Pharmacokinetics and tolerability of artesunate and amodiaquine alone and in combination in healthy volunteers Objectives The WHO recommends artemisinin-based combination therapies for treatment of uncomplicated falciparum malaria . At least 15 African countries have adopted artesunate plus amodiaquine as treatment policy . As no pharmacokinetic data on this combination have been published to date , we investigated its pharmacokinetic interactions and tolerability in healthy volunteers in Africa . Introduction Since Plasmodium falciparum has developed resistance to almost all anti-malarial drugs , the use of combinations of effective antimalarials with different mechanisms of action is required to improve cure rates and delay drug resistance . The World Health Organisation -LRB- WHO -RRB- now recommends treatment of uncomplicated falciparum malaria with one of several artemisinin-based combination therapies -LRB- ACTs -RRB- , including artesunate plus amodiaquine -LSB- 1 -RSB- . To date at least 15 African countries have adopted artesunate plus amodiaquine as first-line malaria treatment policy -LSB- 2 -RSB- . Although the limited data available suggest that the combination of artesunate and amodiaquine is well tolerated as short-course treatment , there are two safety concerns that have not yet been fully addressed . Neutropaenia and hepatitis curtailed the use of amodiaquine as prophylaxis in the 1980s -LSB- 3 -- 5 -RSB- . A recent randomised , controlled trial comparing treatment with amodiaquine alone or in combination with artesunate found marked , asymptomatic neutropaenia on day 28 -LRB- NCI grade 3 -- 4 , absolute neutrophil counts 306 -- 900 μL − 1 -RRB- in 6 % of 153 children with uncomplicated falciparum malaria , all of whom had normal baseline absolute neutrophil counts -LSB- 6 -RSB- . We have previously reported a case of hepatitis in this group of volunteers that developed after the second dose of amodiaquine and was considered probably related to amodiaquine -LSB- 7 -RSB- . A similar report of delayed-onset asymptomatic hepatitis has been described in healthy American volunteers who had received artesunate plus amodiaquine alone followed by artesunate plus amodiaquine plus efavirenz , resulting in early study discontinuation -LSB- 8 -RSB- . In that study , efavirenz was thought to have caused an increase in amodiaquine concentrations by competitive inhibition of the P450 cytochrome enzymes that metabolise amodiaquine . With the widespread use of artesunate plus amodiaquine treatment in Africa , these studies support the need for laboratory monitoring of the treatment 's safety , particularly if antiretrovirals are used concomitantly -LSB- 1 -RSB- . Pharmacokinetic data are still sparse for amodiaquine . After oral administration , amodiaquine hydrochloride is rapidly absorbed and undergoes rapid and extensive metabolisation to desethylamodiaquine , the main active metabolite . Amodiaquine has a short elimination half-life -LRB- approximately 5 h -RRB- , while that for desethylamodiaquine is much longer -LRB- 6 -- 18 days -RRB- -LSB- 9 , 10 -RSB- . There are several pharmacokinetic studies on oral artesunate in healthy volunteers and malaria patients that give broadly consistent results -LSB- 11 , 12 -RSB- . Artesunate is rapidly absorbed , rapidly converted to its main active metabolite dihydroartemisinin and rapidly cleared -LRB- elimination half-life generally < 1 h -RRB- . Dihydroartemisinin has similar pharmacokinetic characteristics but has a slightly longer elimination half-life . There is wide inter-individual variation in artesunate and dihydroartemisinin pharmacokinetic parameters . No pharmacokinetic interaction data have yet been published on amodiaquine when used in combination with artesunate . In view of the forthcoming widespread use of this combination in Africa , our study aimed to investigate potential pharmacokinetic interactions between these antimalarials and assess their tolerability in healthy volunteers in Africa . Methods Study subjects and study design Male or female healthy normal volunteers who provided written informed consent and met the following criteria were eligible to participate in the study : aged 18 -- 45 years ; no abnormalities on medical history , clinical examination , laboratory safety assessment -LRB- full blood count , differential white cell count , routine liver and renal function tests -RRB- or electrocardiogram ; and a negative pregnancy test -LRB- for female volunteers -RRB- . Volunteers were excluded if they were smokers -LRB- > 5 cigarettes/day -RRB- , had taken antimalarials or been in a malarial area in the preceding 8 weeks , had malaria parasites on a thick smear , used recreational drugs , or had ingested any alcohol or any medicines -LRB- including over-the-counter preparations -RRB- in the week preceding study commencement . This was a randomised three-phase crossover study . All volunteers took artesunate -LRB- 4 mg/kg as Arsumax , 50 mg tablets , Sanofi-Aventis -RRB- , the drug with the shorter elimination half-life , in the first phase , prior to the administration of amodiaquine . In the second phase , 7 days later , the volunteers were randomly allocated to one of two treatment groups : group 1 received a single oral dose of artesunate -LRB- 4 mg/kg -RRB- plus amodiaquine -LRB- 10 mg base/kg as Camoquin , 200 mg tablets , Parke Davis -RRB- , and group 2 received a single oral dose of amodiaquine -LRB- 10 mg/kg -RRB- alone . In phase three , 21 days later , the groups received the alternative regimen . All trial drugs were given under direct supervision with 200 ml tap water on an empty stomach after an overnight fast . No caffeinated drinks were allowed during the study period . Standardised meals were offered , commencing with breakfast at 2 h post-dose . Volunteers in phase one had blood samples collected before taking artesunate and at 0.25 , 0.50 , 0.75 , 1.0 , 1.5 , 2 , 3 , 4 , 6 , 8 and 12 h after artesunate administration . In phases two and three -LRB- i.e. volunteers receiving artesunate plus amodiaquine or amodiaquine alone -RRB- venous blood samples were collected pre-dose and at 0.25 , 0.5 , 0.75 , 1 , 1.5 , 2 , 3 , 4 , 6 , 8 , 12 , 24 , 48 and 72 h , and days 4 , 5 , 7 , 10 , 14 and 20 post-dose . At each time two 5-ml venous blood samples were collected in tubes containing sodium heparin anticoagulant , while three 5-ml samples were collected at each time point for the first 12 h from those volunteers receiving both drugs . Each sample was centrifuged within 5 min of collection and the plasma transferred to separate plastic cryotubes and frozen at − 70 °C until analysed for artesunate , dihydroartemisinin , amodiaquine and desethylamodiaquine . Volunteers remained in the study ward for the first 12 h of each phase ; thereafter specimens were collected at outpatient visits . A clinical examination and laboratory safety tests were performed during screening and 6 days after artesunate administration and 20 days after administration of both amodiaquine and amodiaquine plus artesunate . A 12-lead electrocardiogram -LRB- ECG -RRB- was recorded for each volunteer during screening and approximately 2 h after each drug dose . The corrected QT interval -LRB- QTc -RRB- was calculated by dividing the QT interval by the square root of the R-R interval . ECGs were reviewed by C.O. and by a consultant cardiologist . At follow-up visits , the volunteers were asked to report any new symptoms . A targeted physical examination was conducted if indicated . An adverse event together with its relationship to study drugs was defined according to the WHO guidelines -LSB- 13 -RSB- and the severity graded using the National Cancer Institute -LRB- NCI -RRB- Common Toxicity Criteria -LSB- 14 -RSB- . All adverse events and concomitant medications taken were recorded . Any significant clinical or laboratory abnormalities or the use of a concomitant medication with a previously described interaction resulted in the volunteer being withdrawn from the study . Ethical approval This clinical study was conducted in accordance with the principles laid down by the World Health Assembly of 1975 on Ethics in Human Experimentation and the Helsinki Declaration . The study adhered to the standards established for good clinical practice . The protocol was approved by the University of Cape Town Research Ethics Committee and the WHO Secretariat Committee for Research Involving Human Subjects -LRB- SCRIHS -RRB- . Each volunteer