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 (DASP) and disodium ascorbyl campestanol phosphate (DACP).
Introduction
Worldwide, cardiovascular disease (CVD) 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 (LDL-C) lead to an increased risk of atherosclerosis and coronary heart disease [1]. 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 [2–6]. 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 (HDL-C) concentrations [7, 8]. Plant sterols and stanols are thought to decrease plasma cholesterol concentrations by inhibiting cholesterol absorption in the intestine [9]. 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 [10].
A new water soluble plant stanol derivative, disodium ascorbyl phytostanol phosphate (FM-VP4, Fig. 1) 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 [3H]-cholesterol by approximately 50% in human and rat enterocyte cell lines [11, 12]. In vivo, FM-VP4 led to a dose-related inhibition of [3H]-cholesterol absorption in rats, as shown by a maximally 80% reduced area under the concentration time curve (AUC) of orally administered micellar [3H]-cholesterol [13]. Furthermore, the LDL-lowering activity of FM-VP4 was observed in a broad range of LDL-sensitive species, including gerbils [14, 15], hamsters [16], and apolipoprotein E (ApoE)-deficient transgenic mice [17]. In hamsters, FM-VP4 was a more potent LDL-lowering agent than unesterified plant stanols [16], and in ApoE deficient mice, the LDL-lowering effect of FM-VP4 was correlated with retardation of atherosclerotic plaque development [17]. Other effects of FM-VP4 included the decrease of plasma triglyceride levels and the reduction of abdominal fat or body-weight gain in gerbils [14, 15] and mice [18]. 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 (R=CH3) and disodium ascorbyl sitostanol phosphate (R=C2H5)
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 (TG) concentration ≤4.5 mmol/L at the first visit, had a body mass index (BMI) <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 (ULN). 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 (40 days) 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. (Vancouver, Canada). 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 (DASP) and disodium ascorbyl campestanol phosphate (DACP) (Fig. 1), 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 (100, 200, 400, 800, 1,600, or 2,000 mg), and within each group, one subject was randomly assigned to placebo. Once a complete cohort of five subjects was treated and the safety parameters had been reviewed, the following dose was initiated. In the subsequent phase 2 trial, 100 men were treated for 28 days (4 weeks). Twenty-five subjects were assigned to each dose group (100, 200, 400, or 800 mg/day), and within each group, five subjects were randomly assigned to placebo. The first five subjects in each cohort were hospitalized for 5 days. Escalation to the next dosing level was only allowed once these five subjects completed treatment and all results and safety data were evaluated.
Phase 1 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 (EKG) 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 (BP), 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 (6 or 7 days) 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 (NCEP) 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 [alanine aminotransferase (ALT) and aspartate aminotransferase (AST), bilirubin, creatine kinase (CK), creatinine, glucose, and C-reactive protein (CRP)] 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 (TSH) 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 (MRL International, Zaventem, Belgium) by using standardized procedures, and LDL-C was calculated using the Friedewald equation [19]. The results were kept blinded to the investigators.
Plasma was stored at −80°C for further analyses. ApoB, ApoAI, and lipoprotein a [Lp(a)] as well as vitamin E and vitamin A were analyzed in one run after the study had been completed. ApoAI, ApoB, and Lp(a) levels were determined by nephelometry with a Beckman Array (Mijdrecht, the Netherlands) according to the manufacturer’s instructions, and vitamin E and vitamin A were measured by high-performance liquid chromatography (HPLC) with fluorescence detection using a Chromsep Glass, 100*3 mm, inertsil 5, ODS-3 column (Varian-Chrompack, Middelburg, the Netherlands).
All DASP and DACP plasma concentrations were assayed by a validated liquid chromatography/mass spectrometry/mass spectrometry method (LC/MS/MS) based on a previously described method [20]. The lower limit of quantification (LLOQ) 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 (Pharsight WinNonlin, Mountain View, CA, USA) 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 (AUC 0-t) was estimated by the trapezoidal rule [21]. 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 (day 42). In subjects who were hospitalized for the first 5 days of treatment, trough DASP and DACP concentrations were also measured each morning of hospitalization (day 1 through day 5).
