Intramuscular and subcutaneous administration of antiretroviral drugs, compared with oral, enhances delivery to lymphoid tissues in BALB/c mice

Abstract

Background

Multiple tissue reservoirs are established soon after HIV infection, and some tissues may also be pharmacological sanctuaries. Parenteral administration of antiretroviral (ARV) drugs for treatment and prevention of HIV infection is an active area of drug development. The influence of route of administration on ARV tissue pharmacokinetics is not known.

Objectives

To investigate ARV pharmacokinetics in lymphatic and select non-lymphatic tissues (e.g. brain and testes) after intramuscular and subcutaneous administration compared with oral in BALB/c mice.

Methods

Tissue concentrations of cobicistat, efavirenz, elvitegravir, maraviroc, rilpivirine, tenofovir alafenamide and tenofovir disoproxil fumarate were determined. The tissue penetration ratio (TPR) was the primary measure for comparison; a change in TPR arises from factors affecting tissue distribution controlling for changes in systemic bioavailability.

Results

Intramuscular and subcutaneous delivery increased TPRs in the lymph node and spleen for 27 of 28 (96%) drug administration events. Decreased TPRs, however, were found in some tissues such as the brain and testes.

Conclusions

These results demonstrate a change in route of drug administration from oral to intramuscular or subcutaneous can change tissue uptake. This has implications for HIV pharmacotherapy. For example, HIV persists in lymphoid tissues despite long-term oral ARV therapy, and low ARV concentrations have been found in lymphoid tissues. The improved ARV lymphatic tissue bioavailability with intramuscular and subcutaneous administration allows future studies to investigate these routes of drug administration as a therapeutic manoeuvre to limit viral persistence and eliminate viral sanctuaries in the lymphatic tissues, which is a prerequisite for eradication of HIV.

Introduction

The secondary lymphoid tissues, including lymph nodes (LNs) and gut-associated lymphoid tissue, and the brain are among the major reservoirs for HIV with extreme stability even during suppressive ART.^1,2 These same tissue reservoirs may also be pharmacological sanctuaries as evidenced by lower penetration of antiretroviral (ARV) drugs into lymphoid tissues and in some cases evidence for low-level ongoing viral replication, which may contribute to maintenance of the reservoir.^2–4 A need exists, therefore, to investigate strategies to enhance ARV drug penetration into viral reservoirs and determine if more complete viral suppression can be achieved. The determinants of ARV tissue penetration remain poorly understood; the influence of the route of drug delivery, which may influence tissue pharmacokinetics (PK) is not well studied.⁵ The significance of this gap is heightened as parenteral administration of ARV drugs for treatment and prevention of HIV infection is an active area of drug development. Indeed, intramuscular (IM) administration of long-acting formulations of cabotegravir (an integrase strand transfer inhibitor) and rilpivirine (a NNRTI) have recently received regulatory approval for maintenance therapy in persons living with HIV-infection in the United States, Canada and elsewhere.^6,7

Our primary objective was to investigate the PK of IM and subcutaneous (SC) administration, compared with oral, on the penetration of seven different ARV drugs into lymphatic tissues and select non-lymphoid tissues, including the brain, spleen and testes. The ARV drugs selected were cobicistat, efavirenz, elvitegravir, maraviroc, rilpivirine, tenofovir disoproxil fumarate and tenofovir alafenamide. ARV drug selection was based on categorization as high or intermediate bioavailability in an in vitro human lymphoid endothelial cell (HLEC) model.⁸ Preliminary data on the LN penetration of elvitegravir/cobicistat, dolutegravir and raltegravir in HIV-infected persons that showed elvitegravir/cobicistat achieved the highest concentrations, also informed the selection of that agent.⁹

Methods

Materials

We obtained efavirenz, elvitegravir, maraviroc and tenofovir alafenamide from Fisher Scientific Co. LLC, (Pittsburgh, PA, USA); cobicistat from Combi Blocks, (San Diego, CA, USA); rilpivirine from Toronto Research Chemicals (Ontario, Canada); and tenofovir disoproxil fumarate from US Pharmaceuticals (Rockville, MD, USA). DMSO (CAT# D128-500), propylene glycol (PG) (CAT#P355-1) and PBS (CAT#10010023) were purchased from Fisher Scientific; polyethylene glycol 400 (PEG400) (CAT# PX1286B-2), Kolliphor EL (CAT# C5135-500G) and ethanol (CAT# 1117270500) were purchased from Millipore Sigma (Burlington, MA, USA); isoflurane from Halocarbon Product Corporation (Peachtree Corners, GA, USA); and insulin syringes (28 gauge 1/2 inch needle) from BD Biosciences Corporation (Franklin Lakes, NJ, USA).

