Animal Characteristics, Drug Treatment Study.^A

A<p>Sedated with 0.1 cc/kg 10% Ketamine IM.</p>B<p>Prior infection with <i>P. cynomolgi</i>. All others were previously infected with <i>P. knowlesi</i>.</p

Schematic for Drug Treatment Study.

<p>Based on our available pharmacokinetic data and based on the assumption that 4–5 times half-life is required to reach steady state and then to eliminate drug in plasma <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100138#pone.0100138-Birkett1" target="_blank">[14]</a>, drugs levels were assumed to be at steady state (with dosing starting at D −2, and continued through D4) and mostly eliminated by the end of <i>P. knowlesi</i> liver stages (last dose given at 32 hr, and liver stages last about 5.25 days, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100138#pone.0100138-Coatney1" target="_blank">[9]</a>). On D −2, animals were infected IV with 2,500 cryopreserved <i>P. knowlesi</i> sporozoites. Smears and dried blood spots for PCR were collected every day from D5–14 and every other day from D14–D28. Animals were treated if parasites were detected on smear or on D28 (end of study) if smear negative throughout the study.</p

Pharmacokinetics of Lopinavir-ritonavir in Rhesus Monkeys.

A<p>Sedated with 0.1 cc/kg 10% Ketamine IM.</p>B<p>Awake.</p>1<p>Ibarra M, Fagiolino P, Vázquez M, Ruiz S, Vega M, Bellocq B, Pérez M, González B, Goyret A. Impact of food administration on lopinavir-ritonavir bioequivalence studies. Eur J Pharm Sci. 2012 Aug 15;46(5):516–21.</p

Trimethoprim-Sulfamethoxazole, and Lopinavir-ritonavir when used with Trimethoprim-Sulfamethoxazole, but not Lopinavir-ritonavir alone, at clinically relevant concentrations inhibits <i>P. knowlesi</i> liver stage parasites as reflected in absence of parasites in the blood by smear for 28 days (A) and prolonged time to PCR detection (B).

<p>To ensure drug steady state drug and 100% infection, rhesus (<i>Macaca mulatta</i>) monkeys were dosed starting 2 days prior to (D −2) through D4 post infection (with infection defined as D0) with 2,500 purified, cryopreserved <i>P. knowlesi</i> H strain sporozoites IV. Pharmacokinetic modeling used in this experiment predicted all drugs would be reduced to non-active levels prior to parasite emergence from the liver. Animals had Giemsa-stained smear and blood obtained from ear pricks until D28. Prolonged time to detection of parasites in blood was used to gauge liver stage parasite killing. (A) Control- and LPV-RTV-treated animals had positive smears on D10 and 11, and D10 and 12, respectively, but animals treated with TMP-SMX or LPV-RTV+TMP-SMX never became blood smear positive through D28, suggesting reduction of liver stage parasite burden. These findings were confirmed using (B) PCR for detection of parasites in blood (sensitivity, 0.00019 parasites/µl): parasites were detected earlier by PCR on D6 and 7 in controls and LPV-RTV (only)-treated animals, but on D12 in TMP-SMX only or in LPV-RTV+TMP-SMX combination-treated animals. Thus, LPV-RTV had no effect on the time to detection of parasites in blood, but TMP-SMX and LPV-RTV+TMP-SMX-treated animals had significant delays in detection of parasites in the blood, reflecting a reduced liver stage parasite burden.</p