Bioavailability of Sulforaphane Following Ingestion of Glucoraphanin-Rich Broccoli Sprout and Seed Extracts with Active Myrosinase: A Pilot Study of the Effects of Proton Pump Inhibitor Administration.

We examined whether gastric acidity would affect the activity of myrosinase, co-delivered with glucoraphanin (GR), to convert GR to sulforaphane (SF). A broccoli seed and sprout extract (BSE) rich in GR and active myrosinase was delivered before and after participants began taking the anti-acid omeprazole, a potent proton pump inhibitor. Gastric acidity appears to attenuate GR bioavailability, as evidenced by more SF and its metabolites being excreted after participants started taking omeprazole. Enteric coating enhanced conversion of GR to SF, perhaps by sparing myrosinase from the acidity of the stomach. There were negligible effects of age, sex, ethnicity, BMI, vegetable consumption, and bowel movement frequency and quality. Greater body mass correlated with reduced conversion efficiency. Changes in the expression of 20 genes in peripheral blood mononuclear cells were evaluated as possible pharmacodynamic indicators. When grouped by their primary functions based on a priori knowledge, expression of genes associated with inflammation decreased non-significantly, and those genes associated with cytoprotection, detoxification and antioxidant functions increased significantly with bioavailability. Using principal components analysis, component loadings of the changes in gene expression confirmed these groupings in a sensitivity analysis.

Development of rectal enema as microbicide (DREAM): Preclinical progressive selection of a tenofovir prodrug enema.

HIV pre-exposure prophylaxis (PrEP) strategies have the potential to prevent millions of incident HIV infections each year. However, the efficacy of PrEP strategies has been plagued by issues of non-adherence, likely because of the difficulty in motivating otherwise healthy people to adhere to treatment regimens that require significant behavioral changes and daily discipline. An alternative approach to PrEP is to focus on strategies that fit in to normal, and even desirable, sexual behaviors, such as the use of cleansing enemas by men who have sex with men (MSM) prior to receptive anal intercourse (RAI). Here, we describe preclinical efforts toward optimizing a tenofovir (TFV)-based enema formulation for rectal PrEP. Using a murine model, we compared the plasma and tissue pharmacokinetics of TFV and various TFV prodrugs, including tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), and hexadecyloxypropyl tenofovir (CMX157), after dosing as enema formulations with varying osmolality and ion content. We observed that the enema vehicle composition played a more important role than the TFV prodrug properties in achieving rapid and therapeutically relevant tenofovir diphosphate (TFV-DP) concentrations in mouse colorectal tissue. Our results support the next steps, which are further preclinical (non-human primate) and clinical development of a hypo-osmolar TFV enema product for rectal PrEP.

Fecal Coliform Bacterial Detection to Assess Enema Adherence in HIV Prevention Clinical Studies.

Evaluating the efficacy of any HIV prevention strategy is dependent on ensuring and objectively monitoring adherence to the intervention. Medicated rectal enemas are a potential method for providing topical, episodic HIV prophylaxis during receptive anal intercourse. Assessing adherence to recommended enema dosing regimens is essential in evaluating the utility of this strategy. We utilized fecal coliform bacteria on used enema tips as a marker for enema use. Enema tip coliforms were tested by repurposing a microtiter plate-based water quality test designed to detect fecal contamination of water. Coliform detection occurred with 100% sensitivity and specificity when tips were assayed on day of use. The assay performed well post-7 day sample storage at room temperature, yielding a sensitivity of 80% and specificity of 93%. All (n = 64) samples collected in a subset of the DREAM-01 rectal microbicide enema clinical trial tested positive, even when tips were evaluated > 7 days post-reported use. The coliform-based enema tip assay allows monitoring of adherence in interventions involving rectal enemas in a sensitive, specific and inexpensive manner. The test performs well in clinical trial settings.

Rectal Douching Practices Associated with Anal Intercourse: Implications for the Development of a Behaviorally Congruent HIV-Prevention Rectal Microbicide Douche.