Ethics

Mice were housed at the University of Nebraska Medical Center (UNMC) animal facility with 12 h of light and dark cycles, constant temperature and humidity; they had free access to food and water. All animals were cared for under the guidelines of American Association for Laboratory Animal Care (AALAC) as described previously.^8,10 All procedures were approved by the Institutional Animal Care and Use Committee at UNMC according to the guidelines set by the National Institutes of Health, Bethesda, MD, USA.

Mice and drug administration

Healthy, male, adult BALB/c mice, 8–10 weeks old, were purchased from Charles River Laboratories (Wilmington, MA, USA) and allowed to acclimatize within the animal facility for 7 days before the PK studies were initiated. ARV drug oral doses were selected to approximate the human equivalent dose and the same dose was used for all three routes of administration. The drugs were solubilized in the vehicle, DMSO/PEG400/PG/ethanol/Kolliphor EL/PBS (1×) at 12.5%/25%/15%/12.5%/7%/28% v/v ratios.^8,10 The following ARV drug combinations were selected to either reflect clinically used combinations (e.g. elvitegravir/cobicistat) or to avoid any drug–drug interactions (e.g. efavirenz and maraviroc): efavirenz/tenofovir alafenamide, elvitegravir/cobicistat, and maraviroc/tenofovir disoproxil fumarate/rilpivirine. ARV drug doses were: efavirenz/tenofovir alafenamide (30/5 mg/kg), elvitegravir/cobicistat (12/12 mg/kg), and maraviroc/tenofovir disoproxil fumarate/rilpivirine (60/60/5 mg/kg). Each animal received one combination as a single daily dose according to their body weight in a volume of 10 μL/g for oral gavage (OG) and 4 μL/g for IM or SC administration for three consecutive days. The IM dose was administered in the thigh muscle (left or right) and the SC dose was administered into the tissue layer between the skin and muscle below the abdomen.

Tissue collection, processing and quantification of ARV concentrations

Tissue collections were performed at 4 or 24 h after the third dose according to the standard protocol as previously described.^8,10 Briefly, the working area to dissect the animals and dissection sets were cleaned with soap water and then decontaminated with one wash of 70% ethanol (v/v) and followed by bleach to prevent microbial contamination. Blood was collected through the cheek bleeding method. For tissue collection, the animals were euthanized following anaesthesia with isoflurane vapours. Mice were dissected to expose internal organs and then LNs (two each of inguinal, axillary and cervical LNs), brain, spleen, heart, liver, lung, kidneys and testes tissues were excised and weighed before suspending in 70% methanol medium. Collected organs were gently rinsed thrice with HPLC grade water to remove remaining traces of blood and clean the tissues, blotted and air dried, and stored in Eppendorf tubes at –80°C until further processing.^8,11 Plasma samples were prepared as previously described.⁸ Brain, heart, liver, lung, spleen, kidney and testes tissues were homogenized in 70% methanol at 1:5 ratio (wt/v) and LN tissue was homogenized in 70% methanol at 1:20 ratio (wt/v). Tissues were homogenized with a Precellys Evolution Cryolys homogenizer (Bertin Technologies, USA) in a temperature-controlled chamber (<10°C) according to the manufacturer’s protocol and as described previously.⁸ Tissue homogenates were centrifuged at 10 000 rpm for 20 min and supernatants containing drugs were collected into fresh tubes and stored at –80°C until used for drug quantification. Quantitation of plasma and cell-associated cobicistat, efavirenz, elvitegravir, maraviroc, rilpivirine, tenofovir (TFV; parent moiety of tenofovir disoproxil fumarate and tenofovir alafenamide) and TFV-diphosphate (TFV-DP; intracellular anabolite of tenofovir disoproxil fumarate and tenofovir alafenamide) concentrations was performed with validated LC/MS/MS methods as described previously.^2,8,12–14 Standard curves were generated by dissolving standards of each ARV drug in respective blank tissues collected from untreated mice.

