Introduction 1 2 6 7 8 9 10 1 3 11 12 3 3 13 14 15 16 17 16 17 14 15 18 Fig. 1 3 2 5  Preclinical data suggested that FM-VP4 is a potent cholesterol-lowering agent with no significant toxic effects. Here, we report the first human study designed to assess the efficacy, safety, tolerability, and pharmacokinetics of single and multiple doses of this water-soluble plant stanol analogue. Subjects and methods Subjects 2 Drugs 1 Design This single-center, double-blind, placebo-controlled, dose-escalation trial comprised two parts: In phase 1, 30 men received a single dose of FM-VP4. Five subjects were assigned to each dose group (100, 200, 400, 800, 1,600, or 2,000 mg), and within each group, one subject was randomly assigned to placebo. Once a complete cohort of five subjects was treated and the safety parameters had been reviewed, the following dose was initiated. In the subsequent phase 2 trial, 100 men were treated for 28 days (4 weeks). Twenty-five subjects were assigned to each dose group (100, 200, 400, or 800 mg/day), and within each group, five subjects were randomly assigned to placebo. The first five subjects in each cohort were hospitalized for 5 days. Escalation to the next dosing level was only allowed once these five subjects completed treatment and all results and safety data were evaluated. Phase 1 Phase 2 Plasma analyses 9 19 Plasma was stored at −80°C for further analyses. ApoB, ApoAI, and lipoprotein a [Lp(a)] as well as vitamin E and vitamin A were analyzed in one run after the study had been completed. ApoAI, ApoB, and Lp(a) levels were determined by nephelometry with a Beckman Array (Mijdrecht, the Netherlands) according to the manufacturer’s instructions, and vitamin E and vitamin A were measured by high-performance liquid chromatography (HPLC) with fluorescence detection using a Chromsep Glass, 100*3 mm, inertsil 5, ODS-3 column (Varian-Chrompack, Middelburg, the Netherlands). 20 0-t 21 m/z m/z m/z m/z m/z Statistical analyses  P  t  p Results In phase 1, 30 male volunteers completed the study in accordance with the protocol. There were no withdrawals. The majority of subjects (93%) were of Caucasian descent. Mean subject age was 50.4 (range 24–63) years. In phase 2, 101 male subjects were enrolled, but one subject withdrew consent prior to receiving study medication due to personal reasons and therefore contributed no safety or efficacy data. The remaining 100 subjects completed the study. The majority of subjects (89%) were Caucasian, with the remaining being Asian or another race. Mean subject age was 53.7 (range 23–75) years. As calculated from returned tablets, the mean percentage of received tablets was 97%. One subject in the 800-mg group had a compliance <80%. All other patients received >80% of the planned number of tablets during the treatment period. Five subjects received statin treatment within 40 days before the first dose of study treatment (29–35 days). Mean weight ranged between 83.9 ± 13.1 and 84.6 ± 13.0 kg during the entire study. Adverse events In phase 1, 23 treatment-emergent adverse events were reported by 16 subjects. Of these events, five (21.7%) occurred in the 100-mg group, three (13.0%) in both the 200- and 400-mg groups, four (17.4%) in the 800-mg group, two (8.7%) in the 1,600-mg group, and three (13.0%) in both the 2,000 mg and placebo groups. All reported events were mild. The most common events were dizziness, headache, and fatigue. Other adverse events were loose stools, vasovagal attack, influenza, upper respiratory tract infection, elevated bilirubin, elevated BP, arthralgia, difficult micturition, polyuria, and pharyngolaryngeal pain. Three events were considered to be possibly related to the study drug. One was reported by a subject receiving the placebo treatment, whose bilirubin concentration increased from 12 μmol/L on the morning of treatment (baseline) to 25 μmol/L 24 h after treatment but decreased to normal 7 days after