loaded as previously described (ref ISL efficacy) immediately after implantation. The treatment nanofluidic implants loaded with ISL are referred to as nISL_t. ISL was purchased from MedChemExpress.

2.2. Ethics statement

All animal procedures were conducted at the AAALAC-I accredited Michale E. Keeling Center for Comparative Medicine and Research, the University of Texas MD Anderson Cancer Center (UTMDACC), Bastrop, Texas. All animal experiments were carried out according to the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, and the principles of the NIH Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Institutional Animal and Care and Use Committee (IACUC) at UTMDACC (IACUC #00001749-RN00). Indian rhesus macaques (Macaca mulatta: n=8; 2 males and 6 females) of 2–4 years and 2–5 kg bred at this facility were used in the study. All animals had access to clean, fresh water always and a standard laboratory diet. Euthanasia of the non-human primates (NHP) was performed using humane practices (IV pentobarbital) recommended by the American Veterinary Medical Association Guidelines on Euthanasia. Further, the senior medical veterinarian confirmed euthanasia by the absence of heartbeat and respiration.

2.3. Study population

The NHPs used in this study were enrolled in a SHIV prevention study as controls (n=8). Once a week, these NHPs were rectally (males) or vaginally (females) challenged with SHIV_SF162P3 for up to 10 weeks until initial detection of plasma SHIV RNA.[35] NHPs were weekly monitored for SHIV infection and infection was confirmed after a consecutive detection of plasma SHIV RNA. After 5 rectal and 2 vaginal exposure challenges, 100 percent of NHPs, tested SHIV-positive.[35] Upon confirmation of viral infection, animals were transferred to this treatment study. NHPs were implanted with nISL_t a couple of weeks after confirmed infection via a minimally invasive 1 cm dorsal skin incision on the right lateral side of the thoracic spine as previously described.[56]

2.4. Blood collection and plasma and PBMC sample preparation

Blood collection and sample preparation were performed as previously described[24, 25, 35] to assess plasma viral load and intracellular ISL-TP concentrations in PBMC. Briefly, blood was drawn in EDTA-coated vacutainer tubes before implantation and then weekly for up to a month. Plasma was isolated from blood via centrifugation at 1,200 × g for 10 min at 4°C and stored at −80°C until analysis. Afterwards, PBMC were separated from the residual drug by standard Ficoll-Hypaque centrifugation with over 95% cell viability. Subsequently PBMC were counted and centrifuged at 400 × g for 10 min at 4°C. Next, PBMC pellet was lysed in 500 μL of cold methanol/water (70%/30%, v/v) and stored at 80°C until analysis.

All analyses were performed at the CAVP laboratory at University of Colorado Anschutz Medical Campus. A reversed-phase ultra-performance liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) assay for the determination of ISL in NHP plasma was developed. The method utilizes a stable labeled internal standard for ISL and a liquid-liquid extraction. The assay has a quantifiable range of 0.025 ng/mL to 100 ng/mL when 0.2 mL of plasma is analyzed. Intracellular ISL-TP concentrations in PBMCs were quantified using a reversed-phase ultra-performance liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) assay. Typically, 10 fmol/sample was used as the lower limit of quantitation (LLOQ). If additional sensitivity was needed, standards and quality controls were added down to 5 fmol/samples. The following tissues were biopsied: inguinal lymph nodes, rectum and vagina. Tissues were snap-frozen and stored at −80 °C until further analysis of ISL-TP concentrations. Inguinal lymph nodes, rectum, vagina and FC were homogenized, and 50- to 75-mg aliquots were used for ISL-TP quantitation. Tissue ISL-TP concentrations were quantified using a reversed-phase ultra-performance liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) assay.

2.5. Infection monitoring by SHIV RNA in plasma

Infection was monitored by detection of SHIV RNA in plasma using a modification[56] of previously described methods[57, 58]. Infections were confirmed after a consecutive plasma viral load assay. All samples were tested in duplicate reactions and plasma viral loads were reported as viral RNA (vRNA) copies/mL of plasma. Standard curves were generated with ten-fold serial dilutions (1 to 1 × 10⁶ copies per reaction) of an in vitro transcribed SIV gag RNA. The assay was considered positive above the 50 copies/mL limit of detection.

2.6. SHIV drug resistance genotyping

Virion-associated RNA was isolated from plasma of infected animals using the Qiagen Ultrasens viral RNA isolation kit according to the manufacturer’s protocol. First-strand cDNA was then synthesized using ThermoFisher Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR (Cat no. 18080-051) and the manufacturer’s random hexamers protocol.

