Solubility of ARV drugs in N-methyl-2-pyrrolidone (NMP)

Fourteen (14) antiretroviral drugs from different classes were tested for solubility in NMP (Supplementary Table 1). These drugs were selected based on their relevance for HIV treatment or prevention. Out of the fourteen drugs tested in NMP, based on their solubility properties (LogP) six (6) were eventually tested in the ISFI formulation. Both hydrophilic and hydrophobic drugs were included. From the class of integrase inhibitors, we tested MK-2048 and Dolutegravir (DTG). From the class of protease inhibitors (PIs) we tested Darunavir and Atazanavir; and from the class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) we tested Rilpivirine. Ritonavir, a drug often used as a booster for protease inhibitors was also included in some formulations.

From the class of integrase inhibitors, the highest solubility in NMP was recorded for MK-2048, a second-generation integrase inhibitor developed by Bar-Magen et al.31. MK-2048 is highly potent against HIV-1 with in vitro 90% inhibitory concentration (IC90) of ~0.033 μg/mL31,32,33. MK-2048 has low solubility in common biocompatible organic solvents (propylene glycol 5.32 mg/mL, PEG 400 15.6 mg/mL, ethanol 11.3 mg/mL) and poor solubility in water (LogP = 2.67, < 0.1 mg/mL at pH 7), limiting its administration orally or systemically31. In NMP, MK-2048 had a significantly higher saturation solubility of 715 ± 5 mg/mL (Supplementary Table 1). Dolutegravir, is another second-generation integrase strand transfer inhibitor (INSTI) highly potent against HIV-1 with an in vitro protein-adjusted IC90 for HIV-1 of 0.064 μg/mL. Dolutegravir (DTG) had a saturation solubility in NMP of 255 ± 4 mg/mL (Supplementary Table 1). Rilpivirine (RPV), had a saturation solubility in NMP of 228 ± 5 mg/mL. Darunavir, Ritonavir, and Atazanavir are hydrophobic drugs like MK 2048, DTG and RPV. These three drugs also exhibited very high solubility in NMP ranging from 328 ± 2 mg/mL (atazanavir) to 511 ± 2 mg/mL (darunavir). Raltegravir, a hydrophilic drug, exhibited lower solubility in NMP (54 ± 2 mg/mL) compared to MK-2048 and DTG. Lamivudine and Stavudine are also hydrophilic drugs exhibited high solubility in NMP (250 ± 3 mg/mL and 248 ± 3 mg/mL, respectively). The average solubility of the antiretroviral drugs tested in NMP was ~260 mg/mL (Supplementary Table 1). Collectively, these data showed that a wide range of ARVs from multiple classes exhibited high solubility in NMP making it an ideal solvent for ISFI formulations for long-acting delivery of ARVs. MK-2048 and DTG were initially selected to test in the ultra-long-acting injectable formulation.

Density and rheology of ISFI formulations

To measure density and viscosity of ISFI formulations seven different ISFI formulations were prepared by dissolving PLGA (50:50 LA/GA, 27 kDa) in NMP at a range of weight ratios. Placebo formulations containing 1:2, 1:4, 1:9, 1:16, and 1:30 weight ratios (w/w) of PLGA to NMP and ISFI formulation containing MK-2048 (0.9:1:2 w/w/w ratio of MK-2048/PLGA/NMP) and DTG (0.3:1:2 w/w/w ratio of DTG/PLGA/NMP) were evaluated. The density of placebo formulations decreased slightly with increasing concentration of NMP in the formulation. Addition of drug to the formulation had no or minimal effect on formulation density (Table 1). The viscosity of the placebo formulations increased with increasing amount of PLGA and ranged from 2.6 cP for 1:30 w/w PLGA/NMP to 475 cP for 1:2 w/w PLGA/NMP (Table 1). ISFI formulations (1:2 w/w PLGA/NMP) with MK-2048 (300 mg/mL) and DTG (100 mg/mL) had viscosity of 1242 cP and 845 cP, respectively. Compared to placebo formulation with the same PLGA/NMP ratio the viscosity increased 2.6-fold and 1.8-fold for MK-2048 ISFI and DTG ISFI, respectively. Despite the increase in viscosity, both drug ISFIs were syringeable and could be readily administered subcutaneously to mice.