treatment. The other two possibly related events were reported in the 800- and 1,600-mg groups, and both consisted of one episode of loose stools on the day of treatment. In phase 2, 67 subjects reported one or more treatment-emergent adverse events: 12 in the 100-mg group, 14 in the 200-mg group, 11 in the 400-mg group, 15 in the 800-mg group, and 15 in the placebo group. Most events were mild, and four subjects reported a moderate event. The most frequent event was headache, which was reported by a total of 19 subjects and by two (400-mg group) to five subjects (100-mg and 200-mg groups) in each of the five groups. The four adverse events that were moderate included two subjects with headache in the 800-mg group, one subject with an elevated CK level in the 400-mg group, and one subject with epilepsy in the placebo group. No subjects discontinued study treatment due to a treatment-emergent adverse event. One subject in the 800-mg group did not take the study medication for 3.5 days due to nausea and diarrhea, which was not considered to have been related to the study drug. Once the subject recommenced treatment, no further treatment-emergent adverse events were reported. A total of 24 subjects reported events that were considered to be drug-related: eight in the 100-mg group, one in the 200-mg group, two in the 400-mg group, six in the 800-mg group, and seven in the placebo group. The most commonly reported event was flatulence, which was reported by three subjects in the 100-mg groups, one in the 400-mg group, two in the 800-mg group and one in the placebo group. There were no differences in the incidence of treatment-emergent adverse events between active and placebo groups. Blood pressure, heart rate, and EKG analysis There was no effect of FM-VP4 on BP or heart rate during phase 1. In phase 2, the mean systolic BP was slightly decreased after 4 weeks of treatment (a maximum average of 4% in the 100-mg group; data not shown), but there was no relationship between this decrease and the dose of FM-VP4. Diastolic BP did not change. All pre- and postdose EKGs were normal in both phases of the trial. Laboratory analyses 9 9 9 9 9 9 9 9 p  p Overall, there were few abnormal laboratory values, and no trends were observed over time. There appeared to be no relationship between laboratory parameters and the dose of FM-VP4 administered. Efficacy In phase 1, changes in lipids and lipoproteins were not statistically compared between the seven treatment groups, as only one dose of FM-VP4 was administered. 1 2 p 1 n n p  p p Table 1 n   Dose level (mg) P a Placebo 100 mg 200 mg 400 mg 800 mg   mmol/L  Total cholesterol Baseline 6.36 ± 0.65 6.40 ± 0.87 6.40 ± 0.88 6.04 ± 1.05 6.02 ± 0.90  Day 28 6.50 ± 0.72 6.49 ± 1.12 6.26 ± 0.78 5.81 ± 1.05 5.90 ± 0.90  % Change 2.6% 1.2% −1.7% −3.8% −1.7% 0.09 LDL Baseline 4.29 ± 0.63 4.17 ± 0.58 4.40 ± 0.77 4.02 ± 0.93 3.94 ± 0.83  Day 28 4.38 ± 0.79 4.28 ± 0.78 4.18 ± 0.63 3.75 ± 0.88 3.73 ± 0.81  % Change 2.7% 2.9% −4.2% b −4.6% 0.05 HDL Baseline 1.37 ± 0.32 1.34 ± 0.38 1.20 ± 0.25 1.24 ± 0.30 1.21 ± 0.27  Day 28 1.37 ± 0.37 1.29 ± 0.34 1.28 ± 0.31 1.22 ± 0.27 1.26 ± 0.37  % Change −0.1% −3.6% 6.7% −1.2% 4.1% 0.04 TG Baseline 1.56 ± 0.77 1.86 ± 1.30 1.77 ± 0.72 1.73 ± 0.72 1.92 ± 0.68  Day 28 1.65 ± 0.86 1.95 ± 1.16 1.76 ± 1.08 1.86 ± 0.95 1.97 ± 1.00  % Change 8.2% 16.3% 0.7% 7.5% 9.7% 0.8 All values are mean ± standard deviation LDL  HDL  TG a b P Fig. 2 Mean low-density lipoprotein cholesterol levels during 28 days of treatment with placebo or 100, 200, 400, and 800 mg/day FM-VP4 (disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate) and after 14 days of follow-up  Pharmacokinetics 0→t 1/2 max max max 2 1/2 Table 2 Pharmacokinetic parameters of disodium ascorbyl campestanol phosphate (DACP) and disodium ascorbyl sitostanol phosphate (DASP) in 24 subjects after a single dose of 400, 800, 1,600, or 2,000 mg FM-VP4 (DACP and DASP) in phase 1 Dose