Tenofovir administration via rectal douching results in higher rectal-mucosa drug concentration than oral administration. Many who engage in receptive anal intercourse (RAI) use cleansing rectal douches. To inform development of a behaviorally-congruent tenofovir douche, 4751 individuals ≥ 18 years-old, born male, from all US states/territories, who engaged in anal intercourse responded to an online survey. Of those who reported RAI in the prior 3 months, 80% douched beforehand, 82% within 1 h, mean 2.9 consecutive applications; 27% douched afterwards, 83% within 1 h, mean 1.7 consecutive applications. Among multidose users, 78% applied doses within 2 min, and 76% retained liquid < 1 min. Most used tap water (89%) in an enema bottle (50%) or rubber bulb (43%), and douched for cleanliness (97%), to avoid smelling bad (65%), and to enhance pleasure (24%). 98% reported high likelihood of using an HIV-prevention douche. An ideal product will protect within a user's typical number of applications, within 1 h, and be dissolvable in tap water.

MALDI Mass Spectrometry Imaging Reveals Heterogeneous Distribution of Tenofovir and Tenofovir Diphosphate in Colorectal Tissue of Subjects Receiving a Tenofovir-Containing Enema.

Efforts to prevent human immunodeficiency virus (HIV) infection via pre-exposure prophylaxis (PrEP) include the development of anti-HIV drugs as microbicides for topical application to the mucosal sites of infection; however, although understanding the distribution profiles of these drugs in target mucosal tissues is of critical importance to guiding their optimization, data in this regard are largely lacking. With this in mind, we developed a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) approach to visualize tenofovir (TFV), an HIV nucleotide analog reverse-transcriptase inhibitor under investigation for use as a topical microbicide, and its active metabolite TFV-diphosphate (TFV-DP) in colorectal biopsies obtained from healthy volunteers who received TFV-containing enemas. Application of MALDI MSI resulted in sufficient spatial resolution to visualize both TFV and TFV-DP and revealed heterogeneity in the distribution profiles of both analytes, including the presence of regions in which TFV and TFV-DP were undetectable, in colorectal tissue at two different time points and concentrations. Cell-specific staining for CD4 T and CD11c dendritic cells, which are important to the establishment of HIV infection, demonstrated that the TFV and TFV-DP distributions were independent of these cell types. MALDI MSI of endogenous lipids demonstrated that the heterogeneity observed for TFV and TFV-DP was not a function of tissue composition or processing. These data provide unique insight into the spatial distribution of TFV and TFV-DP in human colorectal tissue. In addition, this work establishes an approach that can be leveraged to directly detect and visualize these clinically important analytes more broadly in tissue.

Rectal Douching Associated with Receptive Anal Intercourse: A Literature Review.

To inform the development of HIV-prevention rectal douches, we reviewed the scientific literature and online instructional videos on rectal douching associated with receptive anal intercourse (RAI). Up to 88% of men who practice RAI ever have douched, while 43-64% have douched recently. Of them, 87-97% douche before RAI and 13-48% afterwards. Water, occasionally mixed with soap or salt, is used most often, although up to 31% of men use commercial products. Douching is more common among individuals reporting substance use, sexually transmitted infections, or being HIV-infected. Scant literature is available on women's rectal douching practices, but it is apparently less frequent than among men (32 vs. 70%). Videos advise using 2-3 doses of liquid and retaining it for 10-30 s before expelling. These findings can inform the development of a safe and acceptable rectal douche for HIV prevention.

Hypo-osmolar Formulation of Tenofovir (TFV) Enema Promotes Uptake and Metabolism of TFV in Tissues, Leading to Prevention of SHIV/SIV Infection.

