Spplemental Material

Animal model, drugs, and diets. Male LDLR null (LDLR) mice on a C57BL/6 background and wild-type C57/BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, Maine). At 14 weeks of age, the LDLR mice (n=48) were randomly assigned to one of three treatment groups (vehicle, ritonavir, or ritonavir plus acipimox). The protocol of animal use for this study has been approved by the IACUC of Boston University School of Medicine. Ritonavir was administered by gavages twice daily at a dose of 33 mg/kg/day, similar to the dose used in published mouse studies . In preliminary experiments, the plasma ritonavir concentration, measured by a modified HPLC method , ranged from 3.46 – 7.93 μM (SD 0.86, n=8) in 2 h after drug ingestion, and from 0.23 – 1.83 μM (SD = 1.91, n = 6) in 5 h after drug ingestion. These values are well within the therapeutic range in humans taking ritonavir . Acipimox was supplied in drinking water (0.05% wt/wt) and changed twice a week. The average water consumption was about 4 ml per day, equivalent to 2 mg/day intake of acipimox. This dose has been shown to inhibit adipocyte lipolysis in rodents . In humans, acipimox is well tolerated beyond 2250 mg/day . Ritonavir has been shown to induce aortic lesions in LDLR mice given a standard mouse chow . However, the lesions thus formed are microscopic and unlikely to cause clinical events. In contrast, an ad libitum atherogenic diet induces massive aortic lesions in LDLR, as well as other mouse models of atherogenesis, and hence may mask the pro-atherogenic effects of the antiviral drugs (preliminary studies, data not shown). To overcome this problem, we used a modified dietary regimen in which a high fat, high cholesterol (HFC) diet (Harlan Teklad TD.94509) was fed on either two (28% of the time, n = 8) or three (43% of the time, n = 8) days each week interspersed with standard chow (PurinaOne 5051) on the remaining days. Additional pilot tests showed that the pro-atherogenic effect of ritonavir was properly detected under these dietary conditions (data not shown). The HFC diet contains 36.9% kcal from fat, 20.4% kcal from protein, and 41.2% kcal from carbohydrate, with 1.25% wt/wt cholesterol. These diet regimens better mimic the usual dietary fluctuations in free-living animals and humans and allow efficient detection of atherogenic effects of ritonavir (see below).