Plant sterols and stanols β-sitosterol, campesterol, sitostanol, and campestanol were assessed by selective ion monitoring gas chromatography mass spectrometry (SIM-GC-MS). High-purity solvents were purchased from Merck, Germany. Bis-(Trimethylsilyl)trifluoroacetamide (BSTFA) was obtained from Sigma (Steinneim, Germany) and pyridine from Pierce (Rockford, IL, USA). Beta-sitosterol (24β-ethylcholesterol), β-sitostanol (24α-ethyl-5α-cholestan-3β-ol), and campesterol (24α-methyl-5-cholesten-3β-ol) 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% (w/v) 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 (v/v 5:1) 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 (1:20) leading to a 0.2 mm × 25 m Chrompack CP-sil 19 CB (WCOT Fused Silica) 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 (ANOVA) 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 (TC), HDL-C, and TG. For ApoB, ApoAI, Lp(a), 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 (93%) were of Caucasian descent. Mean subject age was 50.4 (range 24–63) years. In phase 2, 101 male subjects were enrolled, but one subject withdrew consent prior to receiving study medication due to personal reasons and therefore contributed no safety or efficacy data. The remaining 100 subjects completed the study. The majority of subjects (89%) were Caucasian, with the remaining being Asian or another race. Mean subject age was 53.7 (range 23–75) years. As calculated from returned tablets, the mean percentage of received tablets was 97%. One subject in the 800-mg group had a compliance <80%. All other patients received >80% of the planned number of tablets during the treatment period. Five subjects received statin treatment within 40 days before the first dose of study treatment (29–35 days). Mean weight ranged between 83.9 ± 13.1 and 84.6 ± 13.0 kg during the entire study.
Adverse events
In phase 1, 23 treatment-emergent adverse events were reported by 16 subjects. Of these events, five (21.7%) occurred in the 100-mg group, three (13.0%) in both the 200- and 400-mg groups, four (17.4%) in the 800-mg group, two (8.7%) in the 1,600-mg group, and three (13.0%) in both the 2,000 mg and placebo groups. All reported events were mild. The most common events were dizziness, headache, and fatigue. Other adverse events were loose stools, vasovagal attack, influenza, upper respiratory tract infection, elevated bilirubin, elevated BP, arthralgia, difficult micturition, polyuria, and pharyngolaryngeal pain. Three events were considered to be possibly related to the study drug. One was reported by a subject receiving the placebo treatment, whose bilirubin concentration increased from 12 μmol/L on the morning of treatment (baseline) to 25 μmol/L 24 h after treatment but decreased to normal 7 days after treatment. The other two possibly related events were reported in the 800- and 1,600-mg groups, and both consisted of one episode of loose stools on the day of treatment.
In phase 2, 67 subjects reported one or more treatment-emergent adverse events: 12 in the 100-mg group, 14 in the 200-mg group, 11 in the 400-mg group, 15 in the 800-mg group, and 15 in the placebo group. Most events were mild, and four subjects reported a moderate event. The most frequent event was headache, which was reported by a total of 19 subjects and by two (400-mg group) to five subjects (100-mg and 200-mg groups) in each of the five groups. The four adverse events that were moderate included two subjects with headache in the 800-mg group, one subject with an elevated CK level in the 400-mg group, and one subject with epilepsy in the placebo group. No subjects discontinued study treatment due to a treatment-emergent adverse event. One subject in the 800-mg group did not take the study medication for 3.5 days due to nausea and diarrhea, which was not considered to have been related to the study drug. Once the subject recommenced treatment, no further treatment-emergent adverse events were reported.
A total of 24 subjects reported events that were considered to be drug-related: eight in the 100-mg group, one in the 200-mg group, two in the 400-mg group, six in the 800-mg group, and seven in the placebo group. The most commonly reported event was flatulence, which was reported by three subjects in the 100-mg groups, one in the 400-mg group, two in the 800-mg group and one in the placebo group. There were no differences in the incidence of treatment-emergent adverse events between active and placebo groups.
Blood pressure, heart rate, and EKG analysis
There was no effect of FM-VP4 on BP or heart rate during phase 1. In phase 2, the mean systolic BP was slightly decreased after 4 weeks of treatment (a maximum average of 4% in the 100-mg group; data not shown), but there was no relationship between this decrease and the dose of FM-VP4. Diastolic BP did not change. All pre- and postdose EKGs were normal in both phases of the trial.
Laboratory analyses
In phase 1, one subject in the placebo group showed a bilirubin concentration increase from 12 μmol/L on the morning of treatment (baseline) 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 (WBC) count from 6.2 × 109/L on the morning of treatment (baseline) 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 (0.93 mmol/L): 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 (p=0.32 and p=0.38, respectively; data not shown).
