This study demonstrates the feasibility of noninvasive SPECT/CT imaging and direct endoscopic sampling to describe viral surrogate distribution in the lumen of the GI tract after simulated RAI. Imaging with γ-emitting agents has been used to describe drug distribution after rectal administration 19–24
. Previously, we used SPECT/CT or MR with small molecule surrogate, diethylene triamine pentaacetic acid (393 g/mol) mixed into gels to describe colon distribution of microbicide surrogates following simulated RAI 9
. Radiolabeling of isolated leukocytes with 111
In-oxine for intravenous administration is a well established method for diagnosis of infection and the detection of in vivo
inflammation using SPECT/CT 25
. Similarly, 99m
Tc-sulfur colloid (~100 nm) has been used for phagocytic labeling in the imaging of infections 26
. We adapted and combined these methods to use the radiolabeled cells and sulfur colloid to serve as the pathogen, rather than measure host responses to pathogens. Even though direct sampling methods have been used to assess pathogen distribution, use of surrogates of pathogens administered to simulate clinical exposures is unique.
SPECT/CT imaging revealed that our HIV surrogates were largely confined to the rectosigmoid colon, but the distribution was highly variable in this small group of subjects. All subjects had distribution as far as 20 cm within the distal colon (sigmoid colon). The highest concentration of both cell-free and cell-associated surrogates was consistently seen in the rectosigmoid colon, 10 – 20 cm from the anus. This rectosigmoid distribution persisted for up to 24 hours when not lost with defecation. The two subjects who lost most surrogate signal after defecation demonstrate the ability of defecation to clear significant amounts of our HIV surrogates. Quantitative assessment of isotope distribution showed that more than 90% of cell-free surrogates were in the same distribution as cell-associated surrogates and this relationship persisted for 4 hours. The cell-associated surrogate, in contrast, distributed to a larger area and coincided with roughly 50% to 90% of the cell-free surrogate signal, also persisting at least 4 hours.
Based on this luminal surrogate distribution, rectal microbicide gels should provide the greatest concentration of drug in the recto-sigmoid to coincide with the highest concentration of HIV. To cover all possible temporal distributions of HIV seen in our study, microbicide distribution into the sigmoid colon, sustained over 24 h would be necessary. Also, the high percent coincidence of cell-free with cell-associated HIV surrogate distribution suggests that future studies of a candidate microbicide distribution could focus on one or the other form of HIV being targeted, but both surrogates need not be studied. Even where the surrogates didn’t overlap, the luminal distribution was still largely confined to local, contiguous regions of the recto-sigmoid colon. Accordingly, the simpler surrogate to use, namely, the 99mTc-sulfur colloid, may provide a reasonable approximation of the luminal distribution of both forms of HIV. Validation of sulfur colloid as suitable cell-free HIV surrogate remains to be done. Assessment of candidate microbicide vehicle distribution relative to the HIV target may best be assessed by using sequential dosing of radiolabeled microbicide followed by a radiolabeled HIV surrogate using similar luminal distribution and coincidence mapping as were used with two isotopes in this current study.
Even though non-invasive imaging is highly informative, direct sampling of colon tissue may differentiate luminal from tissue distribution, which requires resolution beyond the capacity of SPECT/CT, and provides CD4 cell specific distribution, which was not possible without prohibitively expensive cell sorting prior to labeling and dosing. In our analysis, exogenously administered HIV surrogates – total cells, CD4 cell subsets, and cell-free surrogates – were associated with the tissue biopsies. The presence of the cell-associated surrogate in cells isolated from biopsies suggests a strong association of exogenously administered cells with tissue. The presence of the cell-free surrogate in association with tissue may be due to passive uptake, mucoadhesion, or phagocytosis of the sulfur colloid into the cells extracted from the biopsies. Our methods were insufficient to determine if the HIV surrogates penetrated beyond the surface of the biopsy. Based on distribution and association with tissue, our results suggest that microbicide gels should be capable of preventing infection by both cell-associated and cell-free HIV.
The study had several important limitations related to our surrogates, SPECT/CT, and direct sampling. While the sulfur colloid is a reasonable surrogate for HIV in size and behavior in colloidal suspension, there are other factors that may influence particle behavior. The charged surface of the sulfur colloid may result in different interactions with the superficial mucus compared to HIV and the lack of surface proteins will result in loss of the specific host cell-virus interactions that would be expected with HIV. Furthermore, the sulfur colloid will evade specific host immunological responses, unlike HIV, and be cleared primarily through non-specific phagocytic uptake. These factors may result in differences in both distribution and clearance. Validation of the sulfur colloid as a surrogate for true cell-free HIV distribution is needed.
SPECT acquisition requires the detection within energy windows specific to the emission of the γ-emitting isotopes, and there is an overlap between these energy windows for 111In and 99mTc. We used a model-based correction for this isotope cross-talk to minimize the potential for falsely increasing signal intensity for a single isotope. Imperfect correction would lead to overestimate in distribution of an isotope, but would not affect the overall distribution of both HIV surrogates together. The movement of the sigmoidoscope through the rectosigmoid for direct sampling distends the colon and may carry surrogates with it within the colonic lumen, which may increase the HIV surrogate distribution in subsequent indirect imaging assessments. Because we only passed the scope to an area where we already knew the radiolabel was present (based on prior SPECT/CT) we are not concerned that we distorted the upper limit of proximal distribution. Further, one subject had signal visible in the lumen in the 24 hour imaging that followed the sigmoidoscopic sampling. Given that these limitations would be expected to overestimate the natural, unperturbed HIV distribution which we wanted to better understand, we believe that this method provides a conservative, worst-case scenario for luminal distribution that is highly valuable in guiding rectal microbicide development.
We demonstrated the feasibility of direct and indirect methods to quantitatively assess HIV surrogate distribution within the distal colon. Our results demonstrate that viral surrogates distribute primarily in the recto-sigmoid, and can persist up to 24 h post-exposure in some individuals. These findings provide distribution and clearance criteria for rectal microbicide candidates to maximize likelihood of success. We plan to use similar dual isotope methods to analyze microbicide vehicle distribution, both with and without simulated RAI with an HIV surrogate, to compare the relative distribution of antiviral drug and viral target in situ. Additional work is needed to determine the degree of tissue uptake of exogenously administered CD4 cells to extend our understanding of cell-associated HIV infection following RAI.