Table 1 Density and rheology of ISFI formulations Full size table

Stability studies

To determine the shelf-life of MK-2048 and DTG ISFIs, stability studies were carried out at three different storage conditions (5 °C ± 2 °C, 25 °C ± 2 °C, 40 °C ± 2 °C/75% relative humidity (RH)). Stability was determined for both the ISFI formulation (change in physical appearance) and drug formulated in the ISFI (drug concentration, physical/chemical integrity, activity). ISFI formulations were prepared with MK-2048 (300 mg/mL, 0.9:1:2 w/w/w of MK-2048/PLGA/NMP ratio, PLGA 27 kDa) and DTG (100 mg/mL, 0.3:1:2 w/w/w of DTG/PLGA/NMP ratio, PLGA 27 kDa) and sample aliquots were collected longitudinally over four months to quantify drug concentration and presence of any drug degradation peaks by high-performance liquid chromatography (HPLC). Table 2 (Supplementary information) shows the residual drug at each time point relative to initial drug concentration (time 0 h). For MK-2048 ISFI, the residual drug was 98.5% at day 30 for the formulation stored at 5 °C and to 97.2% and 95.7% after 4 months for formulations stored at 25 °C and 40 °C/75% RH, respectively. For DTG ISFI, the residual drug decreased to 97.8% at day 30 for the formulation stored at 5 °C and to 95.7 and 91.4% after 4 months for formulations stored at 25 °C and 40 °C/75% RH, respectively. ISFI solutions were also monitored for changes in appearance, color, or consistency. No change in physical appearance or consistency was observed after 0, 1, 3, or 4 months of storage at 25 °C or 40 °C/75% RH storage conditions. However, for the formulation stored at 5 °C, the solution turned turbid by day 30 with both MK-2048 and DTG likely due to drug precipitation from the solution or phase separation between PLGA and NMP. These results indicate that both MK-2048 and DTG formulations maintain their stability if stored at room temperature conditions (25 °C) for at least 4 months. The slight decrease in drug concentration (<4% for MK-2048 at 40 °C/75% RH and <7% for DTG at 40 °C/75% RH) over four months was likely due to drug degradation at lower pH as a result of PLGA hydrolysis under the stability study conditions (Supplementary Table 2). However, no degradation peaks were detected by HPLC analysis for MK-2048 and DTG (Supplementary Fig. 1).

Physical stability of MK-2048 and DTG in ISFI formulation

The physical state of the drug in the implant, i.e., crystalline state or molecularly dispersed can influence the extent of burst release and release rate of a drug from the formed implant34. We used differential scanning calorimetry (DSC), a thermoanalytical technique to evaluate the physical state and melting temperature of PLGA and drug in solidified ISFIs. Figure 1a–f shows DSC thermograms of individual neat (reagent only) excipients (PLGA, MK-2048, and DTG), and placebo implant or drug implants prepared by solidification of liquid formulation (20 µl) upon injection to phosphate-buffered saline (PBS). Thermograms of neat PLGA and placebo implant (1:2 w/w PLGA/NMP) showed a broad endothermic peak at 40–50 °C for PLGA and 41–44 °C for placebo implant (Fig. 1a, b) reflecting the amorphous nature of the PLGA 50:50 (LA:GA)34. Neat MK-2048 and DTG gave sharp endothermic peaks at the glass transition temperature 209.3 °C and 189.6 °C, respectively (Fig. 1c, e). MK-2048 and DTG in ISFI formulations (0.9:1:2 and 0.3:1:2 w/w/w drug/PLGA/NMP ratios) showed endothermic peaks for MK-2048 and DTG at 194.5 °C and 187.4 °C, respectively (Fig. 1d, f). This demonstrated that no chemical interaction took place between the drug and polymer indicating that MK-2048 and DTG are both stable in the ISFI formulation. Figure 1d also showed a PLGA endotherm at 42.4 °C which showed that the glass transition temperature of PLGA is maintained in the presence of MK-2048 indicating the stability of PLGA in the presence of drug. Interestingly, we found that the melting peak intensity for PLGA (Fig. 1a, b) and MK-2048 (Fig. 1c, d), but not DTG (Fig. 1e, f), was substantially smaller in the ISFI formulations compared to neat PLGA and neat MK-2048. This suggests that PLGA and MK-2048 were less crystalline and more amorphous when formulated in the ISFI compared to DTG.