was informed of the objectives , nature and possible risks of the trial . Written informed consent was obtained from every volunteer participating in the study . The volunteers were informed that they were free to withdraw consent at any time . Drug assays Artesunate and dihydroartemisinin concentrations in plasma were analysed using the method of Batty and colleagues -LSB- 15 -RSB- with minor modifications . Solid-phase extraction of samples was performed using Bond-Elut phenyl extraction cartridges -LRB- Varian , Harbour City , CA , USA -RRB- . After washing the column with 2 ml of water , samples were eluted from the column with 1.5 ml of acetonitrile , which was then evaporated to dryness under nitrogen . Samples were reconstituted in 100 μl of mobile phase -LRB- 45 % acetonitrile , 0.05 M acetic acid , pH 6.0 -RRB- , and 50 μl was injected onto the column . HPLC was carried out on a Symmetry C 8 , 5 μm , 15 cm × 4.6 mm HPLC column -LRB- Waters , Milford , MA , USA -RRB- using a Waters 600 pump and WISP 712 auto-sampler . The post-column reagent -LRB- 1.2 M KOH in 90 % methanol -RRB- was introduced using a Waters reagent delivery module and the mixture passed through a 1-ml reaction coil -LRB- Waters -RRB- kept at 69 °C . Detection was at 290 nm using a Waters 484 Tunable Absorbance Detector , which produced a linear response over the range of the standard curve -LRB- 20 -- 1,000 ng/ml -RRB- . Quality control samples of 15 , 75 and 150 ng/ml for artesunate and 75 , 450 and 750 ng/ml for DHA were included in each run . Within - and between-day coefficients of variation were below 14 % . The lower limit of quantification was 24 ng/ml for both artesunate and dihydroartemisinin . Amodiaquine and desethylamodiaquine were analysed by LC mass spectrometry using an Agilent 1100 Series LC/MS system . Protein was precipitated from plasma samples -LRB- 200 ul -RRB- using three volumes of acetonitrile . Supernatant -LRB- 5 μl -RRB- was injected onto the HPLC . Chromatography was carried out using a 50 × 4.6 mm C18 Xterra column -LRB- Waters -RRB- . The mobile phase comprised 75 % acetonitrile , 0.02 M ammonia , pH 10.2 . The extracted ion for amodiaquine was m/z 356 and for desethylamodiaquine m/z 328 . The calibration curve for amodiaquine was linear in the range 5 -- 100 ng/ml and for desethylamodiaquine in the range 5 -- 400 ng/ml . Quality control samples of 25 , 75 and 250 ng/ml were used for desethylamodiaquine and 2.5 , 7.5 and 25 ng/ml for amodiaquine . Within - and between-day coefficients of variation were below 12 % . The lower limit of quantification was 5 ng/ml for both amodiaquine and desethylamodiaquine . Statistical analysis Pharmacokinetic parameters were calculated from the artesunate , dihydroartemisinin , amodiaquine and desethylamodiaquine concentration profiles using WinNonLin , version 3.3 -LRB- Pharsight -RRB- , with concentrations outside the specified limits of quantification regarded as missing values . Clearance -LRB- Cl -RRB- was calculated as 0.693 × Vd/T1/2 where Vd is the apparent volume of distribution and T1/2 is the elimination half-life . Because there was no intravenous comparator arm in this study , equivalent bioavailability was assumed for comparisons of apparent volume of distribution -LRB- Vd/f -RRB- and clearance -LRB- Cl/f -RRB- , where f is the oral bioavailability or fraction of the drug absorbed -LRB- unknown -RRB- . All four compounds were first analysed separately . Because both artesunate and dihydroartemisinin are highly parasiticidal in malaria patients , the pharmacokinetic parameters associated with therapeutic response -LSB- area under the plasma concentration time curve -LRB- AUC -RRB- and maximum clearance -LRB- Cmax -RRB- -RSB- are also reported together as dihydroartemisinin equivalents , defined as the sum of the measured dihydroartemisinin concentrations plus the artesunate concentration converted to dihydroartemisinin equivalents -LRB- using molecular weights of 384 and 284 for artesunate and dihydroartemisinin respectively -RRB- -LSB- 16 , 17 -RSB- . As amodiaquine undergoes a rapid and extensive conversion to its active metabolite , desethylamodiaquine was considered the primary analyte -LSB- 9 , 10 , 18 -RSB- . The pharmacokinetic parameters following administration of artesunate and amodiaquine alone and in combination were compared using Stata , version 9.0 -LSB- Stata , College Station , TX , USA -RSB- . Data were log-transformed and then compared using the analysis of variance -LRB- ANOVA -RRB- for a cross-over design to take into account the repeated measures by study subject , treatment period and treatment groups and , for amodiaquine and desethylamodiaquine , a sequence effect . The treatment effects generated from the ANOVA were exponentiated in order to express comparisons between monotherapy and combination therapy as a ratio . Any apparent discrepancies between the difference in the group means and these ratios are due to the fact that the ratios are based on the within-patient differences in log-transformed values and not the group arithmetic means . Given the multiple testing , statistical significance of results should be interpreted with caution . For the safety analysis , means and 95 % confidence intervals -LRB- 95 % CI -RRB- for each haematological parameter -LRB- haemoglobin , haematocrit , platelets , white cell count , absolute lymphocyte and neutrophil count -RRB- were calculated at baseline and at the end of each treatment phase . Changes in these values from baseline were determined using a mixed effect regression model that took into account repeated measures within-patient and were adjusted for the period and period-treatment interaction effects . Results Subject demographics Nineteen volunteers were screened ; two were excluded because of neutropaenia and two withdrew consent . Fifteen healthy normal volunteers -LRB- 10 male , 5 female -RRB- entered the study with a mean age , weight and height of 24.4 years , 67.3 kg and 171 cm , respectively . The mean dose of amodiaquine was 10.72 mg/kg and that of artesunate was 4.26 mg/kg . Safety analysis included all 15 volunteers ; only data from the 13 volunteers who completed the study were included in the pharmacokinetic analyses . A female volunteer was withdrawn due to a new prescription of fluoxetine for depression , as this drug could potentially interact with artesunate and amodiaquine by inhibiting cytochrome P450 enzymes . One male volunteer was withdrawn due to a possible hypersensitivity reaction on his first exposure to amodiaquine . Pharmacokinetic parameters Effect of amodiaquine on artesunate pharmacokinetic parameters Data were analysed using a non-compartmental model . Figure 1a shows the mean plasma concentration time curves of artesunate and dihydroartemisinin following administration of artesunate alone and artesunate plus amodiaquine . The AUC , Cmax and time to Cmax -LRB- Tmax -RRB- of artesunate were similar following monotherapy and combination therapy -LRB- Table 1 -RRB- . Figure 2a illustrates the impact of adding amodiaquine on the dihydroartemisinin AUC . Following the combination therapy , dihydroartemisinin AUC was lower -LSB- ratio 67 % -LRB- 95 % CI 51 -- 88 % -RRB- ; P = 0.008 -RSB- , Cmax was lower -LSB- ratio 51 % -LRB- 95 % CI 33 -- 78 % -RRB- ; P = 0.005 -RSB- , T1/2 was longer -LSB- ratio 157 % -LRB- 95 % CI 115 -- 213 % -RRB- ; P = 0.008 -RSB- and Vd was larger -LSB- ratio 192 % -LRB- 95 % CI 133 -- 275 % -RRB- ; P = 0.003 -RSB- , when compared with artesunate monotherapy -LRB- Table 1 -RRB- . These ratios have been adjusted for repeated measures by subject and period effects . As a measure of the total exposure to artemisinin derivatives , the Cmax and AUC of the combined DHA-equivalents were also analysed and found to be lower following combination therapy -LSB- Cmax ratio 51 % -LRB- 95 % CI 32 -- 84 % -RRB- , P = 0.011 ; AUC ratio 72 % -LRB- 95 % CI 54 -- 95 % -RRB- , P = 0.023 -RSB- when compared with monotherapy . Cl and Tmax of dihydroartemisinin were similar when monotherapy and combination therapy were administered -LRB- Table 1 -RRB- . Fig. 1Profiles of mean drug concentrations over time following monotherapy -LRB- mono -RRB- and combination -LRB- combo -RRB- therapy for a artesunate -LRB- AS -RRB- and dihydroartemisinin -LRB- DHA -RRB- and b amodiaquine -LRB- AQ -RRB- and desethylamodiaquine -LRB- DEAQ -RRB- Table 1Pharmacokinetic parameters for artesunate and dihydroartemisinin when artesunate was