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 (p=0.02). 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 (Table 1). When statistical analysis was carried out on a per-protocol basis and subjects who were noncompliant (n=1) or who received statins within 40 days before study treatment (n=5) 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 (p=0.007 and p=0.01, respectively). The percentage change in ApoB at week 4 compared with baseline differed significantly between treatment groups (p 0.007). 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(a) changes at week 4 compared with baseline (data not shown).
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 (disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate in phase 2 (n=20 per dose) Dose level (mg)P valueaPlacebo100 mg200 mg400 mg800 mgmmol/LTotal cholesterolBaseline6.36 ± 0.656.40 ± 0.876.40 ± 0.886.04 ± 1.056.02 ± 0.90Day 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%0.09LDLBaseline4.29 ± 0.634.17 ± 0.584.40 ± 0.774.02 ± 0.933.94 ± 0.83Day 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%0.05HDLBaseline1.37 ± 0.321.34 ± 0.381.20 ± 0.251.24 ± 0.301.21 ± 0.27Day 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%0.04TGBaseline1.56 ± 0.771.86 ± 1.301.77 ± 0.721.73 ± 0.721.92 ± 0.68Day 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%0.8All 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 (one-sided) 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 (disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate) 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 (t1/2) 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 (tmax), 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 (Table 2). Mean t1/2 of all eligible subjects was 57 h (2–3 days), 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 (DACP) and disodium ascorbyl sitostanol phosphate (DASP) in 24 subjects after a single dose of 400, 800, 1,600, or 2,000 mg FM-VP4 (DACP and DASP) in phase 1Dose level (mg)Numbert1/2 (h)Cmax (ng/mL)tmax (h)AUC 0→∞ (h.ng/mL)AUC 0→t (h.ng/mL)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 ± 4,318DASP  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 ± 14,549Mean ± 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 (7 days) after treatment
AUC0→t strongly correlated with the dose (400–2,000 mg) of FM-VP4 for DACP (R2 = 0.93) and DASP (R2 = 0.90). 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 (Table 2).
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 (Fig. 3). 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 (DASP) and disodium ascorbyl campestanol phosphate (DACP) 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 (DACP and DASP), 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 (baseline), 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 (LLOQ) 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 (data not shown).
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 (Table 3). Campesterol and sitosterol did not change from baseline to day 28 (Table 3).
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 (disodium ascorbyl campestanol phosphate and disodium ascorbyl sitostanol phosphate) or placebo in phase 2Plant stanol or sterolDose level (mg)P valuePlacebo (n = 20)100 (n = 20)200 (n = 20)400 (n = 20)800 (n = 20)ng/mLSitostanolBaseline77.9 ± 33.379.6 ± 30.890.4 ± 32.975.0 ± 28.363.8 ± 19.2Day 28102.5 ± 39.2104.2 ± 38.3132.5 ± 56.3178.3 ± 63.8168.3 ± 44.6% Change41 ± 5837 ± 4648 ± 37159 ± 99178 ± 83<0.0001CampestanolBaseline116.0 ± 84.691.0 ± 36.6104.3 ± 81.7100.7 ± 57.283.4 ± 18.9Day 28140.9 ± 77.3107.1 ± 36.6134.1 ± 78.9156.2 ± 72.5150.2 ± 37.9% Change39 ± 7021 ± 2243 ± 5070 ± 6187 ± 570.002SitosterolBaseline2,998.3 ± 1,842.53,579.3 ± 2,084.32,715.0 ± 10,725.82,704.3 ± 1,071.22,276.3 ± 993.2Day 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 ± 380.61CampesterolBaseline3,275.2 ± 2,017.23,699.1 ± 1,914.82,850.4 ± 1,289.02,791.1 ± 1,403.22,370.8 ± 1,036.6Day 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 ± 350.91Mean ± 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 [8], 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 [22], 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 [23]. In both studies, absorption of plant sterols was measured by intravenous injection and oral administration of labeled isotopes in the fasting state [22, 23]. 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 (Table 3). 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 (FM-VP4). In fact, this new structure allowed self-assembly into micelle structures in aqueous media in the absence of bile salts [24]. 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 [16]. 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 [8]. 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 (LXR) target genes within the enterocyte [25]. 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.

safety
pharmacokinetics
fm-vp4
plant sterols
hypercholesterolemia


Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin

Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls-AChBP–CTD complex than in the Ls-AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.