Oral preexposure prophylaxis (PrEP) has been approved for prophylaxis of HIV-1 transmission but is associated with high costs and issues of adherence. Protection from anal transmission of HIV using topical microbicides and methods congruent with sexual behavior offers the promise of improved adherence. We compared the pharmacokinetics (PK) and ex vivo efficacy of iso-osmolar (IOsm) and hypo-osmolar (HOsm) rectal enema formulations of tenofovir (TFV) in rhesus macaques. Single-dose PK of IOsm or HOsm high-dose (5.28 mg/ml) and low-dose (1.76 mg/ml) formulations of TFV enemas were evaluated for systemic uptake in blood, colorectal biopsy specimens, and rectal CD4+ T cells. Markedly higher TFV concentrations were observed in plasma and tissues after administration of the HOsm high-dose formulation than with all other formulations tested. TFV and TFV diphosphate (TFV-DP) concentrations in tissue correlated for the HOsm high-dose formulation, demonstrating rapid uptake and transformation of TFV to TFV-DP in tissues. TFV-DP amounts in tissues collected at 1 and 24 h were 7 times and 5 times higher, respectively (P < 0.01), than the ones collected in tissues with the IOsm formulation. The HOsm high-dose formulation prevented infection in ex vivo challenges of rectal tissues collected at 1, 24, and 72 h after the intrarectal dosing, whereas the same TFV dose formulated as an IOsm enema was less effective.

Stabilized sulforaphane for clinical use: Phytochemical delivery efficiency.

SCOPE: The isothiocyanate sulforaphane (SF) from broccoli is one of the most potent known inducers of the cytoprotective phase 2 response. Its role in a host of biochemical pathways makes it a major component of plant-based protective strategies for enhancing healthspan. Many nutritional supplements are now marketed that purport to contain SF, which in plants exists as a stable precursor, a thioglucoside hydroxysulfate. However, SF in pure form must be stabilized for use in supplements. METHODS AND RESULTS: We evaluated the stability and bioavailability of two stabilized SF preparations-an α-cyclodextrin inclusion (SF-αCD), and an SF-rich, commercial nutritional supplement. SF-αCD area-under-the-curve peak serum concentrations occurred at 2 h, but six of ten volunteers complained of mild stomach upset. After topical application it was not effective in upregulating cytoprotective enzymes in the skin of SKH1 mice whereas pure SF was effective in doing so. Both of these "stabilized" SF preparations were as potent as pure SF in inducing the cytoprotective response in cultured cells, and they were more stable and as bioavailable. CONCLUSION: Our studies of a stabilized phytochemical component of foods should encourage further examination of similar products for their utility in chronic disease prevention and therapy.

Isoosmolar enemas demonstrate preferential gastrointestinal distribution, safety, and acceptability compared with hyperosmolar and hypoosmolar enemas as a potential delivery vehicle for rectal microbicides.

Rectally applied antiretroviral microbicides for preexposure prophylaxis (PrEP) of HIV infection are currently in development. Since enemas (rectal douches) are commonly used by men who have sex with men prior to receptive anal intercourse, a microbicide enema could enhance PrEP adherence by fitting seamlessly within the usual sexual practices. We assessed the distribution, safety, and acceptability of three enema types-hyperosmolar (Fleet), hypoosmolar (distilled water), and isoosmolar (Normosol-R)-in a crossover design. Nine men received each enema type in random order. Enemas were radiolabeled [(99m)Tc-diethylene triamine pentaacetic acid (DTPA)] to assess enema distribution in the colon using single photon emission computed tomography/computed tomography (SPECT/CT) imaging. Plasma (99m)Tc-DTPA indicated mucosal permeability. Sigmoidoscopic colon tissue biopsies were taken to assess injury as well as tissue penetration of the (99m)Tc-DTPA. Acceptability was assessed after each product use and at the end of the study. SPECT/CT imaging showed that the isoosmolar enema had greater proximal colonic distribution (up to the splenic flexure) and greater luminal and colon tissue concentrations of (99m)Tc-DTPA when compared to the other enemas (p<0.01). Colon biopsies also showed that only the hyperosmolar enema caused sloughing of the colonic epithelium (p<0.05). In permeability testing, the hypoosmolar enema had higher plasma (99m)Tc-DTPA 24-h area under the concentration-time curve and peak concentration compared to the hyperosmolar and isoosmolar enemas, respectively. Acceptability was generally good with no clear preferences among the three enema types. The isoosmolar enema was superior or similar to the other enemas in all categories and is a good candidate for further development as a rectal microbicide vehicle.

