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Multiparametric magnetic resonance imaging to characterize cabotegravir long‐acting formulation depot kinetics in healthy adult volunteers

Aim Cabotegravir long‐acting (LA) intramuscular (IM) injection is being investigated for HIV preexposure prophylaxis due to its potent antiretroviral activity and infrequent dosing requirement. A subset of healthy adult volunteers participating in a Phase I study assessing cabotegravir tissue pharmacokinetics underwent serial magnetic resonance imaging (MRI) to assess drug depot localization and kinetics following a single cabotegravir LA IM targeted injection. Methods Eight participants (four men, four women) were administered cabotegravir LA 600 mg under ultrasonographic‐guided injection targeting the gluteal muscles. MRI was performed to determine injection‐site location in gluteal muscle (IM), subcutaneous (SC) adipose tissue and combined IM/SC compartments, and to quantify drug depot characteristics, including volume and surface area, on Days 1 (≤2 hours postinjection), 3 and 8. Linear regression analysis examined correlations between MRI‐derived parameters and plasma cabotegravir exposure metrics, including maximum observed concentration (C max) and partial area under the concentration–time curve (AUC) through Weeks 4 and 8. Results Cabotegravir LA depot locations varied by participant and were identified in the IM compartment (n = 2), combined IM/SC compartments (n = 4), SC compartment (n = 1) and retroperitoneal cavity (n = 1). Although several MRI parameter and exposure metric correlations were determined, total depot surface area on Day 1 strongly correlated with plasma cabotegravir concentration at Days 3 and 8, C max and partial AUC through Weeks 4 and 8. Conclusion MRI clearly delineated cabotegravir LA injection‐site location and depot kinetics in healthy adults. Although injection‐site variability was observed, drug depot surface area correlated with both plasma C max and partial AUC independently of anatomical distribution.

Conclusion: MRI clearly delineated cabotegravir LA injection-site location and depot kinetics in healthy adults. Although injection-site variability was observed, drug depot surface area correlated with both plasma C max and partial AUC independently of anatomical distribution. Long-acting injectables have been clinically used to sustain therapeutic drug levels at a target site to reduce the frequency of dosing required. 4,5 Despite development of clinically available LA injectables, 6,7 questions remain about mechanisms that control drug release from the depot site, including impact of physiochemical characteristics (e.g., solubility, particle size, particle charge) on dissolution and influence of biologic factors, including depot size, shape, anatomical location, inflammatory response, perfusion and pH. 8 Significant plasma PK variability was noted in prior cabotegravir LA studies, [9][10][11] with potential factors contributing to this variability, including administration technique (i.e., injection into subcutaneous [SC] fat vs gluteal muscle) or dose volume (i.e., split vs unsplit injections).
In preclinical experiments, MRI was used to noninvasively identify drug depot location, volume and surface area changes in IM and SC locations following cabotegravir LA administration in rats. 12 Although ultrasonography is used widely for guidance of LA drug delivery and can be used to assess depot characteristics, 13,14 few reports use MRI to visualize IM injections or quantitatively characterize LA drug depot. Yet, injectable drug depot characteristics were identified, including inflammation and persistence, since the early clinical use of MRI. 15,16 Therefore, this exploratory substudy was designed to investigate use of MRI to clinically image the evolution of the cabotegravir LA injection depot and examine the depot morphometry and its relationship to plasma concentrations and noncompartmental analysis-derived plasma exposure metrics.
In this Phase I study, four men and four women were enrolled in an imaging substudy and underwent noncontrast-enhanced MRI following cabotegravir LA injection (Day 1) on Day 3 and on Day 8 to measure the injection depot following a single cabotegravir 600 mg IM targeted dose. Magnetic resonance imaging was performed ≤2 hours following injection (Day 1) and at Days 3 and 8 postinjection for morphometric assessment (i.e., volume, surface area), transverse relaxation time constant (T2) and apparent diffusion coefficient (ADC) quantification of the depot evolution.
