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Differential Penetration of Raltegravir throughout Gastrointestinal Tissue: Implications for Eradication and Cure
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AIDS: POST ACCEPTANCE, 11 February 2013

Patterson, Kristine B.; Prince, Heather A.; Stevens, Trenton; Shaheen, Nicholas J.; Dellon, Evan S.; Madanick, Ryan D.; Jennings, Steven; Cohen, Myron S.; Kashuba, Angela D.M.

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Objective: To investigate the concentration of the integrase strand inhibitor raltegravir (RAL) throughout gastrointestinal (GI) tissue, especially gut-associated lymphoid tissue (GALT), as an adjunct to current prevention and cure strategies.

Design: Open-label pharmacokinetic study

Methods: HIV-negative men received RAL 400 mg twice daily for 7 days. Seven blood plasma (BP) specimens were collected over 12-hr intervals; timed tissue specimens from terminal ileum, splenic flexure, and rectum were also obtained by colonoscopy following the first dose (FD) and on Day 7 [Multiple Dose (MD)]. RAL concentrations were measured by validated LC-MS assay with 1ng/mL lower limit of detection. Data were analyzed by noncompartmental methods (WinNonlin 6). Tissue exposures are reported as composite medians and tissue density of 1.04 g/mL is assumed for comparisons.

Results: Fourteen men completed evaluations. Median (range) age was 24 (19-49) yrs and BMI 25 (19-31) kg/m2. After the FD, AUC-0-12h was highest in the terminal ileum (594 [mu]g*h/mL). Exposures were 160, 68 and 39-fold greater than BP at the terminal ileum, splenic flexure and rectum, respectively. After multiple doses, exposure was highest at the splenic flexure (2240 [mu]g*h/mL); exposure at the terminal ileum and rectum were equivalent (both 788 [mu]g*h/mL). Following multiple doses, exposures were 160-650-fold greater than BP throughout the colon.

Conclusions: RAL rapidly disseminates into GI tissue and concentrations remain significantly higher than BP. RAL exposure in GI tissue remains higher than any ARV investigated to date. These data suggest that RAL should result in full suppression of viral replication in GI tissue and GALT.


Combination antiretroviral therapy (ART) results in significant reduction in morbidity and mortality in those individuals who achieve sustained virologic suppression [1-3]. Efforts are now focusing on eradication of the HIV epidemic, which require the prevention of new infections and the elimination of HIV from infected individuals.

Two methods of 'cure' are being considered: the complete elimination of HIV from infected individuals or rendering the virus incapable of further replication ('functional cure'). Complete eradication, however, is obstructed by pools of latently infected resting CD4. T cells capable of viral replication [4,5] that are established early in infection [6], and are not protected by the rapid initiation of ART following infection [7]. Gut-associated lymphoid tissue (GALT) is the largest aggregate of lymphoid tissue within the body. GALT contains a high concentration of activated CD4. T cells susceptible to HIV infection, the majority of which die quickly as a result of viral cytopathic effects or host responses [8-10]. However, a minority of infected CD4. T cells survive, reverting to a latent resting state [6,11], and are capable of viral replication when activated [5,12]. These cells may be protected from the effects of potent antiretroviral therapy despite suppression of plasma viremia [4,13].

Numerous strategies aimed towards reversing latency or activating resting infected CD4. T cells are currently being investigated [14-21]. The integrase strand transfer inhibitors such as raltegravir prevent the integration of reverse transcripts into newly infected CD4. T cells, aborting ongoing rounds of viral replication. These drugs are well-tolerated with minimal drug-drug interactions [22]. However to be efficacious, these agents require rapid and high penetration into lymphoid tissue where latent CD4. T cells reside. Our previous investigations demonstrated wide variability in the penetration of ARVs into rectal tissue [23,24]. However, all segments of gastrointestinal tissue may not be equally permissive to ARVs, nor representative of the entire colon. Identifying therapeutics achieving high penetration into viral reservoirs is a key component to the HIV cure initiative. The primary objective of the following investigation was to determine the penetration of raltegravir into three segments of the gastrointestinal tract, including GALT, after single and multiple dosing.