Fig. 1 Drug stability in ISFI formulation by DSC analysis. DSC thermograms: a PLGA (neat; 50:50 LA:GA, MW 27 kDa), b 1:2 PLGA/NMP Placebo ISFI, c MK-2048 (neat), d 0.9:1:2 MK-2048/PLGA/NMP ISFI, e DTG (neat), f 0.3:1:2 DTG/PLGA/NMP; the PLGA peak at ~40–50 °C is not visible due to scale relative to the strong DTG peak. Endodermic peaks with the respective glass transition and melting temperatures are indicated in each panel Full size image

Drug activity after long-term storage in ISFI formulation

DTG-ISFI formulation was stored for 6 months at 25 °C and then solidified in PBS. The concentration of DTG eluted from the solidified implant after 24 h incubation in PBS was determined by HPLC and its HIV inhibitory activity was assessed using TZM-bl cells as described in the methods section. The effective concentration of DTG eluted from the implant required to inhibit virus replication by 50% (IC50) was 2.1 ng/ml (Supplementary Fig. 2). This value is consistent with the IC50 previously reported for DTG35,36 and it was not significantly different from the activity of a freshly prepared solution of DTG (Supplementary Fig. 2).

Scanning electron microscopy (SEM) analysis

The release kinetics of drugs from ISFIs is largely influenced by the microstructure of the implant formed in vitro and in vivo. The microstructures of implants can be influenced by factors including the polymer type, solvent and drug properties, miscibility of the polymer with the solvent, the rate of phase inversion, the effect of the injection site, and the rate of implant degradation24,26,27,28,37,38,39,40,41. Scanning electron microscopy (SEM) imaging was used to investigate the effect of PLGA erosion on the microstructure environment of the implant. Samples were prepared by injecting 20 µL of ISFI formulation (1:2 w/w PLGA/NMP, PLGA MW 27 kDa, 1 mL) into PBS and incubating for 3, 14, and 30 days at 37 °C (n = 3 per time point). Upon injection of the ISFI formulation into PBS, the NMP diffused into the aqueous medium and the ISFI solidified. Cryosectioning of the implant formed in vitro was not possible until day 3 due to implant distortion during the freeze-drying process part of the sample preparation for SEM imaging41,42. As shown in Fig. 2, at day 3, the implant consisted of a central pore caused by the residual NMP. This central pore is surrounded by a thick polymer outer shell of highly interconnected porous structures consistent with an instantaneous precipitation of PLGA from highly miscible solvent like NMP41,42. Erosion of implant was observed by decreasing thickness of the outer shell of the implant from 500–600 μm at day 3 to 200–300 μm at day 30. Additionally, changes in the porous structure of the implant shell were noticed. The diameter of the pores in the shell was in the range of 10–40 μm at day 3. At day 30, large pores were observed with a diameter in the range of 40–100 μm and length of 40–400 μm, respectively. The microstructure of implants at each time point did not differ between samples (n = 3).

Fig. 2 Effect of PLGA degradation on implant microstructure. SEM cross-section images of placebo-ISFIs (1:2 w/w PLGA/NMP, PLGA MW 27 kDa) over a 30 day period (n = 3). a–c low magnification image (×100 ) of the entire implant (scale bar = 100 µm), d–f higher magnification (×200) of the implant shell (shell thickness was measured using SEM scale; scale bar = 50 µm), g–i higher magnification (×500) of the center of the implant (scale bar = 20 µm). a, d, g Implants imaged at day 3 post incubation in 0.01 M PBS pH 7.4 at 37 °C; b, e, h implants imaged at day 14 post incubation; c, f, i implants imaged at day 30 post incubation. Representative image for each magnification and time point is shown (n = 3). Symbols represent implant shell (*) and pore (^) Full size image

