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Differential penetration of raltegravir throughout gastrointestinal tissue: implications for eradication and cure

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

doi: 10.1097/QAD.0b013e32835f2b49
Clinical Science

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 (PK) study.

Methods: HIV-negative men received RAL 400 mg twice daily for 7 days. Seven blood plasma specimens were collected over 12-h intervals; timed tissue specimens from terminal ileum, splenic flexure, and rectum were also obtained by colonoscopy following the first dose and on day 7 [multiple dose (MD)]. RAL concentrations were measured by validated LC–MS assay with 1 ng/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) years and BMI 25 (19–31) kg/m2. After the first dose, area under the time-concentration curve (AUC)0-12h was highest in the terminal ileum (594 μg*h/ml). Exposures were 160, 68 and 39-fold greater than blood plasma at the terminal ileum, splenic flexure and rectum, respectively. After multiple doses, exposure was highest at the splenic flexure (2240 μg*h/ml); exposure at the terminal ileum and rectum were equivalent (both 788 μg*h/ml). Following multiple doses, exposures were 160 to 650-fold greater than blood plasma throughout the colon.

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

aUniversity of North Carolina Department of Medicine

bUniversity of North Carolina Eshelman School of Pharmacy, Chapel Hill, North Carolina, USA.

Correspondence to Kristine B. Patterson, MD, University of North Carolina at Chapel Hill, 130 Mason Farm Road, CB 7215, Chapel Hill, NC 27599-7215, USA. Tel: +1 919 843 2540; fax: +1 919 966 8928; e-mail:

Received 7 November, 2012

Revised 16 January, 2013

Accepted 18 January, 2013

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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 ART 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 in which latent CD4+ T cells reside. Our previous investigations demonstrated wide variability in the penetration of antiretrovirals into rectal tissue [23,24]. However, all segments of GI tissue may not be equally permissive to antiretrovirals, 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 GI tract, including GALT, after single and multiple dosing.

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Study design and participant selection

Healthy HIV-negative male volunteers were enrolled in this 7-day, open-label, investigator-initiated, pharmacokinetic study at the University of North Carolina at Chapel Hill (UNC). The UNC Biomedical Review Board approved the study, which was registered with (NCT01325051). All visits were conducted in the UNC Clinical and Translational Research Unit. All participants provided written informed consent prior to study procedures. Participants were enrolled from April to November 2011.

Participants were screened within 42 days before study medication dosing. Screening procedures consisted of a complete medical history and physical examination, 12-lead electrocardiogram (ECG) with cardiology interpretation, comprehensive laboratory studies (complete blood count with differential, liver function tests, serum chemistries, urinalysis, and urine toxicology). Participants were screened for active hepatitis B and hepatitis C, and HIV (ARCHITECT HIV Ab/Ag Combo; Abbott Laboratories, Saint-Laurent, Quebec, Canada). All testing was performed in the UNC Healthcare McClendon Laboratories. Testing for rectal sexually transmitted infections, including Neisseria gonorrhea, Chlamydia trachomatis was performed in the Microbiology Core Laboratory of the Southeastern Sexually Transmitted Infections Cooperative Research Center (Gen-Probe Aptima, San Diego, California, USA).

Participants were eligible to participate if they were healthy men between 18 and 49 years of age, and had a BMI 18–30 kg/m2 with a total body weight at least 50 kg. Participants were required to have a fully intact GI tract. Participants agreed not to participate in the conception process, abstain from all sexual intercourse and the use of intra-rectal products for 72 h before study entry until study discharge. Participants were excluded for any clinically significant abnormal laboratory tests, physical findings or clinical condition that would interfere with study procedures. Participants were required to stop all prescription and nonprescription medications 7 days before and herbal supplements 14 days before study enrollment, and medications could not be restarted until study completion. Participants were limited to consumption of less than 14 alcoholic beverages per week, and acetaminophen at doses of 1 g per day or less.