Subject demographics, disposition, and safety

Nineteen men screened for this study: 15 were enrolled, and 14 completed the protocol. One subject was withdrawn from participation following the first pharmacokinetic visit due to significant findings on colonoscopy. This subject did not contribute demographic or pharmacokinetic data, and was referred for appropriate follow up. Median (range) age of the 14 evaluable participantswas 24 (19-49) years, weight was 80.1 (56.5-104.9) kg, and body mass index was 25.3 (19.0-30.9) kg/m2. Nine (64%) subjects were Caucasian, and five (36%) were African American. Subjects tolerated the study medication well. One subject had a mild increase in preexisting intermittent elevated alanine aminotransferase (ALT), which returned to normal prior to study discharge. Two evaluable subjects had incidental findings of polyps on initial colonoscopy.


Table 1 summarizes the pharmacokinetic parameters, relative exposures compared to BP, and accumulation over time for each matrix. Figure 1a and 1b represent the pharmacokinetic profiles of all biological matrices following the 1st and 13th dose, respectively, over a 12-hour dosing period. Raltegravir concentrations throughout the GI tract were similar to blood plasma within one hour following the first dose, achieve steady-state concentrations within 12 hours after the first dose, and remain considerably higher than blood plasma.

Blood Plasma

Following the first dose, the AUC0-12h of raltegravir in blood plasmawas 3.7 (1.5-9.5) mghr/mL, and 3.4 (1.5-20.5) mghr/mL after multiple doses. Raltegravir did not accumulate in blood plasma over the study period (MD to FD ratio: 0.9). Overall, the pharmacokinetic profiles in blood plasma following the first dose and multiple doses were consistent with previous results in HIV-negative [26] and HIV-positive [27] women.

Terminal ileum

After the first dose, raltegravir readily penetrated the terminal ileum with concentrations equivalent to blood plasma 1-hour post dose and exceeding blood plasma within 2 hours. Peak concentrations were reached by 4 hours post-dose and remained higher than other segments of the colon for the duration of the first 12-hour dosing period (Fig. 1). Pharmacokinetic parameters are listed in Table 1. The concentration 12 hours after the first dose (C12h) was 64mg/mL. AUC0-12h was 160-fold higher than blood plasma [594 vs. 3.7 (IQR 1.5-9.5) ug*hr/mL]. With repeated doses, tissue exposure increased by 30% (788ug*hr/mL), while plasma exposure remained stable (3.7 vs. 3.4ug*hr/mL).C12h concentrations were lower after multiple doses versus the first dose (7.8 vs. 64ug/mL). However, all C12h concentrations were >80-fold higher than blood plasma concentrations across all doses. Cumulative tissue exposure was essentially unchanged (MD: FD ratio of 0.9) and was 156-fold higher than blood plasma.

Splenic flexure

The maximum concentration at the splenic flexure was reached 10 hours after the first dose and was 49-fold higher than blood plasma. C12h was similar to concentration in the terminal ileum (59 vs. 64ug/mL). Following multiple doses, the AUC0-12h increased from 258 to 2240mghr/mL, which was approximately 650-fold higher than blood plasma. Raltegravir accumulated in the area of the splenic flexure more than any other area within the gastrointestinal tract following multiple doses (accumulation ratio=8.7).


Following the initial dose, the AUC0-12h was lower in the rectum (143ug*hr/mL) than in other segments of the colon, but accumulated 5-fold with repeated dosing (first dose AUC0-12 143ug*hr/mL; multiple dose AUC0-12h 788ug*hr/mL). C12h were 40% lower compared to the terminal ileum (64ug/mL) after the first dose, but remained >200-fold higher than blood plasma concentrations across the multiple dosing period.


Within the terminal ileum, raltegravir rapidly penetrates and surpasses plasma exposure by 160-fold after a single dose. Blood plasma concentrations following 7 days of dosing are achieved in gastrointestinal tissue within 10 hours of dosing. Raltegravir accumulates throughout the GI tissue with exposures 150-600 fold greater than blood plasma following repeated dosing. These concentrations are the highest reported for any orally-dosed antiretroviral in gastrointestinal tissue to date [28]. Previous investigations by our group and others have shown wide variability in colorectal tissue penetration and assumptions between specific antiretroviral agents or classes cannot be made. In HIV-negative men, darunavir exposure in rectal tissue is 2.7 fold higher than blood plasma [24]. The CCR5 antagonist maraviroc exposure in rectal tissue is even higher: 9-fold following a single dose and 27-fold higher than blood plasma following multiple doses [23]. Following multiple doses, etravirine exposure is 2-fold higher than blood plasma [24]. The wide variability in exposure between drugs with similar mechanisms of action and within the same tissue is exemplified in the NRTIs tenofovir and emtricitabine. In rectal tissue, tenofovir exposures are 46-fold higher while emtricitabine exposures are 2.6-fold higher than blood plasma following a single dose [29].