level (mg) Number 1/2 max max 0→∞ 0→t DACP   400 4 79.6 ± 42.3 100.6 ± 25.5 10.7 ± 3.0 8,734 ± 3,864 5,942 ± 2,646   800 4 46.2 ± 21.8 144.5 ± 43.6 12.1 ± 0.1 7,672 ± 2,479 5,787 ± 2,045   1,600 4 35.9 ± 11.1 175.6 ± 106.5 7.9 ± 3.2 11,736 ± 11,455 9,314 ± 10,910   2,000 4 64.0 ± 28.3 190.9 ± 47.2 18.3 ± 6.4 17,179 ± 5,685 11,414 ± 4,318 DASP   400 4 77.3 ± 11.6 247.9 ± 55.1 12.1 ± 0.1 28,142 ± 7,153 21,115 ± 5,255   800 4 52.2 ± 19.0 339.7 ± 86.9 12.1 ± 0.1 28,872 ± 8,183 22,907 ± 9,382   1,600 4 53.0 ± 14.9 344.3 ± 225.8 24.9 ± 21.6 39,029 ± 38,428 30,243 ± 30,624   2,000 4 43.6 ± 21.8 495.9 ± 89.0 18.5 ± 6.6 35,642 ± 16,584 29,204 ± 14,549 Mean ± standard deviation t 1/2  C max  t max  AUC 0→t 0→t R 2 R 2 2 3 Fig. 3  dots  dots  DASP and DACP concentrations increased in a less than dose-proportional manner compared with baseline on days 8 and 28, as well as on day 42, 14 days after treatment. Plant sterol and stanol concentrations In phase 1, concentrations of campestanol and sitostanol did not change within 24 h and 7 days after a single dose of FM-VP4. Also, concentrations of campesterol and β-sitosterol were not affected after a single dose of FM-VP4 (data not shown). 3 3 Table 3 Concentrations of plant sterols and sterols in 100 subjects after 4 weeks of treatment with 100, 200, 400, or 800 mg/day FM-VP4 (disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate) or placebo in phase 2 Plant stanol or sterol  Dose level (mg) P n n n n n   ng/mL  Sitostanol Baseline 77.9 ± 33.3 79.6 ± 30.8 90.4 ± 32.9 75.0 ± 28.3 63.8 ± 19.2  Day 28 102.5 ± 39.2 104.2 ± 38.3 132.5 ± 56.3 178.3 ± 63.8 168.3 ± 44.6  % Change 41 ± 58 37 ± 46 48 ± 37 159 ± 99 178 ± 83 <0.0001 Campestanol Baseline 116.0 ± 84.6 91.0 ± 36.6 104.3 ± 81.7 100.7 ± 57.2 83.4 ± 18.9  Day 28 140.9 ± 77.3 107.1 ± 36.6 134.1 ± 78.9 156.2 ± 72.5 150.2 ± 37.9  % Change 39 ± 70 21 ± 22 43 ± 50 70 ± 61 87 ± 57 0.002 Sitosterol Baseline 2,998.3 ± 1,842.5 3,579.3 ± 2,084.3 2,715.0 ± 10,725.8 2,704.3 ± 1,071.2 2,276.3 ± 993.2  Day 28 3,036.0 ± 1,164.9 3,782.1 ± 2,377.9 2,744.9 ± 1,256.1 3,138.4 ± 1,632.7 2,316.9 ± 734.4  % Change 35 ± 147 5 ± 20 2 ± 23 17 ± 40 10 ± 38 0.61 Campesterol Baseline 3,275.2 ± 2,017.2 3,699.1 ± 1,914.8 2,850.4 ± 1,289.0 2,791.1 ± 1,403.2 2,370.8 ± 1,036.6  Day 28 3,212.3 ± 1,375.9 3,870.2 ± 2,252.6 3,002.7 ± 1,439.2 3,031.5 ± 1,500.1 2,416.9 ± 917.6  % Change 16 ± 80 4 ± 22 6 ± 19 12 ± 35 8 ± 35 0.91 Mean ± standard deviation. Differences between all treatment groups were analyzed by using analysis of variance Discussion In this study, we showed that a single dose of 100–2,000 mg as well as 4-week treatment in doses of 100–800 mg of FM-VP4 administered to moderate dyslipidemic men was well tolerated and safe. Furthermore, 4-week treatment of FM-VP4 reduced LDL-C levels by 6–7% compared with baseline or by 9–11% compared with placebo. The main treatment—emergent adverse events were dizziness, headache, and fatigue after a single administration and headache after multiple-dose administration of the drug. All symptoms resolved spontaneously, and subjects receiving placebo also reported these symptoms. As there was no difference in the incidence between active and placebo groups, it is unlikely that the treatment-emergent adverse events were due to FM-VP4. 8 max 1/2 1/2 1/2 22 23 22 23 3 24 16 8 LXR 25 In conclusion, this study demonstrated that single and multiple doses of FM-VP4 for 4 weeks are safe and well tolerated by moderately hypercholesterolemic subjects. Furthermore, the higher doses of FM-VP4 significantly reduce LDL-C levels by 6–7% compared with baseline or by 9–11% when compared with placebo. The pharmacokinetics showed that DACP and DASP are absorbed and cleared slowly but that the absolute quantity of drug absorbed is low, as suggested by the low plasma concentrations of DACP and DASP. This study suggests that FM-VP4 merits further investigation as an alternative for treating hyperlipidemia.