To clone the reverse transcriptase (rt) gene sequence, nested PCR was performed. For round 1 PCR, 2.5 microliters of each cDNA sample was used per reaction. Each 25 microliter reaction contained 400 nM of each primer (SHIV1598: 5’-ACAGAGGATTTGCTGCACCT-3’; and SHIV2895: 5’-CGTGGCTTCTAATGGCTTGC-3’), 2 mM MgSO₄, 0.2 mM dNTPs, and 1X HiFi PCR buffer, and 1 unit of Platinum Taq DNA polymerase HiFi (ThermoFisher Invitrogen, Cat. no. 11304-011). A 3-step cycle program was used for round 1 PCR amplification of rt: 1 cycle of 94°C for 2 min, followed by 35 cycles of 94°C for 15 sec, 55°C for 30 sec, and 68°C for 2 min; and then a final hold of 68°C for 7 min. Upon completion, 2.0 microliters of the 1^st round PCR samples were used in a second round PCR with primers SHIV1885 (5’-ACAGCTCTGGGGATGTCTCT-3’) and SHIV2618 (5’-GGCCACAATTCGTACCCCAT-3’). Primer concentrations and buffer concentrations were the same as in round 1. PCR cycling for round 2: 1 cycle of 94°C for 2 min, followed by 35 cycles of 94°C for 15 sec, 55°C for 30 sec, and 68°C for 1 min; and a final hold of 68°C for 7 min. PCR was performed using three replicates of each sample. Each PCR sample was analyzed using 0.8% agarose gel electrophoresis in 40 mM Tris-acetate, 1 mM EDTA buffer (1X TAE) for the expected 733 base pair product, and then the fragments were purified using the QIAquick PCR purification kit according to the manufacturer’s protocol (Qiagen, Cat. No. 28104). The DNA sequence of each fragment was determined by Sanger sequencing (Azenta) using the forward and reverse primers SHIV1885 and SHIV2618, respectively, and analyzed using MacVector 18.5 software.

2.7. Residual ISL quantification in drug reservoir

Residual ISL quantification in drug reservoir was done as previously described.[35] Upon explantation, the implants were frozen with dry ice. Prior to drilling, implants were wiped with 70% ethanol. A hole was drilled on the outermost corner on the membrane shell where the filter was not located using a 3/64 titanium drill bit with a stopper. Following drilling, the implants were placed in 50 mL conical tubes with 20 mL 70% ethanol. Each implant was flushed using a 19-gauge needle with 70% ethanol from the sink solution. For sterilization, the implants were incubated in 70% ethanol for 2 days. ISL did not fully solubilize with 70% ethanol, thus ethanol was evaporated under vacuum for 3 days. Afterwards, 5 mL of dimethylsulfoxide was added to each tube and tubes were left shaking at room temperature overnight. An aliquot was transferred to 0.2 μm nylon centrifugal filters and centrifuged at 20,000 × g for 5 minutes at room temperature. An aliquot of the filtered samples was further diluted in 1X PBS and analyzed via high-performance liquid chromatography (HPLC) on an Agilent Infinity 1260 system equipped with a diode array and evaporative light scattering detectors using a 3.5-μm 4.6 × 100 mm Eclipse Plus C18 column and water/methanol as the eluent and 25 μL injection volume. Peak areas were analyzed at 261 nm absorbance.

2.8. Assessment of treatment nISL tolerability

Skin tissues were fixed in 10% buffered formalin and stored in 70% ethanol until analysis. Afterwards, tissues surrounding the implant site were embedded in paraffin, cut into 5 μm sections and stained with hematoxylin and eosin (H&E) at the Research Pathology Core HMRI, Houston, TX, USA. Semiquantitative histopathological assessment of inflammation surrounding the implant site was performed by two-board certified pathologists from two different institutions blinded to the group: Michael Ittmann, M.D. Ph.D. from Baylor College of Medicine and Andreana L. Rivera, M.D. from Houston Methodist Hospital. The pathologists scored all slides using the inflammation scoring system Su et al.[59] adapted from the ISO published standard[60] (Table S1, Supplementary Materials). Briefly, samples are scored based on the presence of inflammatory cell and tissue characteristics. Afterwards, scores reported by both pathologists were averaged per implant (Supplementary Table S1) to calculate the total histological characteristic score for the nISL_t group. Equation 1 from Su et al.[59] was used to calculate the implant reactivity grade. Next, the average placebo-adjusted implant reactivity score (S_pair) was computed by subtracting the implant reactivity grade of nPBS[56] from nISL_t. nPBS was the control implant loaded with PBS used in these same NHPs in the SHIV prevention study.[56] The S_pair classification of nISL_t was determined per the published standard: minimal to no reaction (S_pair from 0.0 to 2.9), slight reaction (S_pair from 3.0 to 8.9), moderate reaction (S_pair from 9.0 to 15.0), and severe reaction (S_pair > 15.1).[59, 60]