Introduction
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast-acting excitatory cholinergic neurotransmission in vertebrates and invertebrates. Each nAChR molecule consists of five subunits; acetylcholine (ACh) binds at extracellular ligand-binding domain (LBD) at the subunit interfaces, and a central, cation-permeable ion channel opens transiently in response to ACh and agonists (Karlin 2002; Sine and Engel 2006). Each subunit possesses an N-terminal extracellular domain with a conserved di-cysteine loop, four transmembrane regions (TM1–TM4 of which TM2 provides most of the channel-lining residues), and a large intracellular loop between TM3 and TM4 (Karlin 2002). Usually, nAChRs are made up of α and non-α subunits (Fig. 1a), the α subunits being defined by adjacent cysteines in loop C of LBD. The ACh binding site is located at the LBD interfaces and formed by loops A, B and C of the α subunit and loops D, E and F which are normally located in non-α subunit (Corringer et al. 2000; Karlin 2002; Lindstrom 2003). However, α7, α8 and α9 subunits can form functional homo-pentamers (Couturier et al. 1990; Elgoyhen et al. 1994; Gerzanich et al. 1994) (Fig. 1a), whereas α9 and α10 subunits form α hetero pentamers (Elgoyhen et al. 2001). When only α subunits are present, they donate not only loops A–C, but also loops D–F.
Fig. 1Nicotinic acetylcholine receptors (nAChRs) and the acetylcholine binding protein (AChBP). a Schematic representation of neuronal nicotinic acetylcholine receptors and acetylcholine binding protein. b Chemical structures of natural nicotinic ligands and neonicotinoids [imidacloprid (IMI) and clothianidin (CTD)] used in this study. c Multiple sequence alignment of AChBP with the ligand binding domain of nAChR subunits. Lymnaea stagnalis AChBP (Ls-AChBP) and N-terminus LBD sequences of insect (species names are colored magenta) and vertebrate (species names are colored black) nAChRs are aligned using ClustalW2 program (Larkin et al. 2007) with default parameters and the details are manually adjusted. Secondary structure elements from the Ls-AchBP–IMI complex are indicated above the multiple alignments. Amino acids involved in the interactions with neonicotinoids are highlighted in bold with yellow and cyan backgrounds in the principal (+)- and complementary (-)-sides, respectively. Amino acid residue number from methionine for each protein was indicated at the top of each sequence
In mammals, nAChRs play central roles in neuromuscular and inter-neuronal cholinergic neurotransmission. Mutations in muscle nAChR subunits account for many cases of congenital myasthenia syndrome (Engel et al. 2003) and also multiple pterygium (Morgan et al. 2006), whereas mutations in neuronal nAChR subunits (α4,β2) can produce epilepsies (Aridon et al. 2006; Steinlein 2004). It has also been shown that cholinergic neurotransmission is reduced in Alzheimer’s (Marutle et al. 1999) and Parkinson’s diseases as well as in schizophrenia (Woodruff-Pak and Gould 2002). Further, in some cases of Alzheimer’s disease, loss of nAChRs has been reported (Court et al. 2001). Thus there is a growing interest in nAChRs as potential drug targets (Arneric et al. 2007; Dani and Bertrand 2007).
In the insect nervous system, where ACh is the primary excitatory neurotransmitter, nAChRs are present at densities comparable to those in the electric organ of the electric fish, Electrophorus electricus (Sattelle 1980), and several classes of insecticides (cartap, bensultap, thiocyclam, spinosad and neonicotinoids) target insect nAChRs (Narahashi 2000; Raymond-Delpech et al. 2005; Millar and Denholm 2007). Of these, neonicotinoids (Fig. 1a) are increasingly used not only for controlling crop pests worldwide, but also for animal health application such as flea and louse controls. Imidacloprid (IMI; Fig. 1b), the first insecticide with billion dollars per annum sales, is a partial agonist of native (Deglise et al. 2002; Ihara et al. 2006; Tan et al. 2007) as well as of recombinant (Ihara et al. 2003) nAChRs expressed in Xenopuslaevis oocytes. On the other hand, clothianidin (CTD; Fig. 1b) and its analog evoke supra-maximal responses (with reference to ACh responses) not only in the Drosophila Dα2/chicken β2 hybrid nAChRs (Ihara et al. 2004) expressed in Xenopus oocytes, but also in native Drosophila central cholinergic neurons (Brown et al. 2006). Single channel recording has shown that a CTD analog induces the opening of the Drosophila nAChR channels at the largest conductance state more frequently than ACh, offering a possible explanation for its super-agonist action (Brown et al. 2006). Contrasting with these actions, some neonicotinoids (Ihara et al. 2006; Salgado and Saar 2004), and bis-neonicotinoids containing two neonicotinoid units joined together by an alkyl chain (Ihara et al. 2007a), antagonize the acetylcholine-induced responses of native insect neurons.