The reported concentrations are dependent on the methods in which the antiretroviral agents are being measured. For example, low or sub-therapeutic concentrations of atazanavir, tenofovir and emtricitabine were found in isolated mononuclear cells from the colorectal tissue of 5 HIV-infected men [30,31]. In contrast our previous [23,24,29,32] and current work quantifies the drug of interest in homogenized tissue. Others have measured intracellular concentrations of raltegravir from peripheral blood mononuclear cells using methods similar to intracellular reverse transcriptase inhibitors [33,34]. However, underestimation of drug concentrations may result from analyzing specific cells from colonic tissue due to the difficulty in obtaining sufficient quantities of in vivo tissue required to accurately identify and isolate enough cells. In contrast, the use of tissue homogenates may result in an overestimation in drug exposure, as both intracellular and extracellular concentrations are being measured. These studies emphasize the importance in developing standard methods for measuring drug concentrations in specific matrices in order to expedite research priorities such that results across investigations can be compared and target concentrations based on specific objections (eg eradication, treatment, or prevention) can be determined.

Eradication priorities have been recently outlined [35]. Embedded within these priorities are strategies aimed towards disrupting proviral quiescence in latently infected CD4. T cells and using a multi-dimensional approach to block production of infectious virions. The viral decay kinetics of raltegravir [36-38] suggests that as resting CD4. T cells are activated, these virions could be quickly rendered non-infectious. Our findings demonstrate that raltegravir achieves maximum concentrations in GALTwithin 12 hours after a single dose, and suggests that an extended dosing period would not be required before interventions aimed at activating the latent reservoir were initiated. Although it could be theorized that the high tissue concentrations quantifiable within 1 hour of dosing are due to rapid gastrointestinal absorption following a complete bowel preparation, plasma concentrations are similar to those in our previous investigations [26,27] suggesting that absorption is not significantly altered by the bowel preparation. Additionally, we have noted rapid drug delivery to colorectal tissue with maraviroc, darunavir, and etravirine in subjects where bowel preparation was not performed [23,24].

Furthermore, a mechanism by which efficacy of potential strategies is easily quantified and reflective of all lymphoid tissue is essential. Rigorous investigations following antiretroviral intensification with raltegravir demonstrate the lack of effect on low-level plasma viremia [39-42], which may not reflect alterations elsewhere in the body. For example, seven HIV-infected individuals were noted to have a reduction in proviral DNA in GALT following raltegravir intensification [43]. In acutely infected individuals in which there is rapid depletion of active CD4. T cells, the prompt initiation of 'megaART'-- including raltegravir and the CCR5-receptor agonist maraviroc to standard ART--results in a reduction in HIV DNA from rectal tissue [44]. Similarly, the marked reduction of rectal tissue HIV DNA in rhesus macaques with chronic simian immunodeficiency virus is also seen with the addition of raltegravir and maraviroc to standard antiretroviral therapy [45]. Depending on the application, as this investigation found, targeting different areas of the colon (eg. GALT vs. colorectal tissue) may be necessary to make precise measures of drug exposure and determine subsequent pharmacokinetic predictors of drug efficacy.

This study reinforces that tissue pharmacokinetics can be unpredictable, and need to be evaluated in the relevant tissues for the appropriate clinical application. We have demonstrated that precisely timed tissue specimens across the gastrointestinal tract can be obtained and data quantified for informative pharmacokinetic information. In this study, we demonstrate that raltegravir can rapidly and efficiently penetrate GALT and colorectal tissue. Identifying antiretrovirals that can enter these viral reservoirs at highly active concentrations can facilitate the selection of optimal therapy for investigations in the prevention and cure of HIV infection.

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