administered alone and with amodiaquine -LRB- ACT -RRB- AUC -LRB- ng · h/ml -RRB- Cmax -LRB- ng/ml -RRB- Tmax -LRB- h -RRB- T1/2 -LRB- h -RRB- Vd/f -LRB- l -RRB- Cl/f -LRB- l/min -RRB- ArtesunateAS alone -LRB- mean ± SD -RRB- 206.4 ± 135.5231.8 ± 155.00.62 ± 0.28 NANANAACT -LRB- mean ± SD -RRB- 183.3 ± 146.5141.6 ± 117.50.86 ± 0.67NANANAACT-to-monotherapy ratio -LRB- % -RRB- -LRB- mean , 95 % CI -RRB- 36 -LRB- 8 -- 173 -RRB- 25 -LRB- 6 -- 105 -RRB- 125 -LRB- 76 -- 202 -RRB- NANANA Significance -LRB- ANOVA -RRB- 0.180.0570.33 DihydroartemisininAS alone -LRB- mean ± SD -RRB- 2,044.4 ± 564.2844.5 ± 309.41.10 ± 0.951.46 ± 0.484.89 ± 1.672.46 ± 0.86 ACT -LRB- mean ± SD -RRB- 1,410.5 ± 543.6446.2 ± 239.52.08 ± 1.722.20 ± 0.859.68 ± 4.163.08 ± 0.82ACT-to-monotherapy ratio -LRB- % -RRB- -LRB- mean , 95 % CI -RRB- 67 -LRB- 51 -- 88 -RRB- 51 -LRB- 33 -- 78 -RRB- 165 -LRB- 82 -- 334 -RRB- 157 -LRB- 115 -- 213 -RRB- 192 -LRB- 133 -- 275 -RRB- 122 -LRB- 96 -- 156 -RRB- Significance -LRB- ANOVA -RRB- 0.0080.0050.150.0080.0030.097 Treatment effects generated from the ANOVA were exponentiated to express within-subject comparisons between monotherapy and combination therapy as a ratio , adjusted for period effectsAUC Area under the plasma concentration time curve , Cmax maximum concentration , Tmax time to Cmax , T1/2 elimination half-life , Vd volume of distribution , f oral bioavailability or fraction of the drug absorbed , Cl clearance , AS artesunate , SD standard deviation , CI confidence interval , NA not available -LRB- too few detectable concentrations for calculation of T1/2 , Cl , Vd -RRB- Fig. 2Stick plot comparing individual patient AUCs following monotherapy and combination therapy of a dihydroartemisinin -LRB- DHA -RRB- and b desethylamodiaquine -LRB- DEAQ -RRB- . -LRB- Note : volunteer A was excluded from the statistical analyses -RRB- Effect of artesunate on amodiaquine pharmacokinetic parameters Amodiaquine samples for one volunteer were missing . Figure 1b shows the mean plasma concentrations of desethylamodiaquine over time following administration of amodiaquine alone and in combination . Figure 2b illustrates the impact of artesunate on the desethylamodiaquine AUC . The volunteer -LRB- subject A -RRB- who had the highest observed AUC for desethylamodiaquine following combination therapy -LSB- 29,504 ng · h/ml compared with the mean of 8,437 -LRB- 95 % CI 5,744 -- 11,131 -RRB- ng · h/ml -RSB- was excluded from the pharmacokinetic analyses of desethylamodiaquine , as this was an extreme outlier and influential observation -LRB- Fig. 2b ; Fig. 3 -RRB- . Fig. 3Stick plot comparing individual patients . Day 7 concentrations of desethylamodiaquine when amodiaquine was administered as monotherapy and in combination with artesunate . -LRB- Note : volunteer A was excluded from the statistical analysis -RRB- The impact of adding artesunate to amodiaquine on the pharmacokinetic parameters of amodiaquine and desethylamodiaquine is reported for the remaining 11 volunteers . The pharmacokinetic parameters of amodiaquine were similar when administered as monotherapy or in combination with artesunate -LRB- Table 2 -RRB- . The desethylamodiaquine AUC is lower -LSB- ratio 65 % -LRB- 95 % CI 46 -- 90 % -RRB- ; P = 0.015 -RSB- , Tmax shorter -LSB- ratio 60 % -LRB- 95 % CI 45 -- 80 % -RRB- ; P = 0.003 -RSB- and clearance more rapid -LSB- ratio 164 % -LRB- 95 % CI 112 -- 243 % -RRB- ; P = 0.016 -RSB- when amodiaquine was administered in combination with artesunate rather than as monotherapy -LRB- Table 2 -RRB- . There was a trend towards a lower day 7 concentration of desethylamodiaquine -LSB- ratio 56 % -LRB- 95 % CI 30 -- 104 % -RRB- ; P = 0.064 -RSB- when amodiaquine was administered in combination -LRB- Fig. 3 -RRB- . These ratios have been adjusted for repeated measures by subject , period and sequence effects . The Vd and Cmax were not significantly different when monotherapy and combination therapy were administered . Table 2Pharmacokinetic parameters for desethylamodiaquine -LRB- DEAQ -RRB- when amodiaquine was administered alone and with artesunate -LRB- ACT -RRB- AUC -LRB- ng · h/ml -RRB- Cmax -LRB- ng/ml -RRB- Tmax -LRB- h -RRB- -LSB- Day 7 -RSB- -LRB- ng/ml -RRB- T1/2 -LRB- h -RRB- Vd/f -LRB- l -RRB- Cl/f -LRB- l/min -RRB- AmodiaquineAQ alone -LRB- mean ± SD -RRB- 162.4 ± 101.429.2 ± 10.92.32 ± 1.16 NA5 .3 ± 4.1361.0 ± 128.369 ± 59ACT -LRB- mean ± SD -RRB- 108.5 ± 56.022.7 ± 9.02.18 ± 1.61 NA3 .9 ± 1.2467.7 ± 180.586 ± 26ACT-to-monotherapy -LRB- % -RRB- -LRB- mean , 95 % CI -RRB- 77 -LRB- 47 -- 127 -RRB- 78 -LRB- 58 -- 103 -RRB- 92 -LRB- 57 -- 147 -RRB- NA74 -LRB- 29 -- 189 -RRB- 128 -LRB- 93 -- 175 -RRB- 172 -LRB- 84 -- 357 -RRB- Significance -LRB- ANOVA -RRB- 0.270.070.680.440.110.11 DesethylamodiaquineAQ alone -LRB- mean ± SD -RRB- 12,041 ± 3,480268.7 ± 70.83.68 ± 1.8519.4 ± 7.3240.8 ± 146.9234.1 ± 97.5768 ± 252ACT -LRB- mean ± SD -RRB- 8,437 ± 4,009301.4 ± 166.12.18 ± 1.0313.3 ± 7.3136.9 ± 83.8210.8 ± 92.91,330 ± 735ACT-to-monotherapy ratio -LRB- % -RRB- -LRB- mean , 95 % CI -RRB- -RRB- 65 -LRB- 46 -- 90 -RRB- 103 -LRB- 73 -- 147 -RRB- 60 -LRB- 45 -- 80 -RRB- 56 -LRB- 30 -- 104 -RRB- 53 -LRB- 25 -- 111 -RRB- 88 -LRB- 51 -- 149 -RRB- 164 -LRB- 112 -- 243 -RRB- Significance -LRB- ANOVA -RRB- 0.0150.820.0030.0640.080.580.016 Treatment effects generated from the ANOVA have been exponentiated to express within-subject comparisons between monotherapy and combination therapy as a ratio , adjusted for period and sequence effectsAUC Area under the plasma concentration time curve , Cmax maximum concentration , Tmax time to Cmax , -LSB- Day 7 -RSB- day 7 concentration , T1/2 elimination half-life , Vd apparent volume of distribution , f oral bioavailability or fraction of the drug absorbed , Cl clearance , AQ amodiaquine , SD standard deviation , CI confidence interval , NA not available -LRB- as too few patients had quantifiable amodiaquine concentrations -RRB- Safety Twenty-nine adverse events were reported over the course of the study by 10 -LRB- 67 % -RRB- of the 15 volunteers . The frequency of adverse events was similar across all three treatment arms -LRB- P > 0.10 -RRB- : 4/15 -LRB- 27 % -RRB- following artesunate alone , 8/15 -LRB- 53 % -RRB- following amodiaquine alone , and 5/15 -LRB- 33 % -RRB- following the combination . All adverse events were consistent with the product information available , resolved spontaneously and , except for the transaminitis -LRB- see below -RRB- were mild or moderate in intensity . A case of asymptomatic , prolonged , severe transaminitis -LRB- NCI grade 3 , AST 5 -- 20 × upper limit of normal -RRB- that developed after phase 3 of the study has been previously published -LSB- 7 -RSB- . This was considered to be probably related to amodiaquine , rather than artesunate , before the pharmacokinetic results were available . The subsequent finding that this volunteer had the highest measured desethylamodiaquine AUC following the administration of artesunate plus amodiaquine -LSB- 29,504 ng · h/ml compared with the combination therapy mean of 8,437 -LRB- 95 % CI 5,744 -- 11,131 -RRB- ng · h/ml -RSB- strengthened this assessment . There were no liver function abnormalities detected in any other volunteers . No renal or electrolyte abnormalities were detected in any volunteers . Significant haematological changes were confined to the white cell counts . During phase 2 of the study , two volunteers -LRB- one following amodiaquine alone , one following the combination -RRB- developed asymptomatic , NCI grade 1 leucopaenia -LRB- 3 to < 4 × 109/L -RRB- and either a grade 1 -LRB- 1.5 to < 2 × 109/L -RRB- or grade 2 -LRB- 1 to < 1.5 × 109/L -RRB- neutropaenia . None of the other observed changes in haematological parameters were outside the normal range . There were no significant changes following treatment seen on the ECGs ; the mean -LRB- 95 % CI -RRB- QTc interval was 398 -LRB- 390 -- 406 -RRB- ms at screening , 401 -LRB- 393 -- 409 -RRB- ms following artesunate alone , 400 -LRB- 385 -- 414 -RRB- ms following amodiaquine alone and 412 -LRB- 400 -- 424 -RRB- ms following the combination . In two patients , the prolongation following treatment with amodiaquine or artesunate plus amodiaquine was considered of borderline clinical significance . Discussion The pharmacokinetic analyses in this randomised , cross-over study showed statistically significant pharmacokinetic interactions resulting in reductions in the AUC of both dihydroartemisinin and desethylamodiaquine when