Data and analyses

Tissue penetration ratio (TPR)

Our primary objective was to investigate the patterns of ARV drug tissue penetration with oral, IM and SC administration. The TPR was the primary measure to compare ARV drug tissue concentrations across the three routes of drug administration. The TPR was calculated as the ratio of ARV drug concentration in tissue (as ng/mL after conversion from ng/g by dividing the values in ng/g by 1.06)⁸ to the concentration in plasma (ng/mL) for the same route of delivery, either oral, IM or SC. The TPR was calculated separately for samples collected at 4 h and at 24 h. We focused on TPR values at 4 h post-dose as those primarily represent tissue penetration, whereas the 24 h post-dose values are strongly influenced by elimination. To compare TPRs with IM and SC administration to that with oral, the change in TPR was calculated as the ratio of TPRs for IM to oral and for SC to oral.

HIV inhibitory quotient (IQ) in plasma and tissues

As a secondary measure of comparison, the HIV IQ was calculated. The IQ is the ratio of the concentration of each ARV drug achieved in plasma or a particular tissue following IM, SC and oral routes of administration divided by the in vitro concentration of that ARV drug required to inhibit HIV replication by 90% (IC₉₀). In these calculations, we used the following, protein-binding adjusted, IC₉₀ values: efavirenz, 470 ng/mL;¹⁵ elvitegravir, 45 ng/mL;¹⁶ maraviroc, 44.6 ng/mL;¹⁷ and rilpivirine, 70 ng/mL.¹⁸ We used the concentration required to inhibit 50% of HIV replication (IC₅₀) for tenofovir alafenamide (1.44 ng/mL)¹⁹ and tenofovir disoproxil fumarate (14.4 ng/mL)^19,20 as IC₉₀ values were not available.

Data obtained from five different animals in each group served as independent biological replicates. The sample size was based on investigator judgement for sufficient rigour and reproducibility. A statistically based sample size justification and hypothesis was not attempted as no prior data existed on tissue penetration with different routes of administration. Summary data were expressed as medians because of the small sample size and change in TPRs and IQ ratios was plotted (Prism 9.0, GraphPad Software, USA). We performed exploratory statistical analyses among the TPRs obtained from IM and SC compared with oral using the Kruskal–Wallis test with Dunn’s multiple comparisons tests (Prism 9.0, GraphPad Software).

Results

The oral ARV drug doses, selected to approximate human equivalent doses, generally achieved plasma concentrations in mice similar to those observed in humans. Table 1 presents the median TPR values at 4 h post-dose for each ARV drug, and Table 2 the median TPR values at 24 h post-dose, with oral, IM and SC administration based on concentrations of the ARV drug quantified in plasma and tissues (Figure S1, available as Supplementary data at JAC Online). Figure 1 and Figure S2 illustrate the patterns of changes in the median 4 and 24 h post-dose TPR values from oral administration to that with IM and SC administration for each ARV drug. IM and SC administration compared with oral more often resulted in an increase in the TPR. For example, at 4 h post-dose, the LN and spleen TPRs were increased in 27/28 (96%) drug administration events with the ARV drugs given by IM and SC. For the brain and testes, the ARV drug TPRs were increased in 18/28 (64%) drug administration events. Among the ARV drugs investigated, rilpivirine administered both IM and SC, efavirenz administered SC and tenofovir alafenamide administered IM resulted in an increased TPR (≥1) in all tissues (8/8). Different magnitudes and patterns of changes in the TPR were observed for some administration events by drug and route. For example, maraviroc administered IM resulted in an increased TPR in all tissues except heart (7/8, 87.5%), whereas when given SC, an increased TPR was found in four (LN, lung, heart and liver) of eight (50%) tissues.

Table 1.