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Study visits

Participants received raltegravir 400 mg orally twice daily on days 1–6 and a single dose on day 7 for a total of 13 doses. Participants participated in two intensive pharmacokinetic (PK) visits, coinciding with the first and last dose of study drug. No less than 24 h prior to both PK visits, participants began a clear liquid diet without solid food, milk, coffee, tea or any product containing red or purple dye. Participants were admitted to the inpatient research unit 18 h prior to each colonoscopy, during which time an observed full bowel preparation with a gallon of polyethylene glycol consumed in 4 h or less was performed. At both PK visits, blood was collected within 30 min before observed dose followed by 1, 2, 3, 4, 6, 8, and 12 h postdose. Each participant was assigned to a colonoscopy procedure time coinciding with a postdose time, which was identical for both first dose and steady state (multiple dose) procedures. Two participants were assigned to one of seven potential times (1, 2, 3, 4, 6, 8, and 12 h postdose). All participants were observed for at least 6 h postcolonoscopy. Participants recorded on a drug diary card the time of all outpatient doses on days 2–6, and were contacted by telephone each day to assess adverse events and compliance with study medication dosing. Participants returned to the clinic for a follow-up safety visit 7–10 days after final dose.

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Sample collections

Blood plasma

Whole blood was obtained using K2EDTA collection tubes (BD Diagnostics, Franklin Lakes, New Jersey, USA) and centrifuged at 800g at 4°C for 10 min. Aliquots of the resulting blood plasma were distributed into labeled cryovials and stored at −80°C until analysis.

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Gastrointestinal tissue

Intestinal tissue samples were obtained during colonoscopy at one of two UNC Gastrointestinal (GI) Procedure Suites according to standard GI procedures. Monitored conscious sedation was provided for all participants. Biopsy sites were identified and rinsed with sterile irrigation water containing simethicone 40 mg oral drops (40 mg/0.6 ml; Major Pharmaceuticals, Livonia, Michigan, USA) before collection. Biopsies were taken from each of three locations: terminal ileum, splenic flexure, and rectum (approximately 15 cm from the anal verge). Five double biopsies were collected at each site using a Radial Jaw 4 Large Capacity Forceps with needle (Boston Scientific, Natick, Massachusetts, USA). Biopsy specimens from each site were pooled into a single labeled cryovial, and immediately snap-frozen in liquid nitrogen. All specimens were stored at −80°C until analysis.

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Sample analysis

Raltegravir concentrations in blood plasma and intestinal tissue were analyzed by a validated liquid chromatography–mass spectrometry assay with a 1 ng/ml lower limit of detection.

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Statistical analysis

Pharmacokinetic parameters were estimated using noncompartmental methods (Phoenix WinNonlin Pro 5.2; Certara, L.P., St. Louis, Missouri, USA). Maximum concentration (C max), time at maximum concentration (T max), and concentration 12 h after dosing (C 12h) were determined by visual inspection of the participant profiles, and used the log-linear trapezoidal method to calculate the area-under-the-time-concentration-curve over the 12-h dosing interval (AUC0-12h). A tissue density of 1.04 g/ml was used to convert ng/g to ng/ml [25]. For tissue, composite concentration–time profiles were created using median concentrations for calculating and reporting pharmacokinetic parameters. Descriptive statistics using WinNonlin were performed. Demographic data are presented as median (range). Pharmacokinetic data are presented as median (25th–75th percentile) for plasma, and as a single composite value from median tissue concentrations. Accumulation ratios were calculated utilizing the equation multiple dose AUC0-12h/first dose AUC0-12h.

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Participant demographics, disposition, and safety

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

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Table 1 summarizes the pharmacokinetic parameters, relative exposures compared to blood plasma, 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-h dosing period. Raltegravir concentrations throughout the GI tract were similar to blood plasma within 1 h following the first dose, achieve steady-state concentrations within 12 h after the first dose, and remain considerably higher than blood plasma.