artesunate and amodiaquine were given in combination to healthy volunteers . Of further concern is that one healthy volunteer failed to reach quantifiable concentration of both artesunate and dihydroartemisinin throughout the initial 12 h . There is insufficient evidence currently available to explain the basis of these interactions . The clinical significance of our findings is unclear because we studied healthy volunteers . In one study comparing pharmacokinetic parameters of dihydroartemisinin in patients and volunteers , patients had a significantly higher Cmax -LRB- 1,948 vs. 1,192 nmol/l -RRB- and AUC0 -- 12 -LRB- 4,024 vs. 1,763 nmol · h/l -RRB- , while their Vd and Cl were both lower . The Tmax and terminal elimination half-life of the patients were similar to those of the volunteers -LSB- 19 -RSB- . Desethylamodiaquine pharmacokinetic parameters in malaria-infected adults in Africa -LSB- 20 -RSB- and children with acute malaria in Papua New Guinea -LSB- 21 -RSB- were similar to those of normal volunteers . In artemisinin-based combination therapy , the artemisinin is given to kill most of the parasites quickly , while the longer-acting partner drug -LRB- e.g. amodiaquine -RRB- is given to kill the residual parasites . For an antimalarial with a long elimination half-life like desethylamodiaquine , the key pharmacokinetic parameter for parasite killing is the AUC , which is a useful predictor of therapeutic response as it captures both the drug concentration and the duration of exposure -LSB- 22 , 23 -RSB- . For such drugs the antimalarial concentration on day 7 is strongly correlated with AUC , and thus treatment response -LSB- 23 -RSB- . An association of desethylamodiaquine concentration on day 7 and amodiaquine treatment response has been found in children with uncomplicated falciparum malaria -LSB- 18 -RSB- . In our study , the AUC -LRB- and probably the day 7 desethylamodiaquine concentration -RRB- was reduced following the artemisinin-based combination ; this may result in reduced parasite killing , particularly in areas where there is reduced amodiaquine sensitivity . Although there is a linear relationship between amodiaquine dose and the desethylamodiaquine AUC -LSB- 24 -RSB- , the wide inter-individual variability in desethylamodiaquine levels -LRB- five-fold in our study -RRB- , and the consequent potential for drug-induced toxicity , could preclude increasing the amodiaquine dose to compensate for the lower desethylamodiaquine AUC observed following co-administration with artesunate . Extensive clinical trial data show that artesunate plus amodiaquine is more effective -LRB- at clearing parasitaemia and preventing recrudescence -RRB- than amodiaquine alone -LSB- 1 , 6 -RSB- . Therefore , if reductions in the Cmax and AUC of desethylamodiaquine and dihydroartemisinin occur in patients , the negative effect on parasite killing may become clinically insignificant due to the additive -LRB- or possibly synergistic -RRB- parasiticidal effects of the combination . However , this may only apply to areas where the level of amodiaquine resistance is low . Although clinically significant resistance to the artemisinins has not yet been documented , amodiaquine resistance is well documented and this is probably the principal explanation for low artesunate plus amodiaquine cure rates observed in a number of African countries -LSB- 1 , 6 -RSB- . Despite our small sample size , we detected the well-known amodiaquine-related adverse drug reactions . Clinically important adverse events were reported by a quarter of these volunteers , even though all were healthy and only two doses were administered 3 weeks apart -LRB- rather than the daily administration for 3 days recommended for malaria treatment -RRB- . When the artesunate plus amodiaquine combination becomes widely used , monitoring of liver function tests and haematological parameters are warranted to define these risks in malaria patients , particularly in patients at special risk such as those at the extremes of age , with HIV co-infection or malnutrition -LSB- 1 -RSB- . However , the feasibility of this recommendation outside of the research setting is of concern . To conclude , the total exposure to the main active metabolites of both artesunate and amodiaquine was significantly reduced when administered in combination to healthy African volunteers ; this might be important clinically . However , because cure rates with this combination are generally higher than with amodiaquine monotherapy , artesunate and amodiaquine could remain in the armamentarium of drugs used to combat falciparum malaria , provided efficacy continues to be monitored and adequate safety precautions can be taken . Further pharmacokinetic research on artesunate plus amodiaquine , when administered concurrently or as a fixed dose combination , is urgently required in patients with malaria to establish the extent and clinical significance of these pharmacokinetic interactions .

B O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O B O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O O O O O B O O B O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O B O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O B O B O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O B O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

amodiaquine
artesunate
pharmacokinetics
tolerability
healthy normal volunteers


Efficacy and safety of disodium ascorbyl phytostanol phosphates in men with moderate dyslipidemia Objective This study investigated the efficacy , safety , tolerability , and pharmacokinetics of a novel cholesterol absorption inhibitor , FM-VP4 , comprising disodium ascorbyl sitostanol phosphate -LRB- DASP -RRB- and disodium ascorbyl campestanol phosphate -LRB- DACP -RRB- . Introduction Worldwide , cardiovascular disease -LRB- CVD -RRB- is the most common cause of death , with atherosclerotic vascular disease as the underlying cause . It has been well established that elevated plasma concentrations of low-density lipoprotein cholesterol -LRB- LDL-C -RRB- lead to an increased risk of atherosclerosis and coronary heart disease -LSB- 1 -RSB- . Dietary modification can improve the lipid profile and the potential risk of CVD , but for a significant portion of the population , treatment with lipid-lowering agents is necessary to reduce blood cholesterol effectively . Statins are the primary class of drugs used for managing LDL-C and are the most potent agents in lowering LDL-C . In fact , the lowering of LDL-C by statin therapy has been shown to decrease the number of fatal and nonfatal myocardial events in both primary and secondary prevention studies -LSB- 2 -- 6 -RSB- . However , statins have not provided the final answer in terms of CVD prevention , and their use is associated with side effects , albeit rare , at higher doses . Therefore , cholesterol-lowering agents that act via other mechanisms are of great interest . Plant sterols , or phytosterols , are naturally occurring compounds in vegetable oil , seeds , and nuts that are structurally related to cholesterol . Daily intake of 2,000 mg of plant sterols or their saturated counterparts , plant stanols , or phytostanols , has been shown to decrease LDL-C by 9 -- 14 % without affecting high-density lipoprotein cholesterol -LRB- HDL-C -RRB- concentrations -LSB- 7 , 8 -RSB- . Plant sterols and stanols are thought to decrease plasma cholesterol concentrations by inhibiting cholesterol absorption in the intestine -LSB- 9 -RSB- . When cholesterol absorption is decreased , the hepatic cholesterol pool is reduced , resulting in enhanced cholesterol synthesis by the liver . At the same time , LDL receptors are upregulated , with ensuing lower LDL-C concentrations in plasma -LSB- 10 -RSB- . A new water soluble plant stanol derivative , disodium ascorbyl phytostanol phosphate -LRB- FM-VP4 , Fig. 1 -RRB- has been developed as a cholesterol-absorption inhibitor . FM-VP4 consists of a mixture of sitostanol and campestanol to which ascorbate is covalently bound via a phosphodiester linkage . It has been shown to inhibit the in vitro uptake of micellar -LSB- 3H -RSB- - cholesterol by approximately 50 % in human and rat enterocyte cell lines -LSB- 11 , 12 -RSB- . In vivo , FM-VP4 led to a dose-related inhibition of -LSB- 3H -RSB- - cholesterol absorption in rats , as shown by a maximally 80 % reduced area under the concentration time curve -LRB- AUC -RRB- of orally administered micellar -LSB- 3H -RSB- - cholesterol -LSB- 13 -RSB- . Furthermore , the LDL-lowering activity of FM-VP4 was observed in a broad range of LDL-sensitive species , including gerbils -LSB- 14 , 15 -RSB- , hamsters -LSB- 16 -RSB- , and apolipoprotein E -LRB- ApoE -RRB- - deficient transgenic mice -LSB- 17 -RSB- . In hamsters , FM-VP4 was a more potent LDL-lowering agent than unesterified plant stanols -LSB- 16 -RSB- , and in ApoE deficient mice , the LDL-lowering effect of FM-VP4 was correlated with retardation of atherosclerotic plaque development -LSB- 17 -RSB- . Other effects of FM-VP4 included the decrease of plasma triglyceride levels and the reduction of abdominal fat or body-weight gain in gerbils -LSB- 14 , 15 -RSB- and mice -LSB- 18 -RSB- . In all of these cases , FM-VP4 showed no side effects to the animals tested . Fig. 1Chemical structure of FM-VP4 , composed of disodium ascorbyl campestanol phosphate -LRB- R = CH3 -RRB- and disodium ascorbyl sitostanol phosphate -LRB- R = C2H5 -RRB- 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 Subjects were recruited via advertisements in local newspapers . The study protocol was carefully explained before subjects were asked to give their written informed consent . The study protocol was approved by the Institutional Review Board of the Academic Medical Centre . Subjects were eligible if they were male , 18 -- 75 years old , healthy as reviewed by medical history and physical examination , had LDL-C concentrations ≥ 3.0 mmol/L during one of the screening visits or at baseline and triglyceride -LRB- TG -RRB- concentration ≤ 4.5 mmol/L at the first visit , had a body mass index -LRB- BMI -RRB- < 35 kg/m2 , and did not use any steroids , β-blockers , corticosteroids , thiazide diuretics , or antiepileptics . Subjects with a history of hypertension , arterial diseases , diabetes mellitus type I or II , hypothyroidism , obstructive biliary disorders , pancreatitis , collagen disorders , or autoimmune disease were excluded , as were subjects with history of malignancy during the previous 3 years , significant hepatic , renal , cardiac , or cerebral disease , or plasma levels of hepatic transaminases higher than two times the upper limit of normal -LRB- ULN -RRB- . Subjects of phase 1 were not allowed to use any lipid-lowering drugs at inclusion , and the use of plant sterol - or stanol-containing products had to be discontinued at the inclusion visit . Subjects of phase 2 had to discontinue the use of plant sterol or stanol products or fish oils at the inclusion visit and statin treatment 6 weeks -LRB- 40 days -RRB- before the start of study treatment . Thirty men participated in the phase 1 trial and 100 men in phase 2 . Drugs Two types of tablets were used : 100 mg oval FM-VP4 tablets or a matching placebo . Both investigational products were supplied through Forbes Medi-Tech Inc. -LRB- Vancouver , Canada -RRB- . FM-VP4 is a semisynthetic esterified plant stanol derivative produced as a disodium salt . The two major components of FM-VP4 are disodium ascorbyl sitostanol phosphate -LRB- DASP -RRB- and disodium ascorbyl campestanol phosphate -LRB- DACP -RRB- -LRB- Fig. 1 -RRB- , which are present in the proportion of approximately 2:1 , respectively . 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 -LRB- 100 , 200 , 400 , 800 , 1,600 , or 2,000 mg -RRB- , 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 -LRB- 4 weeks -RRB- . Twenty-five subjects were assigned to each dose group -LRB- 100 , 200 , 400 , or 800 mg/day -RRB- , 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 A week before treatment , subjects visited the hospital for screening . Informed consent was obtained , and subjects underwent a physical examination . A fasting blood sample was taken to measure lipids , biochemistry , and hematology . Within 3 days before treatment , subjects visited the hospital for another blood sample , and urinalysis and electrocardiogram -LRB- EKG -RRB- were performed . If subjects met all inclusion criteria , they were hospitalized for 24 h on the day of treatment . In the morning , subjects ' weight , supine blood pressure -LRB- BP -RRB- , and heart rate were measured , and a predose fasting blood sample was taken for baseline safety parameters and pharmacokinetics . Subsequently , subjects were administered one to 20 tablets containing 100 mg FM-VP4 each or placebo . Tablets were swallowed with 250 mL of water . Breakfast followed 30 min later . Weight , supine BP , and heart rate were recorded at 3 , 6 , 9 , and 12 h after dosing . A blood sample for pharmacokinetics was taken 6 and 12 h after dosing . Any spontaneous complaints were recorded as adverse events and closely monitored . Subjects were detained overnight under observation , and 24 h after dosing , another blood sample was taken for safety parameters and pharmacokinetics . Subjects ' weight , supine BP , and heart rate were measured , and urinalysis and an EKG were performed . Once the EKG , biochemistry , and hematology of the 24-h postdosing sample had been reviewed , subjects were discharged from the hospital . They returned to hospital 48 , 96 , and 144 or 168 h -LRB- 6 or 7 days -RRB- after treatment for weight , supine BP , and heart rate measurements and for a blood sample to measure safety parameters and pharmacokinetics . At the last visit , a final physical examination was performed . Phase 2 Four to 8 weeks before treatment , subjects visited the hospital for screening . Informed consent was obtained , and subjects underwent a physical examination . A fasting blood sample was taken to measure lipids , biochemistry , and hematology . Subjects were instructed to follow a diet adapted from the National Cholesterol Education Program -LRB- NCEP -RRB- Step 1 diet during the entire study , including the 4 - to 8-week run-in period . Consumption of plant sterol - or stanol-containing products and the use of fish oils had to be discontinued at the screening visit , and statin treatment had to be discontinued 6 weeks before treatment with FM-VP4 . If they met all inclusion criteria , subjects visited the hospital halfway through the run-in period and within 3 days before study treatment for baseline blood lipids , biochemistry , and hematology measurements . At the latter visit , urinalysis was performed and an EKG recorded.The first five subjects per dose cohort , of which four were on active treatment and one on placebo , were hospitalized for 5 days . Each morning , subjects ' weight , supine BP , and heart rate were measured , and a predose fasting blood sample was taken for safety parameters and pharmacokinetics . Subsequently , subjects were administered one to four tablets containing 100 mg FM-VP4 each , depending on the dose cohort , or placebo . Tablets were swallowed entirely with up to 100 mL of water . Breakfast followed 30 min later . All doses were divided and given twice per day ; another one to four tablets were administered 30 min before dinner . The morning and evening doses were packaged in separate bottles . Subjects in the 100-mg group received one FM-VP4 tablet and one placebo tablet . Supine BP and heart rate were also recorded daily after lunch and dinner . Any spontaneous complaints were recorded as adverse events and closely monitored . Subjects were observed overnight and discharged 5 days after the first dosing . All subjects visited the hospital for efficacy and safety measurements once per week during and 14 days after treatment . At the last visit , a final physical examination and an EKG were performed . Compliance was calculated based on the number of tablets supplied to the patient minus the number returned . Plasma analyses Complete blood count , fibrinogen , and the biochemical profile -LSB- alanine aminotransferase -LRB- ALT -RRB- and aspartate aminotransferase -LRB- AST -RRB- , bilirubin , creatine kinase -LRB- CK -RRB- , creatinine , glucose , and C-reactive protein -LRB- CRP -RRB- -RSB- were assessed using the local hospital laboratory . Changes in laboratory parameters were considered abnormal in case of ALT or AST levels greater than three times ULN , a CK level greater than five times ULN , a creatinine increase of ≥ 40 μmol/L compared with baseline , a creatinine level > 177 μmol/L , white blood cell count < 3 × 109/L , and a decrease in hemoglobin of at least 1.5 g/dL compared with baseline . Thyroid-stimulating hormone -LRB- TSH -RRB- was measured at the first screening visit . Urinalysis for blood , glucose , protein , pH , and specific