TPRs at 4 h post-dose

Drug and route	TPR (median) by compartment
	LN	brain	spleen	lung	testes	kidney	heart	liver
Cobicistat
oral	0.207	0.001	0.152	0.351	0.065	0.391	0.143	4.132
IM	4.089a	0.002	0.240	0.495a	0.047	0.522	0.134	2.663
SC	2.304	0.001	0.202	0.383	0.040	0.461	0.153	3.441
Efavirenz
oral	1.875	0.312	0.223	0.388	0.618	0.609	0.293	0.504
IM	2.000	0.279	0.297	0.422	0.654	0.647	0.371	0.656
SC	3.851	0.372c	0.257	0.417	0.656	0.822	0.412e	0.805
Elvitegravir
oral	0.460	0.005e	0.264	0.422	0.081a,b	0.537	0.457	1.761
IM	1.493	0.003	0.253	0.380	0.033	0.518	0.509	1.383
SC	1.660e	0.002	0.267	0.601b,c	0.031	0.605	0.516	1.260
Maraviroc
oral	4.834	0.019	0.477	0.624	0.632	1.100	0.284	1.917
IM	18.588	0.027	0.722	1.045	0.689f	1.898	0.457	2.009
SC	12.564	0.010	0.490	0.603	0.323	0.672	0.266	1.788
Rilpivirine
oral	0.457	0.021	0.029	0.042	0.009	0.096	0.032	0.078
IM	0.959	0.028	0.048	0.067	0.034	0.165	0.056	0.106
SC	0.645	0.028	0.031	0.057	0.024	0.107	0.044	0.103
Tenofovir alafenamide
oral	3.417	0.034	0.769	0.259	0.030	13.571	0.232	46.912
IM	16.487a	0.049f	1.597	0.797a	0.130a	15.891	1.066a,c	65.309
SC	15.016	0.026	1.056	0.487	0.095b	19.117	0.338	59.268
Tenofovir disoproxil fumarate
oral	1.277	0.011	0.277	0.190	0.031	16.057	0.083	40.231
IM	10.826d	0.062a	1.903a	1.784a	0.093a	14.590	0.961a	36.051
SC	30.030b	0.033	0.871	0.894	0.039	11.265	0.230	28.196

^aOral versus IM;

^boral versus SC; and

^cIM versus SC indicate P value <0.05.

^dOral versus IM;

^eoral versus SC; and

^fIM versus SC indicate P value <0.1.

Table 2.

TPRs at 24 h post-dose

Drug and route	TPR (median) by compartment
	LN	brain	spleen	lung	testes	kidney	heart	liver
Cobicistat
oral	3.043	BLQ	0.597	0.014	1.407	0.069	0.223	1.883
IM	23.585	BLQ	0.273	0.965f	7.039	0.070	0.341c	4.905c
SC	5.142	0.425	0.262	0.014	2.138	0.059	0.025	1.353
Efavirenz
oral	0.439	0.018	0.100	0.047	0.464	0.082	0.135	0.019
IM	0.754	0.032	0.050	0.102	0.105	0.142	0.113	0.101
SC	1.378	0.161	0.102	0.096	0.241	0.264	0.102	0.134
Elvitegravir
oral	0.578	0.036	0.031	1.856a	0.116	0.040	0.061	0.030
IM	1.997	0.027	0.014	0.045	0.083	0.069	0.082c	0.043
SC	1.341	0.022	0.221c	0.103	0.079	0.048	0.007	0.043
Maraviroc
oral	1.707	0.067e	0.024	0.198	16.380	0.175e	0.017	0.404
IM	2.602	0.020	0.029	0.099	7.413	0.204	0.041	0.197
SC	4.354	0.012	0.023	0.084	6.698	0.127	0.026	0.172
Rilpivirine
oral	BLQ	0.267a	0.106	0.056a	BLQ	0.056	BLQ	0.063
IM	0.148	0.020	0.020	0.041	0.015	0.132	0.038	0.109
SC	0.497f	0.024	0.027	0.037	0.012	0.148b	0.011	0.093
Tenofovir alafenamide
oral	11.864	0.095	1.600	0.436	0.085	17.726	0.585	54.420
IM	70.257	0.234c	3.406	3.181a,c	0.495a,c	12.452	2.293a,c	91.507d
SC	62.366	0.060	1.571	0.428	0.063	18.980	0.535	71.407
Tenofovir disoproxil fumarate
oral	4.080	0.016	0.687	0.229	0.037	16.706a,b	0.112	44.054
IM	18.978	0.070a	1.705a	0.980a	0.093	5.839	0.933a	32.384
SC	40.274b	0.040	1.540b	0.857e	0.188e	8.185	0.645	33.351

BLQ, all samples were below the limit of quantitation.