FC thickness was assessed via Image J software on Masson trichrome slides. Ten measurements were taken on the membrane side and opposite side for each slide. Masson Trichrome staining was used for analyzing percentage of collagen in an area of the FC. Seven images were taken at 40x magnification in different areas of the FC in contact with the membrane and on the opposite side per slide. Images were analyzed via a program on Matlab Software. Blood vessel quantification in the FC in contact with the membrane and opposite side was done using Image J software on a full slide scan.

2.9. Statistical analysis

All statistical analysis for calculation of the efficacy of ISL were performed with GraphPad Prism 9 (version 9.1.1; GraphPad Software, Inc., La Jolla, CA). Data are represented as mean ± SD with 95% CI between lower and upper limits; median with interquartile range (IQR) between the first (25th percentile) and third (75th percentile) quartiles. Correlations were calculated with Spearman correlation coefficients. Outliers were detected with the Grubbs test (α = 0.05) and removed from descriptive statistics. Paired t test was used to compare logarithmic viral load reductions to baseline. Statistical significance was defined as two-tailed p<0.05. Logarithmic plasma viral load reduction were fitted with a non-linear one phase decay model using GraphPad Prism. Data with fitting R² < 0.9 was excluded from further analysis.

3. Results

3.1. Refillable nanofluidic implant pharmacokinetics in SHIV-positive macaques

The refillable LA ART implant comprises of a titanium casing (20 mm × 13 mm × 4.5 mm) (Figure 1A–B) and a nanofluidic membrane (6 mm × 6 mm × 410 μm) (Figure 1B) diffusive drug release. Drug delivery from the implant is controlled by electrostatic and steric interactions between drug molecules and nanochannels within the membrane, conferring sustained zero-order release kinetics[29–33]. Scanning electron microscopy (SEM) imaging shows the array of nanochannels aligned within the nanofluidic membrane (Figure 1C), while atomic force microscopy (AFM) captures the surface topography of channel inlets (Figure 1D). Two resealable silicone ports are affixed on the upper hemishell of the implant allowing transcutaneous access to the drug reservoir via syringes[34]. A titanium filter is aligned under one of the silicone ports to facilitate solid drug loading and refilling, as needed (Figure 1A–B)[34].

Figure 1. nISL_t implant and plasma, PBMC and tissue distribution of ISL from subcutaneous nISL_t.

(A) Rendering of nISL_t cross-section depicting solid drug loading via syringes. (B) Picture of open implant showing titanium casing, refill ports: drug loading (l) and venting (v), sintered titanium filter (f) and nanofluidic membrane (m). (C) SEM of array of nanofluidic channels. (D) AFM image of the nanochannels. (E) Plasma ISL and (F) intracellular ISL-TP PBMC concentrations in NHP over 4 weeks. (G) Tissue ISL-TP concentrations upon euthanasia after 1 month of nISL_t implantation. NHP, nonhuman primate, ILN, inguinal lymph nodes. N=8, tissue concentration analyzed using mixed effect analysis, *p < 0.05.

The LA ART implant eluting ISL (nISL_t) was assessed for treatment efficacy against SHIV. Nonhuman primates (NHPs) were rectally (n=2; male) or vaginally (n=6; female) challenged via low-dose SHIV once weekly[35]. One week after confirmation of infection via two consecutive positive tests, each animal was subcutaneously implanted with nISL_t in the dorsum (denoted as day 0). The median viral load detected upon implantation was 1.55 × 10⁶ copies/mL (95% CI, 1.64 × 10⁵ to 2.94 × 10⁶ copies/mL). ISL plasma and ISL-TP PBMC concentrations were maintained at a median of 6.38 nM (IQR, 5.17 to 7.82 nM) (Figure 1E) and 0.33 pmol/10⁶ cells (IQR, 0.25 to 0.52 pmol/10⁶ cells) (Figure 1F), respectively, for the duration of the one-month study. A maximum ISL plasma concentration was observed in the first week after implantation at a median of 9.46 nM (IQR, 6.56 to 10.82 nM). Thereafter, we observed median ISL plasma concentrations of 6.66 (IQR, 4.79 to 9.22), 5.54 (IQR, 4.37 to 6.46) and 5.17 (IQR, 4.12 to 6.53) nM at 2, 3, and 4 weeks after implantation, respectively. Accordingly, a maximum ISL-TP PBMC concentration was observed in the first week after implantation at a median of 0.41 pmol/10⁶ cells (IQR, 0.30 to 0.61 pmol/10⁶ cells). Thereafter, we observed median ISL-TP PBMC concentrations of 0.37 (IQR, 0.26 to 0.62), 0.35 (IQR, 0.3 to 1.26) and 0.24 (IQR, 0.20 to 0.27) pmol/10⁶ cells at 2, 3, and 4 weeks after implantation, respectively.