Neonicotinoids show higher affinity for insect nAChRs, accounting, at least in part, for their selective toxicity to insects over vertebrates (Matsuda et al. 2001, 2005; Tomizawa and Casida 2005). Our studies using site-directed mutagenesis combined with two-electrode voltage-clamp electrophysiology have shown that the X residue in the YXCC motif of loop C (Shimomura et al. 2004), the region upstream of loop B (Shimomura et al. 2005) and basic residues in loop D (Shimomura et al. 2002, 2003, 2006) contribute to the high neonicotinoid sensitivity of insect nAChRs (see Fig. 1c for loop positions).
The acetylcholine binding protein (AChBP) from snail, Lymnaea stagnalis (Ls-AChBP) was discovered in glial cells as a water soluble protein modulating synaptic ACh concentration (Smit et al. 2001), and the X-ray crystal structure of the protein was described at the same time (Brejc et al. 2001). Subsequently, X-ray crystal structures of Ls-AChBP homologs from Aplysia californica (Ac-AChBP) (Bourne et al. 2005) and Bulinus truncatus (Bt-AChBP) (Celie et al. 2005b) were reported. Both Ls- and Ac-AChBPs show similarities to the extracellular domain of neuronal α7 nAChRs and form homopentamers in solution (Smit et al. 2001). Notably, all the six regions (loops A–F) that make up the ligand binding site are conserved in AChBPs. Thus AChBPs are considered as surrogates of the LBD in nAChRs and therefore have been employed to elucidate the mechanism for interactions with low molecular weight ligands as well as peptide toxin antagonists (Bourne et al. 2005; Celie et al. 2004, 2005; Dutertre et al. 2007; Hansen et al. 2005; Hansen and Taylor 2007; Ulens et al. 2006). Employing the crystal structures of AChBPs, we have previously modeled LBDs in complex with IMI to show that basic residues in loop D may interact electrostatically with the nitro group of neonicotinoids (Shimomura et al. 2006). Yet, details of neonicotinoid–nAChR interactions are poorly understood. Therefore, we have crystallized the Ls-AChBP in complex with two commercial neonicotinoids, IMI and CTD.
Materials and methods
Materials
Lymnaea stagnalis derived from the stocks of Vrije Universeit (Amsterdam) was provided from Professor Sakakibara at Tokai University. IMI and CTD were gifts from BayerCropscience Co. in Japan. 1-(6-Bromopyridylmethyl)-2-nitroiminoimidazolidine (bromine-substituted imidacloprid; Br-IMI) and N-(2-bromo-thiazol-5-ylmethyl)-N′-methyl-N′′-nitroguanidine (bromine-substituted clothianidin; Br-CTD) were synthesized according to the published methods (Maienfisch et al. 2000; Nishiwaki et al. 2000).
Protein preparation
The cDNA of Ls-AChBP was amplified by RT-PCR from L. stagnalis and was cloned into the pPICZα B vector (Invitrogen) at the Pst I site, with an L1A mutation introduced to facilitate cloning. The Ls-AChBPs were expressed in yeast, Pichia pastris X-33 using EasySelect Pichia Expression Kit (Invitrogen). Secreted proteins were purified with Q-Sepharose column (GE Healthcare) immediately following concentration and buffer-exchange using Vivaflow200 (Zartorius), and then treated with 50 U/mg protein of peptide-N-glycosidase F (Wako Pure Chemical Industries) at 37°C for 24 h to remove the glycosyl-chain at Asn67. The protein samples were subsequently purified with Mono Q and Superdex 200 columns (GE Healthcare). Purified proteins were confirmed to be the samples of interest by N-terminal sequencing and mass spectrometry. Purified proteins were buffer-exchanged against 20 mM Tris–HCl buffer with 0.02% NaN3 (pH 8.0).