gravity was performed by dipstick within 3 days before and 1 day after dosing in phase 1 and within 3 days before , after 4 weeks of treatment , and at the last visit of phase 2 . Urinalysis by wet microscopic slide was performed if the dipstick analysis was abnormal . Lipid analyses were performed by an external laboratory -LRB- MRL International , Zaventem , Belgium -RRB- by using standardized procedures , and LDL-C was calculated using the Friedewald equation -LSB- 19 -RSB- . The results were kept blinded to the investigators . Plasma was stored at − 80 °C for further analyses . ApoB , ApoAI , and lipoprotein a -LSB- Lp -LRB- a -RRB- -RSB- as well as vitamin E and vitamin A were analyzed in one run after the study had been completed . ApoAI , ApoB , and Lp -LRB- a -RRB- levels were determined by nephelometry with a Beckman Array -LRB- Mijdrecht , the Netherlands -RRB- according to the manufacturer 's instructions , and vitamin E and vitamin A were measured by high-performance liquid chromatography -LRB- HPLC -RRB- with fluorescence detection using a Chromsep Glass , 100 * 3 mm , inertsil 5 , ODS-3 column -LRB- Varian-Chrompack , Middelburg , the Netherlands -RRB- . All DASP and DACP plasma concentrations were assayed by a validated liquid chromatography/mass spectrometry/mass spectrometry method -LRB- LC/MS/MS -RRB- based on a previously described method -LSB- 20 -RSB- . The lower limit of quantification -LRB- LLOQ -RRB- was 30.8 ng/mL . Data analysis was carried out using Microsoft Excel 97 , and pharmacokinetic data was analyzed using Pharsight WinNonlin software , version 3.1 build 168 -LRB- Pharsight WinNonlin , Mountain View , CA , USA -RRB- based on noncompartmental kinetics analysis . For phase 1 , concentrations of DASP and DACP in plasma were measured at baseline and 6 , 12 , 24 , 48 , 96 , and 144 or 168 h after treatment . DASP and DACP concentrations were plotted against time , and the half-lives of the compounds were estimated by the method of residuals . The area under the DACP and DASP concentration time curve -LRB- AUC 0-t -RRB- was estimated by the trapezoidal rule -LSB- 21 -RSB- . In phase 2 , trough concentrations of DASP and DACP were measured at baseline , after 8 and 28 days of treatment , and 14 days after treatment -LRB- day 42 -RRB- . In subjects who were hospitalized for the first 5 days of treatment , trough DASP and DACP concentrations were also measured each morning of hospitalization -LRB- day 1 through day 5 -RRB- . Plant sterols and stanols β-sitosterol , campesterol , sitostanol , and campestanol were assessed by selective ion monitoring gas chromatography mass spectrometry -LRB- SIM-GC-MS -RRB- . High-purity solvents were purchased from Merck , Germany . Bis - -LRB- Trimethylsilyl -RRB- trifluoroacetamide -LRB- BSTFA -RRB- was obtained from Sigma -LRB- Steinneim , Germany -RRB- and pyridine from Pierce -LRB- Rockford , IL , USA -RRB- . Beta-sitosterol -LRB- 24β-ethylcholesterol -RRB- , β-sitostanol -LRB- 24α-ethyl-5α-cholestan-3β-ol -RRB- , and campesterol -LRB- 24α-methyl-5-cholesten-3β-ol -RRB- were purchased from Sigma , and stigmasterol from Lacoclau AB , Sweden . For sterol extraction , 500 μl of plasma was mixed with 100 μl 0.01 mg/ml stigmasterol and saponified for 60 min at 60 °C in 1 ml of 4 % -LRB- w/v -RRB- KOH in 90 % ethanol . After saponification , the samples were mixed with 1 ml of water and extracted two times with 2 ml of hexane . The pooled hexane extracts were dried under nitrogen and derivatized with 50 μl BSTFA/pyridine -LRB- v/v 5:1 -RRB- at 60 °C for 60 min . For SIM-GC-MS , 2 μl of derivative mixture were delivered by automatic injection to an HP-5890 gas chromatograph split-injection port -LRB- 1:20 -RRB- leading to a 0.2 mm × 25 m Chrompack CP-sil 19 CB -LRB- WCOT Fused Silica -RRB- capillary column . The injection port contained a glass wool liner . The carrier gas was helium at a linear rate of 1 ml/min . The oven temperature started at 120 °C and was raised to 260 °C at 20 °C / min , then to 280 °C at 2 °C / min , and finally to 300 °C at 40 °C / min and held for 5 min . An HP-5989B mass spectrometer was used as detector . Measurements were done in the electron impact mode at 70 eV with an ion source temperature of 250 °C . The quadropole temperature was 150 °C . Mass spectrometric data were collected in the elected ion mode at m/z = 396 and 357 for β-sitosterol , m/z = 488 and 373 for β-sitostanol , m/z = 382 and 343 for campesterol , m/z = 369 and 384 for campestanol , and m/z = 255 and 394 for stigmasterol . Calibration curves were constructed by mixing 100 μl of 0.01 mg/ml stigmasterol with a series of 0 - to 500-μl samples of a standard solution containing 10 μmol β-sitosterol , 2 μmol/l sitostanol , and 20 μmol/l campesterol . Statistical analyses The primary efficacy variable was the percent change in LDL after 4 weeks of treatment compared with baseline levels in phase 2 . Differences in percentage LDL-C changes between the five treatment groups were calculated using analyses of variance -LRB- ANOVA -RRB- in SAS . P < 0.05 was considered statistically significant . If there was a significant difference between dose groups by ANOVA , each active dose was compared with placebo by a one-sided t test , with p < 0.025 being statistically significant . This procedure was also performed for percentage changes in total cholesterol -LRB- TC -RRB- , HDL-C , and TG . For ApoB , ApoAI , Lp -LRB- a -RRB- , vitamin A , vitamin E , and plant sterols , only ANOVA was performed to test differences in percentage changes between the five treatment groups . Safety data are presented descriptively . Results In phase 1 , 30 male volunteers completed the study in accordance with the protocol . There were no withdrawals . The majority of subjects -LRB- 93 % -RRB- were of Caucasian descent . Mean subject age was 50.4 -LRB- range 24 -- 63 -RRB- 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 -LRB- 89 % -RRB- were Caucasian , with the remaining being Asian or another race . Mean subject age was 53.7 -LRB- range 23 -- 75 -RRB- 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 -LRB- 29 -- 35 days -RRB- . 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 -LRB- 21.7 % -RRB- occurred in the 100-mg group , three -LRB- 13.0 % -RRB- in both the 200 - and 400-mg groups , four -LRB- 17.4 % -RRB- in the 800-mg group , two -LRB- 8.7 % -RRB- in the 1,600-mg group , and three -LRB- 13.0 % -RRB- 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 -LRB- baseline -RRB- 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 -LRB- 400-mg group -RRB- to five subjects -LRB- 100-mg and 200-mg groups -RRB- 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 -LRB- a maximum average of 4 % in the 100-mg group ; data not shown -RRB- , 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 In phase 1 , one subject in the placebo group showed a bilirubin concentration increase from 12 μmol/L on the morning of treatment -LRB- baseline -RRB- to 25 μmol/L 24 h after treatment , as described in `` Adverse events . '' Seven days after treatment , bilirubin concentration was reduced to 13 μmol/L . There were no other clinically significant changes in hematology , biochemistry , plant sterols , or urinalysis measurements , and there were no differences between treatment groups . One subject who received 100 mg of FM-VP4 showed a decrease in white blood cell -LRB- WBC -RRB- count from 6.2 × 109/L on the morning of treatment -LRB- baseline -RRB- to 2.9 × 109/L 24 h after dosing . Seven days after dosing , the WBC count recovered to 7.3 × 109/L . This event was not considered to be clinically significant . Nine subjects had abnormal laboratory values during phase 2 . Four laboratory abnormalities were considered clinically significant and were reported as adverse events . Two subjects showed a decrease of WBC count . One subject in the 800-mg group had a level of 2.2 × 109/L at screening , but those levels were increased to 5.8 × 109/L at the following visit and remained normal during the rest of the trial . This event was not considered clinically significant . A subject in the 100-mg group had a WBC count of 3 × 109/L at screening , and levels fluctuated between 2.8 × 109/L and 4.6 × 109/L during the study . Two subjects showed an increase in CK levels that was greater than five times the ULN . In one of these patients , who was in the 400-mg group , the event was recorded as an adverse event of moderate severity . In the other subject , who was in the placebo group , the event was not recorded as an adverse event , as the patient had performed vigorous exercise the previous day . Both subjects continued with medication , and both had CK levels within normal limits at subsequent measurements . Five subjects showed a decrease in hemoglobin of a least 1.5 g/d -LRB- 0.93 mmol/L -RRB- : one each in the 100-mg , 200-mg , and 400-mg groups , and two in the placebo group . However , those decreases occurred only once , and levels were restored to normal at the subsequent visit . Therefore , those changes were not considered clinically significant . There were no differences between treatment groups in the changes of vitamin A and vitamin E concentrations at week 4 compared with baseline -LRB- p = 0.32 and p = 0.38 , respectively ; data not shown -RRB- . 