We measured ISL-TP concentrations in tissues relevant to HIV replication or reservoir after study termination at week 4 (n=8) (Figure 1G). We assessed inguinal lymph nodes (ILN; n=8), rectum (n=8), cervix (n=6), and adipose tissues (n=8). After a month of continuous subcutaneous ISL delivery, median ISL-TP concentrations were similar across rectal, cervical, and adipose tissues: 6.05 fmol/mg (IQR, 3.81 to 11.75), 5.41 fmol/mg (IQR, 3.52 to 7.47) and 6.99 fmol/mg (IQR, 3.75 to 10.11). Notably, the highest median ISL-TP concentrations were observed in the ILN: 18.02 (IQR, 12.72 to 28.21) fmol/mg.

3.2. nISL_t treatment viral load reduction

To evaluate efficacy of nISL_t, we assessed the logarithmic viral load reduction in SHIV-infected NHP. Figure 2A shows the weekly plasma viral load for individual NHP, while Figure 2B shows the reduction in viral load from baseline. After a week of constant subcutaneous ISL delivery, the first-phase change in plasma viral RNA (vRNA) compared to baseline was assessed individually for each animal. NHP 1 through NHP 8 had a first-phase vRNA decay slope of −2.26, −2.44, −2.06, −2.53, −2.14, −1.07, −1.82 and −1.70 log₁₀ copies/mL, respectively. Overall, the mean first-phase change in vRNA was −2.00 ± 0.47 log₁₀ copies/mL (95% CI, −2.40 to −1.61 log₁₀ copies/mL;).

Figure 2. Plasma viral load in nISL_t (n=8).

(A) Absolute value and (B) change in SHIV RNA from baseline (log₁₀ copies/mL) at weeks 1, 2, 3 and 4 of continuous subcutaneous ISL dosing via nISL_t. Data shows individual NHP.

After 2 weeks of sustained ISL delivery, the mean viral load reduction compared to baseline was −2.30 ± 0.53 log₁₀ copies/mL (95% CI, −2.74 to −1.86 log₁₀ copies/mL; p<0.0001). By week 3, the mean change in vRNA from baseline was −2.34 ± 0.71 log₁₀ copies/mL (95% CI, −2.93 to −1.74 log₁₀ copies/mL; p<0.0001). At study endpoint on week 4, SHIV RNA log₁₀ viral reduction was −2.29 ± 1.13 log₁₀ copies/mL (95% CI, −3.24 to −1.34 log₁₀ copies/mL; p=0.0007). In NHP 7 and 8, the increase in vRNA at week 4 compared to week 3 alluded to viral rebound.

In addition, a negative correlation coefficient between plasma ISL concentration and plasma viral load was observed in all NHP (Figure S1 A–H, Supporting Information). As expected, the viral load decreased at increasing plasma ISL concentration. Four of eight had a statistically significant Spearman correlation coefficient (Figure S1 A–H, Supporting Information). Likewise, a negative correlation coefficient between ISL-TP PBMC concentration and plasma viral load was also observed in all NHP (Figure S1 I–P, Supporting Information), even though not significant. Moreover, as all animals showed a decrease in viral load, we couldn’t determine a cut-off in either ISL plasma concentration or ISL-TP concentration in PBMCs values that elicits a viral load decrease.

The logarithmic reduction of plasma viral load from baseline at week two was correlated with ISL dose (Figure 3A), average plasma ISL concentration (Figure 3B, first 2 weeks) and ISL-TP AUC in PBMCs (Figure 3C, first 2 weeks), where each datapoint belongs to one animal. Plasma ISL concentration showed the strongest correlation (−0.65) with viral load reduction, although not significant (p = 0.081). The vRNA change from baseline (up to week 3) was fitted with a non-linear one phase decay model and the extracted span and rate constant (K) were correlated with ISL dose (Figure 3D and 3G), average plasma ISL concentration (Figure 3E and 3H, first 3 weeks) and ISL-TP AUC in PBMCs (Figure 3F and 3I, first 3 weeks). The span, indicative of the overall vRNA reduction, showed strong correlation with all investigated variables and was significant for ISL dose (p = 0.033) and plasma ISL concentration (p = 0.021).