^aOral versus IM;

^boral versus SC; and

^cIM versus SC indicate P value <0.05.

^dOral versus IM;

^eoral versus SC; and

^fIM versus SC indicate P value <0.1.

Figure 1.

Change in TPR values with IM and SC administration at 4 h post-dose. Heat map of the change in the TPR observed with IM and SC routes of administration compared with oral in LN, spleen, lung, testes, brain, kidney, heart and liver tissues collected at 4 h after the third single daily dose of ARV drug administration. The median change in TPR values is shown in each box and the scale for the colour codes is indicated below. Cobi, cobicistat; EFV, efavirenz; EVG, elvitegravir; MVC, maraviroc; RPV, rilpivirine; TAF, tenofovir alafenamide; TDF, tenofovir disoproxil fumarate. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

We measured the concentration of TFV-DP, the antivirally active metabolite generated from tenofovir alafenamide or tenofovir disoproxil fumarate, in LN and brain tissues (Table 3). Compared with oral administration, increased TFV-DP concentrations were seen in LN and brain with IM and SC administration. SC administration of tenofovir alafenamide and tenofovir disoproxil fumarate produced a greater fold-change increase in TFV-DP LN concentrations than did IM. Conversely, IM administration of tenofovir alafenamide and tenofovir disoproxil fumarate produced greater fold-change increases in TFV-DP concentrations in the brain than did SC.

Table 3.

TFV-DP concentrations in LN and brain tissues and fold change with IM and SC versus oral administration

Drug/route	TFV-DPa (fmole/g) in LN	Fold change over oralb	TFV-DP (fmole/g) in brain	Fold change over oral
Tenofovir alafenamide
oral	0.93		0.09
IM	7.78	8.31	0.59	6.56
SC	19.97	21.33	0.23	2.63
Tenofovir disoproxil fumarate
oral	12.84		0.64
IM	34.83	2.71	29.7	45.90
SC	355.2	27.66	16.5	25.50

^aMedian concentration in n = 5 mice at 4 h after the third daily dose by oral, IM or SC administration.

^bMedian fold change (ratio of IM or SC to oral) in n = 5 mice at 4 h after the third daily dose by oral, IM or SC administration.

The IQ provides information on whether an ARV drug is maintaining a concentration putatively sufficient to inhibit HIV replication. Figure 2 shows the HIV IQ of the ARV drugs in three select compartments (LN, brain and testes tissues) at 4 h post-dose when administered orally, IM and SC. In the LN, IQ values ranged from 5.8 with efavirenz given orally to 1943 for SC administered tenofovir disoproxil fumarate. In contrast, IQ values in the brain were lower reflecting lower tissue concentrations and ranged from 0.05 for maraviroc administered orally to 2.3 with IM delivered tenofovir disoproxil fumarate. Figure S3 shows the HIV IQs at 24 h post-dose.

Figure 2.

HIV IQ in LN, brain and testes at 4 h post-dose. IQ was calculated as a ratio of ARV concentrations in LN, brain and testes following ARV drug administration via oral gavage (OG), IM and SC routes to their respective in vitro IC₉₀ for efavirenz (EFV), elvitegravir (EVG), maraviroc (MVC) and rilpivirine (RPV) or IC₅₀ for tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF). Median IQ values are shown in each box and the scale for the colour codes is indicated below. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Discussion

We investigated ARV PK in lymphoid and select non-lymphoid tissues that are reservoirs for HIV, following oral, IM and SC routes of administration in BALB/c mice. We chose ARV drug doses to approximate those used clinically. The TPR, calculated as the ratio of tissue to plasma ARV drug concentrations for the same route of delivery at 4 h post-dose, was the primary measure for comparison. This metric controls for any change in tissue concentration from only a change in plasma concentration for that route of administration. Thus, an increase or decrease in TPR arises from factors affecting tissue distribution. We observed that IM and SC administration generally enhanced TPRs at 4 h post-dose compared with oral. This was most uniformly seen in the LN and spleen, where TPRs were numerically increased for all ARV drugs and routes of administration except in the spleen for maraviroc given SC. Decreases in the TPR were also observed in some tissues. For example, IM and SC administration of elvitegravir reduced the TPR in the brain, testes and liver. These observations demonstrate that a change in the route of drug administration from oral to IM or SC, separate from a change in plasma bioavailability, has the potential to change the uptake of drug in a tissue. It is understood that oral drug administration may result in reduced plasma concentrations and thereby lower tissue bioavailability due to the influence of multiple factors such as food, slower gastric emptying, intestinal motility, intestinal metabolism, transport and pre-systemic metabolism.^21,22 SC and IM administration may circumvent some of these processes and achieve higher plasma concentrations. However, the effects of SC and IM administration on tissue bioavailability are less well understood. We found no prior data comparing tissue bioavailability following oral, IM and SC administration for these ARV drugs.