The effect of PLGA to NMP ratio on the microstructure of implants was also investigated by SEM imaging. DTG was used as a model drug for DTG-ISFI formulations (100 mg/mL DTG) containing 1:2, 1:4, and 1:8 weight ratios of PLGA to NMP. Solidified implants were incubated for 30 days in PBS at 37 °C and the cross-sectional images of implants were collected. DTG containing implants in Fig. 3a, d, g can be directly compared to the placebo implant of same PLGA/NMP ratio in Fig. 2c, f, i (1:2 w/w PLGA/NMP). Interestingly, the presence of DTG resulted in lower erosion of outer shell and a smaller central pore compared to the placebo implant. DTG formed rose-like shapes within the microstructures of the implant (compare Figs. 2 and 3), likely due to the crystalline nature of DTG in the implant demonstrated by the DSC analysis. Increased amounts of NMP in the formulations with PLGA/NMP 1:4 and 1:8 w/w resulted in smaller and high density rose-like structures that were distributed throughout the implant (compare Fig. 3a, d, g with 3b, e, h and 3 c, f, i). The presence of more NMP also led to greater drug dissolution and mixing within the PLGA matrix, which resulted in smaller microstructures within the implant when the NMP diffused out of the solution and the PLGA/DTG phase-inverted and formed a solid implant. As shown in Fig. 3a–c, the 1:2 w/w PLGA/NMP DTG-ISFI had a thicker outer shell structure at day 30 compared to the 1:4 and 1:8 DTG-ISFI formulations (Fig. 3b, c), suggesting greater erosion of PLGA in implants originated from ISFI formulation with higher content of NMP.

Fig. 3 Effect of PLGA/NMP ratio on the microstructure of the DTG implants. SEM cross-section images of DTG-ISFIs after drug release for 30 days (n = 3). Each column is a representation of ISFIs containing a different ratio of PLGA to NMP. a, d, g 1:2 w/w PLGA/NMP ratio, b, e, h 1:4 w/w PLGA/NMP ratio, and c, f, i 1:8 w/w PLGA/NMP ratio. Row 1 (a, b, c) is a low magnification image (×100, scale bar = 100 µm) of the entire implant, Rows 2 (d, e, f) and 3 (panels g, h, i) are higher magnification images (×200 , and ×500 , respectively, scale bar = 50 µm, 20 µm) focusing on the center of the implant. a 1:2 PLGA/NMP ×100 , b 1:4 PLGA/NMP ×100 , c 1:8 PLGA/NMP ×100 (a–c scale bar = 100 µm), d 1:2 PLGA/NMP ×200, e 1:4 PLGA/NMP ×200 , f 1:8 PLGA/NMP ×200 (d–f scale bar = 50 µm), g 1:2 PLGA/NMP ×500 , h 1:4 PLGA/NMP ×500 , i 1:8 PLGA/NMP ×500 (g–i scale bar = 20 µm). Representative image of each magnification and PLGA/NMP ratio is shown. Symbols represent PLGA (*) and DTG (^) Full size image

In vitro release studies

ISFIs have characteristic release kinetics that include three phases: burst release, diffusion, and degradation41,43. The effect of PLGA to NMP weight ratio on drug release kinetics was investigated with MK-2048 or DTG-ISFIs. As shown in Fig. 4, both drugs exhibited the same relationship correlating release kinetics to the ratio of PLGA to NMP. Formulations containing a higher amount of NMP exhibited higher burst release and faster release kinetics. For both drugs, burst release occurred over the first 24 h post formulation injection into the release medium. For MK-2048-ISFIs, burst release ranged between 95 and 16% for formulations containing 1:30 w/w PLGA/NMP and 1:2 w/w PLGA/NMP respectively. For DTG-ISFIs, burst release ranged between 85 and 8% for formulations containing 1:16 w/w PLGA/NMP and 1:2 w/w PLGA/NMP respectively. When comparing the burst release of the two drugs, MK-2048 exhibited a larger burst release across all formulations compared to DTG. This could be attributed to the higher solubility of MK-2048 in NMP (715 ± 5 mg/mL) compared to DTG (255 ± 4 mg/mL) (Table 1, Supplementary Information), and to the amorphous nature of MK-2048 in the ISFI formulation as per the DSC analysis (Fig. 1d). For MK-2048, 100% release was reached by day 40 or earlier for three of the PLGA/NMP ratios investigated (1:30, 1:9 and 1:4 w/w). At the lowest ratio of PLGA/NMP (1:2 w/w), MK-2048 release reached 100% by day 208 (Fig. 4a). The release rate of MK-2048 within the zero order kinetics ranged between 17.4 μg/week (1:2 w/w PLGA/NMP) to 43.6 μg/week (1:8 w/w PLGA/NMP). For DTG, formulations containing 1:16 and 1:8 w/w PLGA/NMP reached 100% release by day 30 and day 120, respectively. At day 120, the remaining DTG-ISFIs (1:4 and 1:2 w/w PLGA/NMP) reached 60 and 34%, respectively (Fig. 4b). The release rate of DTG within the zero order kinetics ranged between 9.4 μg/week (1:2 w/w PLGA/NMP) and 33 μg/week (1:8 w/w PLGA/NMP). Overall, DTG exhibited slower release kinetics compared to MK-2048 which could be attributed to its crystalline nature in the ISFI (Fig. 1f) and its lower solubility in NMP compared to MK-2048. The release kinetics of the various PLGA/NMP ratios for both MK-2048-ISFI and DTG-ISFIs (in the 1:2 and 1:4 PLGA/NMP ISFIs) were statistically significantly different (Kruskal–Wallis test, p < 0.0001). For both drugs, the data showed the ability to fine-tune the release kinetics in vitro by changing the ratio of PLGA to NMP. The difference in the release kinetics of MK-2048 and DTG was attributed to factors including the difference in drug loading (MK-2048 300 mg/mL, DTG 100 mg/mL), the affinity of the drug to NMP and PLGA, the solid state of drug in the ISFI (crystalline vs. amorphous), and the dissolution of the drug from the ISFI over time. Both drugs exhibited long-term release profiles from the ISFI demonstrating the promise of using these formulations as ultra-long-acting drug delivery systems.