Figure 3. Viral load correlation with ISL dose, plasma and ISL-TP PBMC concentration.

Logarithmic viral load reduction from baseline at week 2 correlated with (A) ISL dose (mg/kg/day), (B) average plasma ISL (first 2 weeks), (C) ISL-TP AUC in PBMCs (first 2 weeks). Logarithmic viral load reduction from baseline up to week 3 were fitted using a one phase decay model: rate constant was correlated with (D) ISL dose, (E) average plasma ISL (first 3 weeks), and (F) ISL-TP AUC in PBMCs (first 3 weeks); Span was correlated with (G) ISL dose, (H) average plasma ISL (first 3 weeks), and (I) ISL-TP AUC in PBMCs (first 3 weeks). Data analyzed with Pearson correlation two-tailed P-value, *P<0.05. Each point in the graph represents one animal.

3.3. SHIV resistant mutations

Two SHIVSF162P3 infected animals (NHP 7 and NHP 8) showed increase in plasma viral load at 3- and 4-weeks post-nISL_t implantation (Figure 2B). These results suggested the possibility of emergent drug resistant variants in response to ISL monotherapy. To determine if the rebound in plasma viral RNA were associated with drug resistance, we examined the sequence of a 733 base pair region of the rt gene that includes highly conserved motifs (Y183, M184, D185, D186), which constitutes part of the polymerase active site of reverse transcriptase (RT). At the 4-week post-implant, when the plasma viral RNA was 124,164 and 22,752 copies/mL for NHP 7 and NHP 8, respectively, we observed nucleotide changes of A to G at position 550 of rt in both animals. This substitution results in an amino acid change from M to V at position 184 of RT (M184V). Of relevance, M184V mutation was detected in SIV-infected NHP treated with weekly oral ISL[36]. Specifically, oral dosing occurred on days 0 and 7, and M184V mutation emerged on day 14 regardless of the administered dose (1.3 mg/kg, 3.9 mg/kg, 13 mg/kg or 18.2 mg/kg) These results further indicate that M184V mutation is associated with viral rebound during ISL monotherapy.

3.4. Residual ISL in drug reservoir

To evaluate the release rate of nISL_t throughout one month of subcutaneous implantation, we measured residual drug within the implant (Table 1). Residual drug within the implant ranged 14–59% of the initial loaded amount. The nISL_t implants had a mean release rate of 0.79 ± 0.26 mg/day and achieved the highest mean viral load reduction of 2.30 ± 0.53 vRNA log₁₀ copies/mL at week 2.

Table 1.

nISL_t drug residual after 1-month implantation.

nISLt (NHP #)	Sex	Weight (kg)	Loaded ISL(mg)	Residual ISL (mg)	ISL release rate (mg/day)	Dose (mg/kg/day)	Plasma concentration (nM)	ISL-TP in PBMC (pmol/106 cells)	Viral load decay at week two
1	F	3.6	31.55	12.12	0.64	0.18	7.89	0.62	−2.15
2	F	4.1	33.74	20.02	0.45	0.11	5.65	0.33	−1.58
3	F	4.1	36.00	10.16	0.85	0.21	4.58	0.30	−1.70
4	F	3.5	34.69	13.10	0.71	0.20	8.17	0.42	−2.41
5	M	4.0	41.11	10.77	0.99	0.25	8.48	0.67	−3.25
6	F	3.6	36.49	8.29	0.92	0.26	5.89	0.18	−2.65
7	M	3.9	43.83	6.21	1.23	0.32	7.91	1.26	−2.30
8	F	4.1	35.43	19.74	0.51	0.12	5.54	0.30	−2.37
Mean ± SD		3.86 ± 0.26	36.61 ± 3.99	12.55 ± 5.00	0.79 ± 0.26	0.21 ± 0.07	6.76 ± 1.50	0.51 ± 0.35	−2.30 ± 0.53

3.5. Histological assessment of nISL_t tolerability

To assess nISL_t tolerability, we histologically examined the tissue and FC surrounding the implants (n=8) after a month of subcutaneous ISL delivery via hematoxylin and eosin (H&E) analysis (Figure 4). nISL_t had a total histological characteristic score of 11.50 ± 2.35 (scale from 0 to 32) (Figure 4A, Table S1, Supporting Information), which was statistically different than that of PBS implants (p<0.0001). Further, nISL_t had an average implant reactivity score of 18.69 ± 3.80 (scale 0 to 56). The placebo-adjusted implant reactivity score (S_pair)[37] was calculated by subtracting the placebo (PBS) reactivity score from the treatment (nISL_t) reactivity score (Table 2). nISL_t had an S_pair score of 11, indicating a moderate reaction (Figure 4B). Of relevance, these results are congruent with our previous finding in healthy non-SHIV infected rhesus macaques: after 4 months of implantation, we observed histologically moderate reactions to ISL-eluting nanofluidic implants in male (S_pair: 9.21) and female (S_pair: 14.05) animals[35].