We observed enhanced distribution of ARV drugs into LN when administered IM and SC, likely due to their absorption into lymphatic vessels at the site of injection and further distribution into lymphatics.^23–25 In lymphoid tissues, small therapeutic molecules gain entry into the lymphatic system through lymphatic vessels, which are lined by a lymphoid endothethial cell monolayer.^26,27 This is similar to the brain, where a monolayer formed with brain endothelial cells or the blood–brain barrier limits entry of drugs.²⁸ The increased TPRs in the LN and spleen with IM and SC administration (Table 1 and Figure 1) show a concordance between these tissue studies in mice and the in vitro HLEC model.⁸ Additional reasons for the increase in ARV drug LN concentrations may include direct targeting of drugs into the lymphatics or prolonged drug exposure from IM and SC administration that may follow slow-release kinetics as compared with the oral route of administration.^23–25,29 We also observed increased brain tissue concentrations of several ARV drugs with IM and SC administration. The brain, like lymphoid tissues, is a persistent HIV reservoir.³⁰ Rilpivirine and tenofovir disoproxil fumarate administered IM and SC, efavirenz given SC, and maraviroc and tenofovir alafenamide given IM showed an increase in brain tissue concentrations compared with oral. However, efavirenz and elvitegravir administered IM and cobicistat, elvitegravir, maraviroc and tenofovir alafenamide given SC showed lower brain concentrations than oral. This variability in tissue distribution may be attributed, in part, to physicochemical properties of drugs such as degree of lipophilicity, size and their ability to bind to protein or RBCs or due to involvement of drug efflux transporters.^31–33 Finally, interesting patterns of tissue delivery and activation to TFV-DP were observed for tenofovir disoproxil fumarate and tenofovir alafenamide. IM and SC administration of tenofovir disoproxil fumarate and tenofovir alafenamide increased TFV-DP concentrations in LN and brain versus those achieved with oral administration. SC administration led to greater increases in the LN than did IM; the fold change was 10.2-times greater for tenofovir disoproxil fumarate and 2.6-times greater with tenofovir alafenamide. Conversely, IM administration led to greater increases in TFV-DP brain concentrations. The fold-change increase with tenofovir disoproxil fumarate was 1.8-times greater with IM versus SC and was 2.5-times greater for tenofovir alafenamide. These findings, particularly striking for the increase in TFV-DP LN concentrations when tenofovir disoproxil fumarate was given SC and brain concentrations when tenofovir disoproxil fumarate was given IM, indicate differences in tissue and cell uptake and phosphorylation. Potential explanations for differences in tissue delivery with IM and SC administration, in addition to physicochemical properties of drugs themselves, include greater drug spreading with an IM injection providing a larger surface area for absorption, higher perfusion of muscle than SC tissue, and regional variation in lymphatic flow. Collectively, these findings highlight the need to determine mechanisms of tissue distribution of ARV drugs with different routes of administration. The choice of route of delivery can be clinically relevant. For example, the Bacillus Calmette–Guérin TB vaccine was markedly more effective in preventing infection and in generating high levels of antigen-responsive T cells across lung parenchymal tissues when given IV compared with intradermal or aerosol delivery to non-human primates (NHPs).³⁴