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 . In the phase 2 run-in period , mean LDL-C levels were 3.94 ± 0.71 mmol/L at 4 -- 8 weeks before baseline , 4.19 ± 0.78 mmol/L halfway through the run-in period , and 4.16 ± 0.76 mmol/L at baseline . The absolute lipoprotein levels at baseline and after 4 weeks of treatment per dose group and the mean percentage change in LDL-C levels are presented in Table 1 . The percent changes of LDL-C by visit are also depicted in Fig. 2 . ANOVA analyses showed a borderline statistically significant difference in the percentage change in LDL-C and HDL-C , and pairwise comparisons between each active dose and placebo revealed that 400 mg of FM-VP4 reduced LDL-C significantly -LRB- p = 0.02 -RRB- . In the placebo group , LDL-C increased by 2.7 % , and in the 400-mg/day group LDL-C reduced by 6.5 % . There were no statistically significant differences in percentage change of LDL-C changes between the other active doses and placebo , and there were no statistically significant differences in percentage change of HDL-C between any of the active doses and placebo -LRB- Table 1 -RRB- . When statistical analysis was carried out on a per-protocol basis and subjects who were noncompliant -LRB- n = 1 -RRB- or who received statins within 40 days before study treatment -LRB- n = 5 -RRB- were excluded , a dose-response in percentage change from baseline in LDL-C was observed : changes in the placebo group and in the 100 - , 200 - , 400 - , and 800-mg/day groups were 3.7 % , 0.9 % , − 4.1 % , − 6.9 % , and − 6.2 % , respectively . When using these per-protocol data , decreases in the 400 - and 800-mg/day groups were significantly different from placebo -LRB- p = 0.007 and p = 0.01 , respectively -RRB- . The percentage change in ApoB at week 4 compared with baseline differed significantly between treatment groups -LRB- p 0.007 -RRB- . Absolute changes in ApoB were 0.017 , 0.063 , − 0.058 , − 0.047 , and − 0.038 g/L in the placebo , 100 - , 200 - , 400 - , and 800-mg/day group , respectively . There were no differences between treatment groups in ApoAI and Lp -LRB- a -RRB- changes at week 4 compared with baseline -LRB- data not shown -RRB- . Table 1Plasma lipid and lipoprotein concentrations before and after 4 weeks of treatment with placebo or with 100 mg , 200 mg , 400 mg , or 800 mg/day of FM-VP4 -LRB- disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate in phase 2 -LRB- n = 20 per dose -RRB- Dose level -LRB- mg -RRB- P valueaPlacebo100 mg200 mg400 mg800 mgmmol/LTotal cholesterolBaseline6 .36 ± 0.656.40 ± 0.876.40 ± 0.886.04 ± 1.056.02 ± 0.90 Day 286.50 ± 0.726.49 ± 1.126.26 ± 0.785.81 ± 1.055.90 ± 0.90 % Change2 .6 % 1.2 % − 1.7 % − 3.8 % − 1.7 % 0.09 LDLBaseline4 .29 ± 0.634.17 ± 0.584.40 ± 0.774.02 ± 0.933.94 ± 0.83 Day 284.38 ± 0.794.28 ± 0.784.18 ± 0.633.75 ± 0.883.73 ± 0.81 % Change2 .7 % 2.9 % − 4.2 % − 6.5 % b − 4.6 % 0.05 HDLBaseline1 .37 ± 0.321.34 ± 0.381.20 ± 0.251.24 ± 0.301.21 ± 0.27 Day 281.37 ± 0.371.29 ± 0.341.28 ± 0.311.22 ± 0.271.26 ± 0.37 % Change − 0.1 % − 3.6 % 6.7 % − 1.2 % 4.1 % 0.04 TGBaseline1 .56 ± 0.771.86 ± 1.301.77 ± 0.721.73 ± 0.721.92 ± 0.68 Day 281.65 ± 0.861.95 ± 1.161.76 ± 1.081.86 ± 0.951.97 ± 1.00 % Change8 .2 % 16.3 % 0.7 % 7.5 % 9.7 % 0.8 All values are mean ± standard deviationLDL low-density lipoprotein , HDL high-density lipoprotein , TG triglyceridesaDifferences between all treatment groups were analyzed by using analysis of variance . If the difference between treatments was statistically significant , then each active treatment group was individually compared with placebobP < 0.025 -LRB- one-sided -RRB- as compared with the change in the placebo groupFig . 2Mean low-density lipoprotein cholesterol levels during 28 days of treatment with placebo or 100 , 200 , 400 , and 800 mg/day FM-VP4 -LRB- disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate -RRB- and after 14 days of follow-up Pharmacokinetics In the 100 - and 200-mg cohorts of phase 1 , the majority of plasma samples had DASP and DACP concentrations below detection level . Therefore , the AUC 0 → t and the plasma elimination half-live -LRB- t1/2 -RRB- values were not evaluable , and for that reason , those data are not presented . For the groups of 400 mg or higher , the peak DACP level was reached 6 -- 24 h after FM-VP4 administration -LRB- tmax -RRB- , and DASP tmax was reached 12 -- 24 h postdose with the exception of one subject in the 1,600 mg group who had a tmax of 49 h -LRB- Table 2 -RRB- . Mean t1/2 of all eligible subjects was 57 h -LRB- 2 -- 3 days -RRB- , which ranged from 16 to 134 h for DACP and from 29 to 90 h for DASP . Table 2Pharmacokinetic parameters of disodium ascorbyl campestanol phosphate -LRB- DACP -RRB- and disodium ascorbyl sitostanol phosphate -LRB- DASP -RRB- in 24 subjects after a single dose of 400 , 800 , 1,600 , or 2,000 mg FM-VP4 -LRB- DACP and DASP -RRB- in phase 1Dose level -LRB- mg -RRB- Numbert1/2 -LRB- h -RRB- Cmax -LRB- ng/mL -RRB- tmax -LRB- h -RRB- AUC 0 → ∞ -LRB- h.ng / mL -RRB- AUC 0 → t -LRB- h.ng / mL -RRB- DACP 400479.6 ± 42.3100.6 ± 25.510.7 ± 3.08,734 ± 3,8645,942 ± 2,646 800446.2 ± 21.8144.5 ± 43.612.1 ± 0.17,672 ± 2,4795,787 ± 2,045 1,600435.9 ± 11.1175.6 ± 106.57.9 ± 3.211,736 ± 11,4559,314 ± 10,910 2,000464.0 ± 28.3190.9 ± 47.218.3 ± 6.417,179 ± 5,68511,414 ± 4,318 DASP 400477.3 ± 11.6247.9 ± 55.112.1 ± 0.128,142 ± 7,15321,115 ± 5,255 800452.2 ± 19.0339.7 ± 86.912.1 ± 0.128,872 ± 8,18322,907 ± 9,382 1,600453.0 ± 14.9344.3 ± 225.824.9 ± 21.639,029 ± 38,42830,243 ± 30,624 2,000443.6 ± 21.8495.9 ± 89.018.5 ± 6.635,642 ± 16,58429,204 ± 14,549 Mean ± standard deviationt1/2 half-life in plasma , Cmax peak concentration , tmax time to reach peak concentration , AUC0 → t area under the concentration time curve where t is the last time of blood sampling at 168 h -LRB- 7 days -RRB- after treatment AUC0 → t strongly correlated with the dose -LRB- 400 -- 2,000 mg -RRB- of FM-VP4 for DACP -LRB- R2 = 0.93 -RRB- and DASP -LRB- R2 = 0.90 -RRB- . However , AUC increased in a lower than dose-proportional manner . Between 400 mg and 2,000 mg , DACP and DASP AUC increased approximately twice and 1.3 times , respectively , when the dose was increased fivefold -LRB- Table 2 -RRB- . In phase 2 , trough DACP and DASP concentrations were at or near steady-state levels by day 8 in the subset of subjects per active dose group who were hospitalized and sampled during the first week of dosing -LRB- Fig. 3 -RRB- . The mean concentrations for all subjects in each cohort on day 8 were about the same as the mean concentrations on day 28 , at the end of the treatment period . At the end of the follow-up period , 14 days after the final dose , DASP was present in the plasma of most or all of the subject plasma samples in the 200 - , 400 - , and 800-mg groups . DACP was mainly present in plasma samples of most subjects in the 800-mg group . The majority of plasma samples for DACP in the 100 - and 200-mg groups and for DASP in the 100-mg group were below detection limit 14 days after follow-up . Fig. 3Mean trough concentrations of disodium ascorbyl sitostanol phosphate -LRB- DASP -RRB- and disodium ascorbyl campestanol phosphate -LRB- DACP -RRB- during 28 days of dosing and 14 days postdosing follow-up . The dots of days 2 -- 5 represent the 16 hospitalized subjects on FM-VP4 -LRB- DACP and DASP -RRB- , whereas the dots of days 8 , 28 , and 42 represent all 80 subjects on FM-VP4 . DASP and DACP were not present in plasma at day 1 -LRB- baseline -RRB- , and DACP concentrations were mostly below detection limit during the first 5 days of intake in the 100-mg group . The mean levels of DASP in the 100-mg group and DACP in