Figure 4. Histological characterization of inflammatory response to nISL_t in NHPs.

(A) Total histological score of fibrotic capsule (FC) surrounding PBS and nISL_t implant. (B) Placebo-adjusted implant reactivity score (S_pair) of nISL_t FC after a month of subcutaneous ISL delivery. S_pair values 0.0–2.9 (no reaction, green), 3.0–8.9 (slight reaction, yellow), 9.0–15.0 (moderate reaction, orange), and >15.1 (severe reaction, red). (C) Representative Masson Trichrome image of FC showing the membrane side in contact with nISL implant (“M”) and the opposite side (“O”). (D) 40 × magnification of red box outline of M FC. Comparison of (E) FC thickness, (F) percentage of collagen and (G) quantification of blood vessel lumens in M and O of PBS and nISL_t. (H) Fluorescence recovery after photobleaching (FRAP) Fluorescein isothiocyanate (FITC) diffusivity coefficient between PBS, nISL_t, subcutaneous tissue and free FITC groups. Data presented as median ± IQR.

Table 2:

nISL_t fibrotic capsule histopathological scoring

Implant	Total histological score (0–32)	Average implant reactivity score (0–56)	Spair	Reactivity grade
nISLt	11.5 ± 2.35	18.69 ± 3.80	11	Moderate reaction
PBS	4.90 ± 2.76	7.69 ± 4.58	-	-

The FC was stained with Masson Trichrome for further characterization (Figure 4C–D). The FC in contact with the membrane is designated as “M”, whereas the section opposite to the membrane is designated as “O” (Figure 4C). In terms of FC thickness, we noted M was significantly thicker than O for both nISL_t (M: 730.46 ± 248.63 μm versus O: 235.80 ± 166.11 μm; p<0.0001) and PBS (M: 239.73 ± 114.91 μm versus O: 86.73 ± 52.27 μm; p<0.0001) (Figure 4E). These results indicate that the outlet holes in the implant likely contributed to a higher degree of fibrotic response. Further, continuous drug exposure also likely triggered a more pronounced inflammatory response, as M was significantly thicker in nISL_t than PBS (p<0.0001).

In addition, the FC was assessed for increased collagen production, an indication of augmented foreign body reaction. No statistical difference (p=0.86) was observed in the percentage of collagen in M between nISL_t (48.09 ± 10.62%) and PBS (50.12 ± 16.86%) (Figure 4F). However, there was a significantly lower percentage of collagen in O: 34.21 ± 13.73% (nISL_t, p<0.0001) and 36.82 ± 17.06% (PBS, p<0.0001) compared to M in their respective groups.

The number of blood vessel lumens in M are 116.6 ± 50.85 lumens (nISL_t) and 75.67 ± 36.48 lumens (PBS) (p=0.097) (Figure 4G). Although not statistically significant, the increase in blood vessel lumens could be attributable to an inflammatory response to the drug. Fewer blood vessel lumens were observed in O: 43.75 ± 22.64 lumens (nISL_t, p=0.0023) and 42.08 ± 36.15 lumens (PBS, p=0.14) compared to M in respective group.

Fluorescence recovery after photobleaching (FRAP) was performed on FCs surrounding PBS and nISL_t to determine the diffusion coefficient. Fluorescein isothiocyanate (FITC) was used as a surrogate for ISL to analyze diffusivity through the FC. Mean diffusion coefficient values of 0.28 (SD, ± 0.047 × 10⁻⁶ cm²/s) and 0.20 (SD, ± 0.010 × 10⁻⁶ cm²/s) were obtained for PBS and nISL_t, respectively (Figure 4H). These values were compared to mean diffusivities in subcutaneous tissue, 1.09 (SD, ± 0.059 × 10⁻⁶ cm²/s), and free FITC, 0.34 (SD, ± 0.033 × 10⁻⁶ cm²/s) (Figure 4H). Statistical significance was observed between free FITC and all groups (p<0.0001). These results are consistent with our previous finding in healthy non-SHIV infected NHP[35]. ISL release is not impeded by FC formation as shown by similar diffusivity values to subcutaneous tissue (Figure 4E).