What is already known about this subject • As an intramuscular injection, long-acting (LA) cabotegravir is promising for HIV-1 preexposure prophylaxis given its potent antiretroviral activity and infrequent dosing requirements  Table 1). The study consisted of a screening period, a 28-day oral cabotegravir leadin phase at a dose of 30 mg day À1 followed by a 14-to 42-day washout period to accommodate flexibility in clinic scheduling, and a single 3 mL IM dose of cabotegravir LA 600 mg (200 mg mL À1 cabotegravir dose concentration; Figure 1). Following dosing, participants returned for safety assessments and PK sampling of the blood plasma periodi- Review Board (Baltimore, MD) approved the study protocol and conduct. All participants provided written informed consent. This study was sponsored by ViiV Healthcare.   Both individual participant T2-weighted and ADC MRI parameters were quantified to determine if any unique physiologic characteristics to the depot could be identified (e.g., differentiating drug vs leukocyte infiltration; Figure 4A,B). In addition, investigating the depot heterogeneity using these measurements could be of value to detect drug agglomeration vs diffuse localization. Mean T2 values were lower in IM than in SC tissue compartments on Day 1, and a slight reduction in this parameter value was observed in both locations by Day 3 that was maintained through Day 8 ( Figure 4C). Conversely, ADC values were lower in SC than IM tissue on Day 1, and the SC depot ADC value dropped by Day 3 and was maintained at this level until Day 8 ( Figure 4D).  Table 2.

| Nomenclature of targets and ligands
Linear regression analysis was performed to assess the relationship between the MRI-derived parameters and plasma cabotegravir  (Table 3; Figure 6A,B). Interestingly, total depot surface area strongly correlated with several exposure metrics, including cabotegravir plasma C max and AUC 0-8wk (Table 3; Figure 6A). Although a trend towards positive correlations was also observed between total depot volume with cabotegravir exposure metrics, this trend was not as strong as with the total depot surface area. The stronger relationship between PK and depot surface area vs PK and depot volume is consistent with the Noyes-Whitney equation, which expresses drug-dissolution rate as a function of surface area. 19 Figure 6B); however, the T2 values for both RP and SC tissue depots were considerably higher than that observed in the IM depot at Day 1 ( Figure 4C). Therefore, these high values skewed the correlations.
No correlations were observed between total depot ADC values and cabotegravir exposure metrics.    26,28 However, needle track-associated retrograde drug spillage of cabotegravir LA into the SC location was observed after IM administration in some participants (e.g., participant 7; Figure 2E). In addition, the participant who was misinjected in the RP cavity exhibited a very different PK profile than the participants whose route of administration was either IM, IM and SC, or SC. The C max was significantly lower (18-41%) 9 its rates of dissolution and absorption are expected to be a function of depot surface area. 19 The dissolved amount of cabotegravir will be higher with increased volume of interstitial fluid and thus, associated with an increased level of inflammation. 9 At Day 8, the depot volume and inflammation level were the lowest for SC and RP injections, an observation that may explain the longer T max .
However, because of the small sample size in this study, definitive PK conclusions with respect to anatomical site are limited.
Noninvasive imaging approaches to assess LA drug distribution in humans for durations of weeks to months have been sparingly applied, because detection techniques to directly assess drug substance are not readily available. [29][30][31][32][33] Computed tomography has been used to assess LA injectable depot location variability between gluteal muscle and SC adipose fat and, in doing so, has been used to determine the association between BMI and IM misinjections. 28,34 Ultrasonography has traditionally been used to guide injectable therapies to the intended locations and has also been used in conjunction with elastography to characterize the dispersion and physiochemical properties of the drug. 13,14 Although MRI cannot be used to visualize the active drug substance in a solid-suspension formulation like cabotegravir LA, traditional excipients (e.g., oil, polyethylene glycol, mannitol, water) generate adequate MRI signal for detection and discrimination from biologic tissue-based signal. 30,32,35 Magnetic resonance imaging was also used to assess drug depot volume expansion in a patient receiving an intragluteal injection of diclofenac 2 mL in which a rapid increase in depot volume over the initial 24 hours was observed. 33 In a previous rodent imaging study, cabotegravir LA IM depots appeared to manifest a subacute inflammatory response caused by the active drug particles that led to increased oedema in the depot region, thereby resulting in additional high-intensity MRI signal. 12 Although the drug depot volume also increased in the SC-administered animals, the expansion observed in these animals was less than that observed for IM administration. Interestingly, the vehicle-administered depot volume appeared to quickly decrease below baseline, reflecting rapid dissipation of the excipients from the depot region and minimal oedema in the absence of drug substance.