the 200-mg group were below the lower limit of quantification -LRB- LLOQ -RRB- because some of the subjects had levels below the LLOQ , which were regarded as 0 ng/ml 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 -LRB- data not shown -RRB- . In phase 2 , sitostanol and campestanol apparently rose in a dose-dependent fashion over the 28-day time period , with the maximal percent change from baseline being 87 % for campestanol and 178 % for sitostanol in the 800-mg group -LRB- Table 3 -RRB- . Campesterol and sitosterol did not change from baseline to day 28 -LRB- Table 3 -RRB- . Table 3Concentrations of plant sterols and sterols in 100 subjects after 4 weeks of treatment with 100 , 200 , 400 , or 800 mg/day FM-VP4 -LRB- disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate -RRB- or placebo in phase 2Plant stanol or sterolDose level -LRB- mg -RRB- P valuePlacebo -LRB- n = 20 -RRB- 100 -LRB- n = 20 -RRB- 200 -LRB- n = 20 -RRB- 400 -LRB- n = 20 -RRB- 800 -LRB- n = 20 -RRB- ng/mLSitostanolBaseline77 .9 ± 33.379.6 ± 30.890.4 ± 32.975.0 ± 28.363.8 ± 19.2 Day 28102.5 ± 39.2104.2 ± 38.3132.5 ± 56.3178.3 ± 63.8168.3 ± 44.6 % Change41 ± 5837 ± 4648 ± 37159 ± 99178 ± 83 < 0.0001 CampestanolBaseline116 .0 ± 84.691.0 ± 36.6104.3 ± 81.7100.7 ± 57.283.4 ± 18.9 Day 28140.9 ± 77.3107.1 ± 36.6134.1 ± 78.9156.2 ± 72.5150.2 ± 37.9 % Change39 ± 7021 ± 2243 ± 5070 ± 6187 ± 570.002 SitosterolBaseline2 ,998.3 ± 1,842.53,579.3 ± 2,084.32,715.0 ± 10,725.82,704.3 ± 1,071.22,276.3 ± 993.2 Day 283,036.0 ± 1,164.93,782.1 ± 2,377.92,744.9 ± 1,256.13,138.4 ± 1,632.72,316.9 ± 734.4 % Change35 ± 1475 ± 202 ± 2317 ± 4010 ± 380.61 CampesterolBaseline3 ,275.2 ± 2,017.23,699.1 ± 1,914.82,850.4 ± 1,289.02,791.1 ± 1,403.22,370.8 ± 1,036.6 Day 283,212.3 ± 1,375.93,870.2 ± 2,252.63,002.7 ± 1,439.23,031.5 ± 1,500.12,416.9 ± 917.6 % Change16 ± 804 ± 226 ± 1912 ± 358 ± 350.91 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 . No drug-related abnormalities could be identified by laboratory safety tests . In phase 1 , one subject receiving 100 mg of FM-VP4 showed a decrease in WBC count 24 h after treatment , but this was considered unrelated to the study drug and returned to normal 7 days after treatment . In phase 2 , one subject in the 100-mg group and one in the 800-mg group also showed low WBC counts , but these abnormalities were already present at baseline . One subject in the placebo group but also one in the 400-mg group showed increased CK levels at one visit . However , we think this is no reason for concern . First of all , the patient in the 400-mg group had no complaints . Second , the levels had returned to normal by the next visit without medication discontinuation . Because no adverse events have been reported for plant sterols in general -LSB- 8 -RSB- , we think this single CK increase was likely to be a chance finding . In addition , five subjects from various dose groups showed a hemoglobin decrease at one of the visits . These decreases were nonpersistent , and levels had returned to normal by subsequent visits . No EKG or hemodynamic abnormalities were detected after administration of FM-VP4 . Also , no liver or kidney abnormalities were identified . Thus , in terms of safety tests , there was no evidence that treatment with FM-VP4 caused any acute or delayed toxicity . In phase 1 , DACP and DASP were absorbed slowly into plasma , with a tmax of approximately 12 h for both components . Clearance was also rather slow , with the average elimination t1/2 of 57 h. Furthermore , t1/2 from phase 1 can be used to estimate the time to reach steady-state concentration levels by calculating three times t1/2 , which is 3 × 57 = 171 h , or 7.1 days . This estimated time to reach steady state corresponds with phase 2 data that showed a steady state after approximately 8 days , as the mean trough concentration at day 8 was similar to the mean trough concentration after 4 weeks of treatment . The low plasma concentrations of DACP and DASP suggest low bioavailability of FM-VP4 in humans . This is supported by findings with other plant sterol structures . The bioavailability from 600 mg unesterified soy plant stanols was only 0.04 % for sitostanol and 0.15 % for campestanol -LSB- 22 -RSB- , and in another study , the absorption of campestanol from a margarine spread containing 540 mg campestanol as fatty acid esters was 5.5 % in healthy subjects -LSB- 23 -RSB- . In both studies , absorption of plant sterols was measured by intravenous injection and oral administration of labeled isotopes in the fasting state -LSB- 22 , 23 -RSB- . As we have not administered labeled FM-VP4 intravenously , we were not able to estimate the bioavailability of FM-VP4 in humans and to investigate whether the esterified ascorbate group affects its absorption . Future studies with FM-VP4 are needed to assess the bioavailability of FM-VP4 in humans . Repeated doses of FM-VP4 in phase 2 appeared to increase sitostanol levels in plasma by up to 178 % and campestanol by 87 % , whereas corresponding sterol components did not change . This rise in campestanol and sitostanol appeared to increase with dose but in a less than proportional manner . Furthermore , the difference in increase from baseline between sitostanol and campestanol was consistent with the difference in plasma concentrations for DASP and DACP of approximately 2:1 , which was most clear in the 400 - and 800-mg groups . We speculate that conversion of DASP and DACP to free stanols may have occurred in vivo or that FM-VP4 alters the metabolism of plant stanols . Nevertheless , the absolute increase in total stanol levels remains relatively small , as the stanol contribution is < 5 % of total levels -LRB- Table 3 -RRB- . Therefore , the clinical relevance of such increases is probably limited . Based on our data , we may speculate on the mechanism by which FM-VP4 exerts its LDL-C-lowering effect . Plant sterols and stanols are thought to compete with cholesterol for incorporation into mixed micelles , the vehicles that transport sterols to the enterocyte . In the case of FM-VP4 , the solubility of plant stanols for the mixed micelles has been improved by the addition of the hydrophilic ascorbyl residue to the hydrophobic campestanol and sitostanol tail through a phosphodiester linkage -LRB- FM-VP4 -RRB- . In fact , this new structure allowed self-assembly into micelle structures in aqueous media in the absence of bile salts -LSB- 24 -RSB- . Although the assembly of micelles by means of FM-VP4 has never been directly compared with other types of plant sterols , the incorporation of plant stanols and sterols as well as cholesterol into mixed micelle normally requires the addition of bile salts in vitro and in vivo . Furthermore , in animals , FM-VP4 was shown to be more potent than unesterified plant stanols in a direct comparison when administered in the diet -LSB- 16 -RSB- . The results of the current clinical trial in humans showed that FM-VP4 lowered LDL-C by 6 -- 7 % compared with baseline , whereas the control group showed a 3 -- 4 % increase in plasma LDL-C levels . This means that compared with placebo , FM-VP4 showed LDL-C decreases of 9 -- 11 % when administered at 400 -- 800 mg/day over 4 weeks . This effect was also reached with plant sterol and stanol esters , but at doses that were two to three times higher than the currently used doses of FM-VP4 -LSB- 8 -RSB- . Thus , the higher solubility of FM-VP4 into mixed micelles may lead to a more efficacious competition with cholesterol for incorporation into the micelles , and as a consequence , a lower dose of FM-VP4 is required to realize an equal LDL-lowering effect compared with plant sterol and stanol esters . However , the optimal treatment dose and duration , schedule and formulation for FM-VP4 have yet to be determined . On the other hand , it has also been suggested that , in addition to competition with cholesterol for incorporation into the micelles , plant sterol and stanol esters also may activate liver X receptor -LRB- LXR -RRB- target genes within the enterocyte -LSB- 25 -RSB- . It is unknown whether DACP and DASP activate such genes within the enterocyte or whether there may be systemic effects or additional mechanisms to decrease cholesterol absorption . Further research is needed to elucidate the mechanism by which several types of plant sterol and stanol esters lower LDL-C levels . 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 .

O O B O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O