Evaluation of equilibrium binding of neonicotinoids
The equilibrium binding of neonicotinoids with Ls-AChBP was determined by quenching of intrinsic tryptophan fluorescence. Ls-AChBP at 600 nM in binding site concentration in 20 mM Tris–HCl buffer with 0.02% NaN3 (pH 8.0) was incubated with each ligand at various concentrations on a 96-well plate. Ls-AChBP was excited at 280 nm, and emission intensity was recorded at 342 nm using Varioskan microplate reader (Thermo Fisher Scientific) at room temperature. Data were fitted by non-linear regression according to the following equation using Prism Software version 4.03 (GraphPad Software): where ΔF and ΔFmax represent quenching of fluorescence at a ligand concentration X and maximum quenching of fluorescence at saturation, respectively. F0 and [Protein] are fluorescence measured in the absence of ligands and the concentration of Ls-AChBP, respectively.
Crystallization and X-ray data collection
Purified Ls-AChBPs (6.0 mg/mL) were incubated with 0.5 mM neonicotinoids at 4°C for 1 h prior to crystallization. Ls-AChBP-neonicotinoid complex crystals were obtained by vapor diffusion method at 20°C with 1:1 ratio of protein to reservoir solution containing 0.2 M Na citrate pH 5.7, 15–22% PEG3350 and about 0.5 mM of either IMI, CTD, Br-IMI or Br-CTD. The crystals were flash-cooled in liquid nitrogen after soaking into the cryoprotectant solutions containing 0.2 M Na citrate pH 5.7, 25% PEG3350, 20% glycerol and about 0.5 mM of each neonicotinoid. X-ray diffraction data sets were collected at 90 K using either Bruker AXS DIP6040 detector at BL44XU (Yoshimura et al. 2007) or ADSC QUANTUM 210 detector at BL44B2 (Adachi et al. 2001) beamlines in SPring-8, and processed with Mosflm (Leslie 1992) or HKL2000 (Otwinowski and Minor 1997). In order to identify the positions of the sulfur atom at the thiazole ring of CTD as well as the bromine atom of Br-IMI and Br-CTD, anomalous data from the crystals complexed with these neonicotinoids were collected at wavelength of 1.75 Å and 0.919 Å (Table 1), respectively.
Table 1X-ray diffraction data collection and refinement statistics for Ls-AChBP–neonicotinoid complexesIMICTDBr-IMIBr-CTDCTD (S ano)aData collection BeamlinebBL44XUBL44B2BL44B2BL44B2BL44B2 Wavelength (Å) 0.9000.9190.9190.9191.75 Space groupP65P65P65P65P65 Cell dimensions a, c (Å)75.0, 351.074.6, 351.074.8, 350.974.5, 351.274.6, 351.3 Resolution (Å)15.8–2.5650.0–2.7050.0–2.7050.0–2.9050.0–2.90 Rsym (%)c, d8.0 (41.3)8.5 (38.9)9.7 (34.1)11.8 (34.1)8.7 (39.5) I/σc8.1 (1.7)20.7 (4.0)47.0 (10.7)45.5 (10.9)60.8 (9.32) Completeness (%)c94.8 (96.7)100 (100)100 (100)100 (100)99.5 (96.2) Redundancyc2.6 (2.6)5.7 (5.7)23.0 (23.3)22.5 (23.2)21.6 (20.5)Refinement Resolution (Å)15.8–2.5850.0–2.70 No. of reflections3303130121 R/Rfree (%)e20.3/27.720.2/27.0 Bond length (Å)/angles (deg)f0.008/1.30.008/1.3 Average B factor41.841.6 PDB accession codeg2ZJU2ZJVSee text for abbreviations of the neonicotinoid namesaData set for detection of anomalous peaks at sulfur atomsbAll data sets were collected at SPring-8cValues in parentheses refer to data in the highest resolution shellsdRsym = Σ|I- < I>|/ΣI, where I is the observed intensity and <I> is the average intensity from multiple observations of the symmetry-related reflectionseR = Σ||Fo| – |Fc|| / Σ|Fo|. Rfree is an R factor of the CNS refinement evaluated for 5% of reflections that were excluded from the refinementfRMS deviations from ideal values were calculatedgThe atomic coordinates and structure factors have been deposited in the Protein Data Bank
Structure determination and refinement
The structure of the IMI complex was solved by molecular replacement with Phaser (McCoy 2007) using the coordinates of an Ls-AChBP in complex with nicotine (PDB entry: 1UW6) (Celie et al. 2004) as a search model. Subsequently, the refined coordinates of the IMI complex were used to solve the structure of the CTD complex. Refinements and manual model building were performed with CNS version 1.2 (Brünger et al. 1998) and Coot (Emsley and Cowtan 2004), respectively. The non-crystallographic-symmetry restraints between the subunits were initially applied for the refinements and removed at later steps. The details of data collection and refinement statistics are provided in Table 1. Figures are generated by PyMOL (DeLano Scientific LLC), Bobscript (Esnouf 1999) and Raster3D (Merritt and Bacon 1997).