Table 1

Table 1

Fig. 1

Fig. 1

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Blood plasma

Following the first dose, the AUC0-12h of raltegravir in blood plasma was 3.7 (1.5–9.5) μg*h/ml, and 3.4 (1.5–20.5) μg*h/ml after multiple doses. Raltegravir did not accumulate in blood plasma over the study period [multiole doses (MD) : first dose (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.

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Terminal ileum

After the first dose, raltegravir readily penetrated the terminal ileum with concentrations equivalent to blood plasma 1-h postdose and exceeding blood plasma within 2 h. Peak concentrations were reached by 4 h postdose and remained higher than other segments of the colon for the duration of the first 12-h dosing period (Fig. 1). Pharmacokinetic parameters are listed in Table 1. The concentration 12 h after the first dose (C 12h) was 64 μg/ml. AUC0-12h was 160-fold higher than blood plasma [594 vs. 3.7 (IQR 1.5–9.5) μg*h/ml]. With repeated doses, tissue exposure increased by 30% (788 μg*h/ml), whereas plasma exposure remained stable (3.7 vs. 3.4 μg*h/ml). C 12h concentrations were lower after multiple doses vs. the first dose (7.8 vs. 64 μg/ml). However, all C 12h concentrations were more than 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.

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Splenic flexure

The maximum concentration at the splenic flexure was reached 10 h after the first dose and was 49-fold higher than blood plasma. C 12h was similar to concentration in the terminal ileum (59 vs. 64 μg/ml). Following multiple doses, the AUC0-12h increased from 258 to 2240 μg*h/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 GI tract following multiple doses (accumulation ratio = 8.7).

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

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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 GI tissue within 10 h 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 GI tissue to date [28].

Previous investigations by our group and others have shown wide variability in colorectal tissue penetration reinforcing that 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: nine-fold following a single dose and 27-fold higher than blood plasma following multiple doses [23]. Following multiple doses, etravirine exposure is two-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 whereas 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 five 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 (e.g. 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 multidimensional 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 noninfectious. Our findings demonstrate that raltegravir achieves maximum concentrations in GALT within 12 h 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 h 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 participants in whom 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 ART [45]. Depending on the application, as this investigation found, targeting different areas of the colon (e.g. 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 GI 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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The authors appreciate the efforts of the UNC Gastrointestinal Procedure nurses in coordinating the precise timing of colonoscopies.

Supported in part by National Institutes of Health grants R37 DK49381 (M.S.C.), R34 AI087065 (A.D.M.K.), K23 AI773355 (K.B.P.), U01 AI095031 (A.D.M.K.), P30 AI50410 (UNC Center for AIDS Research), and UL1 RR025747 (UNC TraCS Clinical Translational Research Center). Supported in part by the Merck Investigator Initiated Research Program. Rectal sexually transmitted disease screening was performed in the Microbiology Core Laboratory of the Southeastern Sexually Transmitted Infections Cooperative Research Center under the direction of Dr Marcia Hobbs and supported by NIH grant U19 AI31496.

Author Contributions: K.B.P., M.S.C. and A.D.M.K. conceived and designed the experiments. H.A.P. and T.S. coordinated the specimen collections. N.J.S., E.S.D., R.D.M. performed the colonoscopies and obtained the biopsy specimens. S.J. analyzed the specimens. K.B.P. and A.D.M.K. analyzed the data. K.B.P. and A.D.M.K. created the presentation of some of the data for the 13th International Workshop on Clinical Pharmacology of HIV Therapy April 16–18, 2012 Barcelona, Spain. K.B.P. and A.D.M.K. wrote the article. All authors reviewed the article.

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Conflicts of interest

K.B. P. and A.D.M.K. have received investigator initiated research grants from Merck and GlaxoSmithKline. The remaining authors have no competing interests.

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gut-associated lymphoid tissue; HIV cure; latency; pharmacokinetics; raltegravir

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