Prostratin (Pr) is Inactive in the Mixed Lymphocyte Reaction Assay for Immunosup-pression:  Implications for Regulatory Strategy and Patient Selection for Pr as a Drug for HIV.

Paul E. Driedger, PKC Pharmaceuticals, Inc. New Boston Street   Woburn, MA  01801(USA)

Background: Prostratin (Pr) was discovered in 1976 as the active agent causing poisoning of cattle that had eaten Pimelea prostrata weed. It blocks HIV replication, down-regulates CD4 and activates latent HIV.  Pr is in preclinical development as a drug to reduce latent HIV reservoirs in patients (pts) receiving HAART, but several problems attend this approach.  Pr at 3 mg/kg i.p. is lethal to mice, it causes mouse ear erythema, and its active/toxic conc. ratio is narrow in animal tests.  Since Pr has substantial toxicities, it may be difficult to justify its risks in Phase I trials for activation of latent HIV in healthy pts on HAART with undetectable virus.  In analogy to typical initial testing of toxic anti-cancer compounds, commonly restricted to end-stage pts who have failed other treatments, an alternative approach for initial Pr regulatory approval would pursue its activity as an inhibitor of HIV replication in severely compromised AIDS pts who have failed other treatments.  Pr is attractive as an HIV inhibitor because its target is likely cellular rather than viral, making viral resistance less likely.  However, Pr is known to downregulate CD4, raising concerns for its use in immunocompromised pts.  To address this concern, Pr has now been tested for activity in the mixed lymphocyte reaction (MLR), a standard test routinely used to identify and characterize immunosuppressants such as cyclosporin A (CsA).. Material and Methods: Human buffy coat PBMCs from two donors were centrifuged, adherence-purified to remove monocytes, and plated at 20,000 cells/donor/well.  Drugs were added on Day 0; on Day 4 [3H]thymidine was pulsed, and [3H] was counted 18 hours later. Results: Donor A + Donor B cells grew 3.9-fold above the same # of cells plated separately.  Anti-CD4 blocked A+B growth by 93%; CsA completely blocked growth at 100 nM and showed an IC50 of 4 nM.  Pr at all concentrations (10 nM-10 µM) did not inhibit A+B growth.  Pr slightly or partially restored growth inhibited by Cs A or anti-CD4, respectively.  When tested against A or B cells plated alone, Pr at 10 nM and 100 nM was inhibitory relative to untreated controls; at 1 µM and 10 µM, Pr was slightly (1.8-fold) stimulatory. Conclusion: Pr did not show any antiproliferative activity in the standard MLR assay for immunosuppressants.  Results of control tests indicated that the unchanged cell numbers in the A+B test with Pr cannot be attributed to a simple mitogenic effect on each donor’s cells alone, but rather support a genuine lack of immunosuppressant activity for Pr, in spite of its ability to downregulate CD4.  Although more extensive and detailed confirmatory testing is needed, these results support the idea that immunosuppression is not an undue risk for testing Pr as an HIV replication inhibitor in immunocompromised AIDS pts with advanced disease.