Fig. 4 In vitro release kinetics. Cumulative release of MK-2048 over the course of 220 days (a) and DTG over the course of 120 days (b) from ISFI formulations containing varying ratios of PLGA to NMP. Drug release studies were carried out under sink conditions in 0.01 M PBS pH 7.4 + 2% solutol at 37 °C. Drug concentration was maintained constant across all PLGA/NMP ratios for each drug. All error bars are equivalent (s.d. positive and negative values) and represent standard deviation with n = 3. Release kinetics were significantly different across various PLGA/NMP ratios for both MK-2048 and DTG-ISFIs and across drugs at the same PLGA/NMP ratio (1:2 and 1:4 w/w PLGA/NMP) (Kruskal–Wallis test, p < 0.0001). Source data are provided as a Source Data file Full size image

In vivo ultrasound imaging

Ultrasound (US) imaging24,39,40,41 was used to assess implant bioerosion by measuring the volume of the four different ISFI formulations (1:2 and 1:8 w/w PLGA/NMP placebo and DTG-ISFIs) in vivo over 30 days. The effect of the PLGA/NMP ratio (1:2 vs. 1:8 w/w PLGA/NMP) and the effect of the presence of drug (placebo vs. DTG ISFIs) on depot degradation in vivo were assessed by measuring volume change of implants over 30 days. In the first 3 days, the US images depicted the natural influx of fluid and hardening of the implant over time from the outside to the inside, with a corresponding increase in ‘white’ contrast caused by the PLGA polymer coming out of solution (Fig. 5a, b). Over time all four formulations exhibited a reduction in implant volume (Fig. 6). Data in Fig. 6 indicated a large variance in implant volume measurements across animals. Notably, the 1:8 PLGA/NMP placebo formulation had a significantly smaller volume than the 1:8 DTG-ISFI formulation at all time the points analyzed except for the 48 hr and 7-day time points (p < 0.05). Also, the 1:8 DTG-ISFI formulation was significantly smaller than the 1:2 DTG-ISFI formulation at the 1 h, 72 h, and 14 day time points. A plot of implant volume change over time for individual mice is included with the supplementary information (Supplementary Fig. 4). In addition, a plot of raw and model-estimated mean % volume of implant, by group and time is included in the supplementary information (Supplementary Fig. 5). When comparing the volume change at day 30 for the 1:2 PLGA/NMP ISFI formulation with and without DTG, the mean volume at day 30 (n = 7) relative to day 0 (100%) was 48 and 39% for placebo and DTG ISFIs respectively (Supplementary Fig. 5). This data shows that the volume decrease due to PLGA degradation was ~1.73%/day for placebo implants compared to 1.37%/day for DTG implants. This was calculated based on the 52% (V day30 = 48% V day0 ) and 41% (Vd ay30 = 39% V day0 ) volume reduction over 30 days for placebo and DTG ISFIs, respectively. These data support the hypothesis that higher solvent content leads to greater change in implant volume, and that the placebo ISFI erodes more rapidly than ISFI with drug. When an ISFI containing a higher amount of NMP is injected, a larger amount of solvent diffused out leaving behind a smaller implant of PLGA (placebo) or PLGA/drug (DTG-ISFI). In addition, when more solvent diffused out, a larger volume of water diffused into the polymer implant, which in turn accelerated the ester hydrolysis of the PLGA implant. The ability to control the rate of implant erosion by changing the ratio of PLGA/NMP demonstrates the flexibility to fine-tune the formulation.