Results and discussion
Structure analysis
In this study, we have crystallized the Ls-AChBP in complex with IMI and CTD to elucidate the mechanism underpinning their selective and diverse actions on nAChRs. Both complexes were crystallized in the same space group with one pentamer in an asymmetric unit. Ligand-omit and anomalous electron density maps clearly indicated AChBP in the crystals bound neonicotinoids (see Fig. 3a, b). The positions and orientations of the 2-chloropyridine ring in IMI and 2-chloro-1,3-thiazole ring in CTD were modeled to satisfy the observation of Br- and S-anomalous peak positions. The X-ray crystal structures of the Ls-AChBP complexed with IMI and CTD were refined at 2.58 and 2.70 Å resolutions, respectively (Table 1).
The neonicotinoid-bound Ls-AChBPs form the same homopentameric structure (Fig. 2a, b) as those determined for the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-bound apo form (Brejc et al. 2001) and the other ligand-bound forms (Celie et al. 2004, 2005; Hansen et al. 2005; Ulens et al. 2006). Typically, each subunit has a secondary structure composed of an N-terminal 12-residue α-helix followed by β-strand-rich parts as shown previously for other Ls-AChBPs. Root mean square differences (rmsd) of Cα atoms between the IMI- or CTD-complex and the nicotine-complex (Celie et al. 2004) were observed as 0.975 and 0.758 Å, respectively. In two structures determined in the present study, neonicotinoids occupied all five ligand-binding sites that are formed between the two adjacent subunits (Fig. 2a, b). The neonicotinoids occupy broadly the same position as HEPES and (-)-nicotine within the pocket formed by “A-F loop” regions. Although “loops A, B, D and E” are mainly composed of β-strands and only loops C and F have no canonical secondary structure, we use these designations to facilitate comparison with previous studies on nAChRs (Corringer et al. 2000; Matsuda et al. 2001, 2005). The principal side [(+)-chain] of the ligand-binding site possesses residues from loops A, B and C which are located in α subunits, whereas the complementary side [(-)-chain] contains loops D, E and F, which are donated by non-α subunits in α/non-α heteropentamers. Overall, the structures of the IMI and CTD complexes were very similar with Cα rmsd of 0.597 Å. However, the loop C region has a specific conformation in each complex. For example, compared with the IMI complex, loop C of the CTD complex takes a “closed conformation”, showing approximately 4 Å shift of the positions of the Cα atoms (Fig. 2c). The variations in loop C conformation were often observed between different chains within the same Ls-AChBP pentamer in complex with neonicotinoids (e.g. subunits A and C of the CTD complex, data not shown). Conformational change in loop C is evident when comparing structures with the apo form Ls-AChBP complexed with HEPES (PDB entry code: 1UX2) (Fig. 2c). The Thr155-Asp160 loop region of the (-)-chain upstream of loop F also indicates a conformational change in the ligand-binding site. These results suggest that induced-fit movement of loop regions is essential for recognition of ligands including neonicotinoids. The detailed binding interactions of neonicotinoids will be considered in relation to their key components, the aromatic ring and the guanidine/related moieties.
Fig. 2Overall view of crystal structure of Ls-AChBP complexed with imidacloprid (IMI). The pentameric structure of Ls-AChBP is viewed from the top (a) corresponding to an extracellular surface of nAChRs and from the side (b). IMI molecules bound to Ls-AChBP are shown in space-filling model, whereas carbons, nitrogens and oxygens are colored green, blue and red, respectively. Two subunits, A and B chains, are colored by pink and blue, respectively. c The structure of A–B domain boundary of the Ls-AChBP complexed with IMI (red) is compared with that of the Ls-AChBP complexed with CTD (blue) and the apo form (light brown) complexed with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (PDB entry code: 1UX2) (Celie et al. 2004). Ligands are presented by stick models
Interactions with aromatic rings of insecticides
When complexed with Ls-AChBP, the pyridine ring of IMI was buried deep in the binding site, interacting with loop E segments from the (-)-chain (Fig. 3c). Recently, Tomizawa et al. (2008) has reported that two azido-neonicotinoid probes containing the photolabile group in the pyridine ring labeled Tyr164 in loop F of the (-)-chain and Tyr192 in loop C of the (+)-chain in the Ls-AChBP. However, these two distinct binding modes were not observed in our models, neither in the IMI complex, nor in the CTD complex of the Ls-AChBP. The nitrogen atom of the pyridine ring forms a hydrogen bond with the amide group of Met114 and the carbonyl group of Leu102 in loop E via a water molecule, resembling the observations for both the Ls-AChBP-nicotine complex (Celie et al. 2004) and Ac-AChBP-epibatidine complex (Hansen et al. 2005).