The potential clinical significance of changes in ARV drug tissue PK following IM and SC routes over oral was explored with the IQ. The IQ is the ratio of drug concentration in any biological matrix divided by an in vitro inhibitory concentration (i.e. the amount of drug available to the amount of drug needed to inhibit HIV replication). The IQ has utility in ARV drug development, as discussed by the FDA because a high IQ indicates sufficient drug concentrations can be achieved that may minimize the emergence of viral resistance.³⁵ To calculate IQ values, we used IC₉₀ values for efavirenz, elvitegravir, maraviroc and rilpivirine adjusted for plasma protein binding. It is not known, however, whether tissue binding of these drugs is the same as plasma protein binding. IQ values are shown in Figure 2 for LN, brain and testicular tissues at 4 h post-dose. As an illustration, at 4 h post-dose, the LN IQ of maraviroc increased from 14.9 with oral to 290.4 with IM and 418.7 with SC administration. In contrast, the brain tissue IQ of maraviroc, though increased from 0.05 with oral to 0.29 with IM and 0.25 with SC, remained <1, indicating concentrations of drug in brain tissue were less than the IC₉₀, raising questions of whether sufficient anti-HIV activity could be achieved. In the brain, the highest IQ observed was 2.3 with tenofovir disoproxil fumarate given IM, and IQ values for most drugs and routes of administration (11/18) remained <1. This indicates, at least for these ARV drugs, that sufficient brain concentrations may not be achieved and that more targeted brain delivery approaches may be necessary. IQ values at 24 h post-dose are shown in Figure S3. In the LN and brain, 59% and 89% of IQ values, respectively, were <1, compared with none and 61% at 4 h post-dose. These changes reflect the effect of drug elimination and illustrate challenges if maintaining an IQ above 1 is as important for tissue PK as it is in plasma, e.g. with integrase strand transferase inhibitors.³⁶

Our primary interest in this investigation was tissue distribution of ARV drugs to lymphoid tissues. This is based on, e.g. a study in NHPs that showed lymphatic tissues contained more than 98% of the SIV RNA+ and DNA+ cells in the absence of ART.¹ Over 6 months of suppressive ART, low ARV drug concentrations were measured in lymphatic tissues and the numbers of SIV RNA+ cells in lymphatic tissues remained detectable; SIV RNA+ levels in the brain actually increased suggesting ongoing replication.^1,2 Studies in HIV-infected persons have reported lower ARV drug concentrations in LN mononuclear cells compared with PBMCs and associations between low ARV drug LN concentrations and measures of persistent viral production despite long-term suppression of plasma HIV-RNA to <50 copies/mL.^2,4,14 Therefore, strategies that can increase ARV exposure in lymphoid tissues may represent therapeutic manoeuvres to achieve more potent viral suppression. In addition to a change in route of drug administration as shown in the present work, drug formulation approaches have also been shown to increase LN concentrations. SC administered drug combination nanoparticles (NPs) have been shown to increase LN concentrations. Kraft et al.³⁷ demonstrated that a targeted long-acting ARV product containing lopinavir, ritonavir and TFV in a 4:1:3 molar ratio enhanced LN concentrations in NHPs. Perazzolo et al.³⁸ confirmed these results with NPs containing atazanavir, ritonavir and TFV, which led to increased LN concentrations in NHPs. An improved understanding and application of route of administration and drug formulation on tissue concentrations is necessary because parenteral administration is moving into human therapeutics for HIV prevention and treatment. Results from two Phase 3 trials of long-acting, IM cabotegravir plus rilpivirine demonstrated high levels of viral suppression, with approximately 93% of total participants maintaining plasma HIV-RNA <50 copies/mL at 48 weeks, which was non-inferior compared with oral ART.^6,7 However, no data are available on suppression of HIV replication other than in plasma and such investigations in other compartments are needed.

In summary, to the best of our knowledge, this is the first study to investigate the comparative tissue PK of ARV drugs with oral, IM and SC routes of administration in a rodent model. Our results demonstrate a change in route of administration from oral to IM or SC delivery may improve ARV drug concentrations in lymphoid tissues, but can decrease concentrations in some tissues such as the brain and testes. The mechanisms of these changes in tissue penetration now need to be determined. Further studies are warranted in other animal models such as humanized mice and NHPs to study tissue pharmacodynamic properties and then translational human clinical investigations. The ability to increase ARV drug concentration in lymphoid tissues may achieve concentrations optimal to limit viral replication and reduce viral reservoirs in HIV-infected persons and ultimately lead to strategies for remission or eradication of HIV/AIDS.