Fig. 5 Ultrasound (US) images of the ISFI after subcutaneous injection. ISFI formulation containing 1:2 w/w PLGA/NMP was injected (65 µL) s.c. into nude (nu/nu) mice (n = 7) and imaged using volumetric ultrasound. Images illustrate 2D slices from one plane through the same subcutaneous implant volume at days 0 and 2 (a, b, respectively). The black center region (yellow arrow) indicates the portion of the implant remaining in the liquid state (presumably, NMP solvent), which decreased after initial injection. c is the same implant as b, except with green arrows delineating the outer implant boundary, indicated by a fine grayscale demarcation in the ultrasound image. Volume measurements were obtained by a reader who measured the approximate implant dimensions on two orthogonal image planes, resulting in length, width and depth measurements to estimate ellipsoid volume (volume of the liquid core was not separately quantified in this study). Scale bars indicate 1 mm Full size image

Fig. 6 Analysis of the change in implant volume over time. Measurement of the implant volume decrease that occurs over 30 days calculated from 2D in vivo ultrasound images for four different ISFI formulations (n = 7 mice per group, see also Supplementary Figs. 4 and 5). Boxes represent 25th to 75th percentiles, and whiskers the range from the minimum to maximum values. Source data are provided as a Source Data file Full size image

In vivo pharmacokinetic studies

To assess drug release kinetics in vivo using the ISFI formulations, initial pharmacokinetic studies were carried out using single-drug ISFIs (Figs. 7a and 8). Formulations were prepared with six individual antiretroviral drugs in 1:2 w/w PLGA/NMP (PLGA 27 kDa). For each formulation, the indicated dose of the individual drug was injected subcutaneously into NOD scid gamma chain knockout mice (NSG) mice (n = 4) based on their body weight, and plasma samples were collected on day 1, 3, 7, 14, 21, and 30 and analyzed for drug concentration (Figs. 7a and 8). Dolutegravir and darunavir, together with ritonavir were administered at two different doses each to assess the effect of administered dose on drug release kinetics (Fig. 7b). Initial estimates for PK parameters were obtained through non-compartmental analysis (NCA) using WinNonlin Phoenix 6.1 (Pharsight, Mountain View, CA) on the composite median PK profile. Plasma concentrations were quantified using a validated high-performance liquid chromatography-tandem mass spectrometry LC/MS-MS method44 and were plotted over time in Fig. 7. The median composite concentration vs time profiles suggested a multiphasic elimination for all six individual drug ISFI formulations. After an initial rapid decline in plasma concentrations, drug release approached zero order kinetics (Fig. 7). For dolutegravir, darunavir, and ritonavir, administration of higher dose led to higher plasma concentration, however, a formal assessment of dose proportionality was not conducted (Fig. 7b). Comparing dolutegravir, rilpivirine and darunavir when administered at equal dose (100 mg/kg), dolutegravir exhibited the highest plasma concentrations throughout the entire duration of the study. These results demonstrated that in vivo pharmacokinetics were drug dependent and exhibited different profiles when administered in the same ISFI formulation and equivalent dose. More hydrophobic drugs like atazanavir (LogP 4.5), rilpivirine (LogP 4.86) and ritonavir (LogP 3.9) exhibited a faster decrease in plasma concentration within the first 24–48 h (Fig. 7a). This can potentially be attributed to their high solubility in NMP and lower affinity for the PLGA matrix resulting in a greater burst release.

Fig. 7 Plasma concentration of various ARVs formulated as ISFI. ISFI formulations were administered subcutaneously to NSG mice (n = 4) and plasma samples were collected longitudinally over 30 days. a Plasma concentrations of individual ARVs, b comparison of ISFI made using two different concentrations of dolutegravir (250 and 100 mg/kg), darunavir (300 and 100 mg/kg), and ritonavir (33.8 and 11.4 mg/kg). Mean plasma concentrations + s.e.m. are shown. Protein-adjusted IC90 (PA-IC90) values are: dolutegravir 64 ng/ml, MK-2048 33 ng/ml, rilpivirine 12 ng/ml, darunavir 2.4 ng/ml, and atazanavir 14 ng/ml. Since ritonavir is used as a booster in these formulations its IC90 is not indicated. Source data are provided as a Source Data file Full size image