Our findings of cabotegravir LA drug being distributed along IM fascial planes is consistent with previous clinical observations and with our previous preclinical MRI study findings, which were confirmed using imaging mass spectroscopy showing cabotegravir LA drug colocated within interfacial planes of muscles. 12,31,33 When examining which key imaging endpoints (e.g., volume, surface area, T2-weighted, ADC) were key markers associated with drug absorption and PK exposure, the drug depot surface area assessed within 2 hours of administration was highly correlated with early plasma cabotegravir PK, C max and with partial AUC 0-4wk . This is unsurprising because insoluble drug particle dissolution rates are directly related to specific surface area of the drug particle, 36,37 and dissolution studies showing surface area of insoluble drugs used as LA injectables correlated well with in vivo drug exposure. 21,38 However, in addition to drug particle size or surface area driving dissolution, the increased surface area of the depot has been shown to be associated with increased C max and AUC when administering two cabotegravir LA depots instead of a single depot to healthy volunteers. 9 Although all participants were administered the identical 3-mL volume in the present study, independent of injection-site location, the depot surface area rapidly increased during the first week postdose, perhaps reflecting less agglomeration and resulting in increased particle surface area exposure. Although total depot volume on Day 1 showed similar trends regarding correlations with early exposure metrics, these relationships were not as strong as with the total depot surface area on Day 1, suggesting that the shape of the depot could be irregular ( Figure 2D) and exhibit a greater surface area by volume ratio than theoretically possible assuming a spherical drug deposition. When examining relationships between IM and SC depot surface area or volume and plasma cabotegravir exposure metrics, there was less statistical power in the analysis given the fewer data points. However, although IM depot volume and surface area on Day 1 positively correlated with partial AUC 0-4wk , the SC contribution to the depot volume and surface area tended to negatively correlate with exposure metrics at this early time point. These disparate findings between IM and SC may suggest the ideal location for cabotegravir LA depot is in muscle as intended. Correlations between IM and SC depot volume and surface area and exposure metrics were lost after Day 1, possibly reflecting the depot size and shape being driven more by drug substance at Day 1, rather than by retrograde drug leak and oedema resulting from local subacute inflammatory response, which was more prevalent at later time points.
T2-weighted and ADC MRI parameters were assessed to examine whether they could differentiate between physiochemical agglomeration of drug substance and physiologic subacute inflammation.
Although these MRI-derived parameter assessments have been used previously to examine drug dissolution ex vivo, no reports exist of these applications for in vivo assessment of LA injectable properties. 39,40 If drug agglomeration occurred during the initial week postinjection, then either patterns of reduced T2 and ADC or an overall reduction in these values might have been detected. Nevertheless, no patterns or regions of significance were observed in T2 or ADC change in the images, and this was also reflected in the quantitative T2 and ADC values measured in total and IM and SC regional depots.
Differences existed in T2 between IM and SC depot locations as expected given that endogenous T2 values are higher in SC than IM tissue. 41 For example, the total depot T2 in participant 8 increased over time, simply reflecting the larger contribution of the SC compartment throughout the imaging week. Alternatively, ADC values tend to be higher in IM than SC tissue, 42 so total depot ADC in the same participant decreased over time. The strong negative correlations between Day 1 total depot T2 values and exposure metrics are due to the significantly higher T2 in both SC and RP depots, which were also associated with lower cabotegravir LA drug levels at this time.
MRI was used to clearly delineate injection-site location and assess cabotegravir LA depot kinetics. Although injection-site variability was associated with the current study, surface area of the cabotegravir LA depot appeared to strongly correlate with both plasma C max and partial AUC independently of anatomical distribution. Imaging has potential for interrogating the physiological processes involved in LA drug depot formation and its evolution. These imaging data may help inform the development of LA therapeutics and how to optimize LA injectable physicochemical properties, including drug substance particle size distribution and LA injectable formulation variables, such as selection of excipients (i.e., surfactants, stabilizers).