4. Discussion

In this study, we demonstrated that our subcutaneous ISL-eluting implant achieved viral load reduction in treatment-naïve SHIV-infected macaques. The implant rapidly achieved and sustained steady plasma ISL and ISL-TP PBMC concentrations throughout the study. The implant had a mean delivery dosage of 0.21 ± 0.07 mg/kg/day, which yielded a median ISL-TP PBMC concentration of 0.37 pmol/10⁶ cells (IQR, 0.26 to 0.62) by week 2. As a result, we observed a median viral load reduction of −2.30 ± 0.53 log₁₀ copies/mL (95% CI; −2.74 to −1.86 log₁₀ copies/mL).

In comparison, oral ISL delivered once-daily (QD, 0.19 mg/kg/day) or once-weekly (QW, 3.9 mg/kg/week) in NHP resulted in a mean ISL-TP PBMC concentration of 0.53 pmol/10⁶ cells[36]. Despite a three-fold difference in dosing, similar log₁₀ vRNA reductions of −1.67 and −1.54, were achieved for QD and QW dosing, respectively. Although the median ISL-TP PBMC concentration was lower in our study, we noted a higher median viral load reduction than oral QD and QW.

The drug potency of nISL_t, calculated as the ratio between log₁₀ vRNA reduction and ISL dosage (mg/kg/day), was 12.058 ± 3.72. Compared to similar treatment studies of infected NHP (Figure 5), nISL_t showed higher potency than oral ISL QD (p = 0.12, +37%) or QW (p = 0.0003, +337%), which were 8.78 ± 3.089 and 2.76 ± 1.041, respectively[36]. Considering complete bioavailability for both subcutaneous and oral delivery, these NHP results suggest that ISL potency increases at increasing frequency of administration.

Figure 5. Drug potency comparison in SHIV-infected NHP.

Drug potency was calculated as vRNA reduction / dose (mg/kg/day). The nISL_t subcutaneous (SQ) implant is compared to daily oral ISL (QD), weekly oral ISL (QW), Cabotegravir (CAB) LA intramuscular (IM) injections (3 injections 4 weeks apart) and, tenofovir alafenamide fumarate (TAF) SQ nanofluidic implant (nTAF_t). Data presented as mean ± SD.

In humans, a single oral ISL dose (0.5 mg) achieved a median ISL-TP concentration of 0.1 pmol/10⁶ PBMCs, which resulted in a median viral load reduction of −1.26 log₁₀ copies/mL (range −0.60 log₁₀ copies/mL; −1.62 log₁₀ copies/mL) on day 7[38]. Based on pharmacokinetic modelling from the literature[39], the same ISL-TP PBMC concentration could be achieved and maintained with a daily oral dose of 0.022 mg/day, corresponding to a three times lower total dose over a week. This is consistent with our NHP findings that increasing the frequency of ISL administration provides enhanced potency.

Next, we compared ISL potency to other ARVs. Specifically, we evaluated LA monotherapy studies where infected NHP were treated with cabotegravir (CAB)[40] or tenofovir alafenamide (TAF).[41] CAB LA intramuscular injection (50 mg/kg) achieved a maximum log₁₀ vRNA reduction of −4.1 on day 28, resulting in a calculated potency of 2.30, which is 5-fold lower than nISL_t. In our previous study, TAF was subcutaneously delivered using the nanofluidic implant (nTAF_t) at a dose of 0.83 mg/kg/day. nTAF_t achieved a maximum log₁₀ vRNA reduction of −1.37 at 2 weeks post-implantation[41]. nTAF_t potency was almost 9-fold lower (p<0.0001) than nISL_t.

The NHP in the studies above (Figure 5) were infected with different strains of virus, namely SHIVSF162P3 (in nISL_t and nTAF_t treatment studies[41]), SIVmac239 (in CAB LA study[40]) or SIVmac251 (in ISL QD and QW oral studies[36]). However, SIVmac239 was cloned from the biological isolate of SIVmac251, thus the RT sequence is nearly identical. Moreover, SHIVSF162P3 was generated by replacing the env gene of SIVmac239 with a cloned subtype B and C env, respectively; the RT of these strains are derived from SIVmac239. While SIVmac251 is considered to be more fit than either SHIV strains, a comparative study showed that at three weeks after infection (the time at which our device was implanted), SIVmac251 and SHIVSF162P3 achieved comparable plasma vRNA[42]. Although comparisons between these studies show a greater drug potency with nISL_t, further investigations using different strains are warranted to substantiate this finding.