Fig. 8 In vivo release of various ARVs formulated individually or in combination. Indicated dose of antiretrovirals formulated individually (a, b) or in combination (c–e) as ISFI was injected subcutaneously into NSG mice and plasma concentrations were analyzed longitudinally. Plasma concentrations for each individual mouse are shown. a MK2048, n = 4. b Rilpivirine (RPV), n = 4. c Co-formulated darunavir (DRV) and ritonavir (RTV), n = 4. d Co-formulated atazanavir (ATV) and ritonavir (RTV), n = 4. e Co-formulated dolutegravir (DTG), darunavir (DRV) and ritonavir (RTV), n = 3 PA-IC90 of drugs in ISFIs are indicated with dotted lines in each panel and correspond to the following values: MK-2048 33 ng/ml (a), rilpivirine 12 ng/ml (b), darunavir 2.4 ng/ml (c, e), atazanavir 14 ng/ml (d), dolutegravir 64 ng/ml (e). Since ritonavir is used as a booster in these formulations its IC90 is not indicated Full size image

The ultra-long-acting release of drugs beyond 30 days and the ability to formulate multiple drugs in a single ISFI formulation are illustrated in Fig. 8. This data shows that all drugs exhibited sustained plasma concentrations when administered alone or in combination with other drugs. Plasma concentrations of MK-2048 and dolutegravir dosed at 550 mg/kg and 250 mg/kg respectively were 3× and 10× greater than the protein adjusted (PA)-IC90 respectively for at least 5 months post-administration. Both MK-2048 and dolutegravir exhibited sustained plasma concentrations for four months (DTG-ISFI) to up to one year (MK-2048-ISFI). Rilpivirine at a dose of 100 mg/kg exhibited a faster clearance with plasma concentrations dropping below the PA-IC90 by day 120 post-administration (1 of 4 mice had RPV below IC90 at day 60 post-administration). Darunavir was dosed at 300 mg/kg and exhibited an initial first order decline in plasma followed by sustained release of plasma concentrations at or above the PA-IC90 for at least 30 days. Fast clearance was also observed for atazanavir dosed at 300 mg/kg with plasma concentrations dropping below the PA-IC90 at day 10 post-administration. To establish the ability of the ISFI to deliver multiple drugs in a single injection, we combined in one formulation darunavir with ritonavir, in a second formulation we combined atazanavir with ritonavir, and in a third formulation we combined the integrase inhibitor dolutegravir with darunavir and ritonavir. Ritonavir in these formulations was included as a booster for the protease inhibitors45. Figure 8c, d, e show that combination of multiple ARV in single injection results in sustained release of all three drugs. These results demonstrate the ability to co-formulate multiple drugs into a single ISFI solution and to maintain high concentrations of drug in the plasma for several months.

Termination of drug delivery by ISFI removal

To determine the efficiency and kinetics of termination of drug delivery after ISFI removal, we administered DTG-ISFI (250 mg/kg) subcutaneously to five NSG mice. Four months after ISFI administration, the median concentration of DTG in plasma was 629 ng/ml (range 529–822 ng/ml) (Fig. 9). Implants were then removed by making a small incision into the mouse skin next to the implant injection site (Fig. 9a). Plasma DTG concentration was measured at 1, 3, 7, 14, and 21 days after implant removal (Fig. 9b). Interestingly, 24 h after implant removal (first time measured), DTG plasma concentrations dropped 36-fold to a median concentration of 17.3 ng/ml (range 5.4–132.0 ng/ml). At this time point, four out of the five animals had DTG plasma levels below its PA-IC90 (64 ng/ml). By day 3, the median DTG concentration was reduced to 1.8 ng/ml (range 1–27.6 ng/ml) (Fig. 9c). By day 7, DTG plasma concentrations were below the limit of detection in all but one mouse that had a DTG concentration of 8.0 ng/ml. This mouse had DTG plasma levels below the limit of detection by day 14 (Fig. 9b). Analysis of residual DTG in the removed implants and comparison to the dose administered showed that 4 months post ISFI administration, 30% of the original amount of DTG (range 28.6–33.7%) was still available in the implants. These data show that sustained release of drug from the ISFI can be efficiently stopped by removal of the implant.