Induction of the phase 2 response in mouse and human skin by sulforaphane-containing broccoli sprout extracts.

The isothiocyanate sulforaphane was isolated from broccoli extracts in a bioactivity-guided fractionation as the principal and very potent inducer of cytoprotective phase 2 enzymes and subsequently shown to inhibit tumor development in animal models that involve various carcinogens and target organs. Because broccoli and broccoli sprouts are widely consumed, extracts obtained from them are viewed as convenient vehicles for sulforaphane delivery to humans. In relation to our current interest in devising strategies for protection against UV light-induced skin cancer, it was necessary to examine the safety and efficacy of topical application of sulforaphane-containing broccoli sprout extracts as single and multiple doses in both mice and humans. Topical application of an extract delivering 100 nmol sulforaphane/cm(2) increased the protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase A1, and heme oxygenase 1, three representative phase 2 enzymes, in mouse skin epidermis. Quantitative assessment of the activity of NQO1 24 h after dosing showed increases of 1.5- and 2.7-fold after application of single and multiple (thrice, every 24 h) doses, respectively. A dose-escalation safety study in healthy human subjects revealed no adverse reactions when doses as high as 340 nmol of sulforaphane in the form of broccoli sprout extracts were applied topically to the center of a 1-cm-diameter circle drawn on the volar forearm. A subsequent efficacy study showed that despite the interindividual differences in basal levels, the enzyme activity of NQO1 in homogenates of 3-mm full thickness skin punch biopsies increased in a dose-dependent manner, with maximum increases of 1.5- and 4.5-fold after application of 150 nmol doses, once or three times (at 24 h-intervals), respectively, thus providing direct evidence for induction of the phase 2 response in humans.

Hyperosmolar sexual lubricant causes epithelial damage in the distal colon: potential implication for HIV transmission.

BACKGROUND: Many sexual lubricants are hyperosmolar. Hyperosmolar enemas induce epithelial damage, and enema use has been associated with an increased risk of HIV infection. To inform the development of rectal microbicide formulation, we evaluated the effects of hyperosmolar gels on the rectal mucosa. METHODS: Two commercial lubricants were compounded into iso-osmolar and hyperosmolar mixtures (283 and 3429 mOsm/kg, respectively). Each gel was radiolabeled with 500 micro Ci of (99m)Technetium-diethylene triaminepentaacetic acid, and 10 mL was given rectally to 10 subjects in random sequence. Sigmoidoscopy by an endoscopist blinded to treatment assignment was performed 90 min later to obtain luminal and mucosal samples. Urine radiolabel detection was used to assess mucosal permeability. RESULTS: Epithelial denudation 10 cm from the anus occurred to a greater degree with the hyperosmolar gel than with the iso-osmolar formulation (median toxicity grade, 2.50 vs. 1.17 out of 3, respectively; P=.009). The hyperosmolar gel was also associated with lower isotope luminal concentration at 10 cm, compared with the iso-osmolar gel (median, 8.9% vs. 54.6% of administered concentration, respectively). Mucosal permeability measured through 12 h was reduced with the hyperosmolar gel (P=.037). CONCLUSION: Rectally applied hyperosmolar gels induce greater epithelial denudation and luminal secretion than iso-osmolar gels. Because denudation plausibly increases the risk of HIV transmission, hyperosmolar gels make poor rectal microbicide formulations, and hyperosmolar sexual lubricants may increase susceptibility to HIV infection.