This study demonstrates that the RG formulation of TFV gel is safe and acceptable when administered for up to seven days in sexually abstinent participants with a history of RAI. Gastrointestinal AEs such as abdominal pain, rectal urgency, diarrhea, and flatulence were the most common AEs seen but generally mild or moderate. The preparatory enema and subsequent flexible sigmoidoscopy may have contributed to these symptoms. However, the overall rates of these symptoms were less than those seen in the RMP-02/MTN-006 study which evaluated a more hyperosmolar formulation of TFV gel 
. Use of TFV gel did not appear to be associated with mucosal damage as assessed by a broad range of histological, immunological, and microbiological parameters. Significant changes in gene expression were identified in the TFV gel arm using microarray technology. The biological significance of these changes, and more importantly whether they represent a mucosal safety signal, is not clear and will require further evaluation. A recent preclinical study identified intestinal injury associated with in vitro
exposure of colorectal explants to the hyperosmolar vaginal formulation of TFV 
. However, similar histopathological changes were not seen in the TFV gel arm of the MTN-007 study.
The inclusion of N-9 as a positive control was associated with evidence of mild mucosal injury but was not associated with significant changes in epithelial sloughing. Indeed epithelial sloughing was seen at baseline in several participants. Non-human primate studies of rectal exposure to N-9 also failed to demonstrate epithelial sloughing 
. Fecal calprotectin was also not elevated throughout the study. This assay is useful in discriminating between irritable bowel syndrome and IBD 
but appears to have limited utility in the evaluation of RM. Based on these data, the MTN will not use these two assays in future Phase 1/2 studies of candidate RM.
It is unclear whether the changes in mucosal gene expression assessed by microarray are related to the TFV or its associated formulation. Future RM studies may answer this question. The CHARM-01 study (NCT01575405) will explore the impact of three different TFV formulations on rectal safety and acceptability. Each formulation has a different osmolality ranging from 479 to 3111 mOsmol/kg) and the study should be able to evaluate the impact of osmolality on mucosal safety. The MTN-017 study (NCT01687218) will be a Phase-2 RM study in which participants will receive 8 weeks of TFV gel (either daily or with sex) followed by 8 weeks of oral Truvada®. This study will provide information on the consequences of extended exposure to rectal TFV as well as a crossover comparison of oral and topical exposure to TFV. Both studies are expected to start in 2013.
Importantly, 31% of the participants in MTN-007 were women; emphasizing the need for RM for both men and women. We observed some modest gender specific differences in mucosal safety biomarkers but a larger study would be required to provide more definitive data on the impact of gender on the gastrointestinal mucosa. Differences in mucosal safety parameters between the 9 cm and 15 cm samples were more marked and in keeping with previous studies demonstrating regional heterogeneity in colonic mucosal biology 
This study demonstrated that it is possible to collect adequate mucosal samples for Phase 1 RM studies via anoscopy. This observation will potentially simplify the design and execution of future Phase 1 RM studies although it is difficult to collect more than 7 rectal biopsies via anoscopy and so studies requiring collection of rectal samples for mucosal safety, pharmacokinetic, and pharmacodynamic assessment may still require flexible sigmoidoscopy.
It is encouraging that the RG formulation of TFV is well tolerated as the vaginal formulation used in previous studies was associated with a high frequency of gastrointestinal side effects when given rectally 
. This safety profile together with evidence from the RMP-02/MTN-006 study showing that rectal use of TFV is associated with ex vivo/in vitro
inhibition of HIV-1 viral replication 
provides a compelling rationale for progression of this product into Phase 2 development. Collectively, these data will determine the longer term safety profile of this product and help decide whether it is a suitable agent to take into Phase 3 effectiveness studies.