Despite ISL high potency and its known intrinsic resistance to mutation, development of resistance can still occur. Resistance to ISL is caused by the M184V substitution, as shown in in vitro drug selection studies with HIV-1[43, 44]. However, other studies suggest that ISL remains effective against RT inhibitor resistant viruses.[45, 46] Here, we discovered in vivo selection of the ISL resistance mutation M184V in two rhesus macaques with therapeutic blood concentrations of ISL. This was not unique to our study as the emergence of M184V after 2 weeks of weekly oral ISL treatment was also noted in SIV-infected NHP[36]. Moreover, the emergence of M184V with high viral loads suggests the potential for this mutation, which is also selected by lamivudine (3TC) and emtricitabine (FTC), to impact the antiviral activity of ISL.

Studies have documented a progressive increase in the prevalence of transmitted drug resistance across several geographical regions[47–50]. This reinforces the need for therapeutic regimens where multiple ARVs targeting different pathways in the mechanism of infection are used in combination. In line with this, daily oral ISL and doravirine, a non-nucleoside RT inhibitor (NNRTI) with favorable resistance profile, demonstrated HIV-1 suppression in ARV treatment-naïve participants for 96 weeks in a phase 2b clinical trial (NCT03272347).[51] Nevertheless, for the rational design of ARV combinations, it is important to fully assess the therapeutic profile of individual ARVs, which is the purpose of the present study on ISL. Here we highlighted that ISL potency is substantially influenced by the route and frequency of administration. Favorable potency results were obtained with the subcutaneous nISL_t implant as compared to oral administration.

In SHIV-infected macaques, the nISL_t implant exhibited tolerability with no reported observed adverse events (AEs) such as impaired healing, swelling, edema, or irritation at the site of implantation. The foreign body reaction (FBR) at the tissues surrounding the implant was moderate. As expected, a thin FC developed around the device. While thicker at the membrane biotic/abiotic interface, the FC did not impede the ISL diffusion as shown by similar diffusivity values to subcutaneous tissues. This is important for LA implants designed for long-term deployment. Overall, these findings show similarities to the safety, tolerability and FBR obtained with nISL implant in healthy non-infected NHP as well as with an investigational polymeric ISL-releasing implant assessed in clinical trial in healthy individuals[19]. This is relevant as HIV infection leads to progressive immune system impairment, with depletion of CD4+ T cells, which are crucial for coordinating the immune response. HIV also affects macrophages, B cell, monocytes among other cells, which are involved in the FBR to implants[52].

While drug potency and safety profiles are key factors for translation, clinical impact depends on patient acceptability. In this context, LA platforms such as the nISL_t can eliminate barriers to the success of ARV treatment, including pill fatigue, forgetfulness, and lack of adherence. Of relevance, the nISL_t has demonstrated capability to release and maintain ISL concentrations for 20 months in NHP[35]. Additionally, the success of HIV treatment depends on life-long viral suppression. In this context, the refillability of the nISL_t implant could improve long-term treatment adherence and outcome[6]. The ease of refilling renders the technology superior to other LA implants, which require periodic invasive surgical replacement procedures. Further, the nanofluidic implant technology was previously validated for LA delivery of other ARVs (CAB, TAF, and FTC) and therapeutics, either alone or in combination [27, 53–55]. In line with this, future investigations will entail assessment of the nanofluidic technology for co-delivery of multiple ARVs for HIV treatment.

5. Conclusion

In this study, we present the potential of our SQ LA nanofluidic implant for HIV-1 treatment through constant and sustained release of ISL. Currently, ISL is administered either daily or weekly via oral dosing in clinical trials. The focus of our study is to investigate the potency, efficacy, and tolerability of ISL when subcutaneously administered at a low dose in a continuous manner. Viral load reductions were achieved in SHIV-infected macaques upon subcutaneous ISL release, with a higher potency than daily or weekly oral dosing. Despite initial viral load reduction, we noted the emergence of M184V mutation with ISL monotherapy in 2 out of 8 animals. This result underscores the importance of combination ARV for effective viral suppression. Building on this data, future investigations will leverage our drug-agnostic nanofluidic platform for investigations of combination ARV delivery to achieve effective long-term viral suppression.

Highlights for:

Antiviral potency of long-acting islatravir subdermal implant in SHIV-infected macaques

• Sustained subQ delivery of islatravir shows significant viral load reduction in NHP

• SubQ delivery of islatravir shows higher potency than daily or weekly oral dosing

• 25% of NHP showed emergence of M184V mutation with islatravir monotherapy

• SubQ delivery of islatravir was well tolerated in SHIV-infected NHPs