The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program (NTP). The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by the NTP on March 2009 (see http://ntp.niehs.nih.gov/ntp/htdocs/levels/09-3566%20NTP-ITOX-R1.pdf). The findings and conclusions for this study should not be construed to represent the views of the NTP or the US Government.
Saquinavir (SQV) is a protease inhibitor targeted to interfere late in the viral life cycle during cleavage of the final viral protein element. The protease inhibitors bind to the catalytic site of the viral protease enzyme, which plays a critical role in cleaving the large polyproteins of the immature virus. The protease inhibitors represent a new treatment for AIDS patients and, since the protease inhibitors act at a totally different site in the viral cycle, combination treatment with nucleoside analogues may be effective. Reasons for concern are seen in the potential cross resistance between different protease inhibitors and the potential for drug interactions since the protease inhibitors are metabolized in the liver and some are potent inhibitors of cytochrome p4501.
Saquinavir was nominated to the NTP for toxicological evaluation and was selected for immunotoxicity studies by the project officer. Thus, the purpose of these studies was to determine the potential effects of SQV on the immune system and the doses to be used in a full immunotoxicology protocol.
The study reported here is an initial range-finding study conducted in female B6C3F1 mice. The animals were treated twice daily for 28 days by oral gavage. Saquinavir was prepared weekly as a solution in the vehicle 0.5% methylcellulose.
The baseline toxicology studies are summarized in Table ES-1. In this range-finding study of orally administered SQV at total daily doses of 100, 300, 600, 1000 and 1200 mg/kg/day for 28 days, there were no effects on body weight. Beginning on the second week of the study, a decrease in weight gain was observed in the high dose animals. In the third week a significant increase in body weight gain was observed in the low-dosed animals (100 mg/kg) as compared to the vehicle controls, while a significant decrease in weight gain was observed in dose levels greater than or equal to 300 mg/kg. Over the total study period (Day 29-Day1), only the high dose, 1200 mg/kg, had a significant weight change with a decrease of 39%. There were few notable changes among the other standard toxicological parameters that were assessed, including organ weights and hematological parameters.
Table ES-2 summarizes the immunology studies. No significant effects were observed on the absolute number of the various spleen subpopulations evaluated. Evaluation of the humoral immune response in the plaque assay to the T-dependent antigen, sheep erythrocytes (sRBC) demonstrated a significant increase at the high dose when evaluated as either specific activity or total spleen activity. The increase (91%) in the 1200 mg/kg dose level, when evaluated as AFC/106 spleen cells reached the level of statistical significance at p < 0.01. When evaluated as AFC/Spleen, the increase was 121% compared to vehicle control animals; however, the increase was only statistically significant when evaluated using parametric analysis. When evaluated in the sRBC ELISA, the high dose animals did not differ significantly from the vehicle controls. Evaluation of cell-mediated immunity using the mixed leukocyte response (MLR) indicated that treatment with SQV did not modulate this component of the immune system. The most interesting findings where SQV's effect on innate immunity when evaluated by Natural Killer (NK) cell activity. At the low dose levels, 100 and 300 mg/kg, a decrease in NK activity was observed while the higher dose levels enhanced NK activity.
In conclusion, treatment with SQV at the high dose of 1200 mg/kg had a significant effect on humoral immunity as measured by the antibody-forming cell response to the T-dependent antigen, sRBC. A statistically significant enhanced response was observed at the 1200 mg/kg dose level; however; the lower doses failed to enhance the humoral immune response as compared to the vehicle controls. Cell-mediated immunity was not affected. SQV appears to be an immunomodulatory drug when administered twice a day for 28 days in female B6C3F1 mice at dose levels up to and including 1200 mg/kg. Since there were marked effects on the NK activity, i.e., suppression at the low dose levels and enhancement of NK activity at the higher dose levels, and the fact that NK cells play a major role in killing viral-infected cells germane to SQV's use in the treatment of AIDS, it would seem appropriate to consider saquinavir for evaluation in a full protocol study, utilizing doses of 300, 600 and 1200 mg/kg.
|Parameter||Overall Effect||Specific Results||Maximum Effect||Dose||Comments|
|Body Weight||No Effect|
|Day 8||No Effect|
|Day 15||No Effect|
|Day 22||No Effect|
|Day 29||No Effect|
|Body Weight Changes||Decreased|
|Day 8-1||No Effect|
|Day 15-1||Decrease||61%||1200 mg/kg|
|Day 22-1||Decrease||53%||1200 mg/kg|
|Day 29-1||Decrease||39%||1200 mg/kg|
|Gross Pathology||No Effect|
|% Lymphocytes||No Effect|
|% Neutrophils||No Effect|
|% Eosinophils||Decrease||72%||300 & 600 mg/kg|
|Abs Lymphocytes||No Effect|
|Abs Neutrophils||Decrease||51%||300 mg/kg|
|Abs Eosinophils||Decrease||81%||600 mg/kg|
a NBS = not biologically significant
|Parameter||Overall Effect||Specific Results||Maximum Effect||Dose||Comment|
|Splenic Phenotypesa||No Effect|
|IgM Humoral Immune Response to sRBC||Increased|
|IgM AFC/106 Cells||Increase||91%||1200 mg/kg|
|IgM AFC/Spleen||Increase||121%||1200 mg/kg||Parametric Analysis|
|Serum Titer Log2||Increase||11%||600 mg/kg|
|Mixed Leukocyte Response||No Effect|
|Responders Only||No Effect|
|Total Cells x 107||Increase||19%||1200 mg/kg|
|NK Cell Activity||Mixed|
|LU/107 Cells||Increase||435%||1200 mg/kg|
a Absolute Values