Fig. 3Electron density maps of bound ligands and their interactions with the loop E region. The annealed Fo–Fc omit maps (blue) for imidacloprid (IMI) (a) and clothianidin (CTD) (b) bound to the Ls-AChBP are contoured at 4σ and drawn with the final models of the native and non-Br-neonicotinoids. In addition, the anomalous maps of bromine (purple) from the Br-neonicotinoid-complexes and sulfur atoms (yellow) from the native-neonicotinoid-complexes for ligands, which are contoured at 5 and 1.5σ, respectively, are overlaid. Interactions of the loop E region with pyridine moiety of IMI (c) and thiazole moiety of CTD (d) are shown in the binding site of the Ls-AChBP. In each ligand and amino acid, chlorine, nitrogen, oxygen and sulfur atoms are colored purple, blue, red and yellow, respectively. The hydrogen bonds are presented by orange broken lines
The chlorine atom was located in the vicinity of a hydrogen bond bridge between the backbone C = O of Leu112 and the backbone amide NH of Arg104. As a result, it made van der Waals contact the peptide backbones of these two amino acids. The aromatic ring of CTD also contacts with the loop E segment in a manner similar to IMI (Fig. 3d). The nitrogen atom of the thiazole ring of CTD, in a similar fashion to the nitrogen atom of the pyridine ring of IMI, formed hydrogen bond with the backbone carbonyl of Leu102 and the backbone amide group of Met114 via water molecule (Fig. 3d).
Nicotine, which is active not only on insect, but also on vertebrate nAChRs, has also been shown to interact with the backbone of loop E in a similar manner to neonicotinoids (Celie et al. 2004). Therefore interactions with the backbone are insufficient to explain the selective actions of neonicotinoids. On the other hand, Met114 in loop E was located in the vicinity of the nitroimine group (Fig. 3c, d) which plays a key role in selective insect nAChR–neonicotinoid interactions (Ihara et al. 2003). Some insect α subunits possess either basic or hydrogen bondable residues favorable for interactions with the nitro group such as those corresponding to Met114 (Fig. 1c). Hence, if two adjacent α subunits form a ligand-binding site, such residues may contribute to the neonicotinoid sensitivity of insect nAChRs.
Interactions with guanidine and related moieties
The crystal structures of the Ls-AChBP bound with nicotine or carbamylcholine (Celie et al. 2004) and Ac-AChBP in the presence of epibatidine (Hansen et al. 2005), as well as quantum chemical calculations (Cashin et al. 2005), suggested that the protonated nitrogen atom in nicotinic agonists undergoes cation–π interactions with Trp143 in loop B. In line with this notion, it was earlier demonstrated that the electron-deficient guanidine moiety of neonicotinoids is likely to contact with Trp143 (Matsuda et al. 2005; Tomizawa and Casida 2005). However, the guanidine moiety of IMI and CTD was found to stack with the aromatic ring of Tyr185 in the crystal structures of CTD- as well as IMI-Ls-AChBP complexes (Fig. 4a, d). Since Tyr185 is conserved throughout vertebrate and invertebrate nAChR α subunits (Fig. 1c), this residue itself is not involved directly in determining the selective interactions with neonicotinoids. Yet the interactions with Tyr185 contribute to the affinity for neonicotinoids. The stacking level for CTD appears to be weaker than that for IMI (Fig. 4a, b). In addition, the stacking interaction resulted in CH–π contacts with Trp143 of the methylene (CH2–CH2) bridge in IMI, which was not seen in CTD, accounting, at least in part, for the higher binding potency of IMI (Kd = 1.57 ± 0.21 μM, n = 3) compared to that of CTD (Kd = 7.26 ± 1.13 μM, n = 3) on the Ls-AChBP as evaluated by quenching of the protein fluorescence.
Fig. 4Imidacloprid (IMI) and clothianidin (CTD) binding to Lymnaeastagnalis AChBP (Ls-AChBP). a IMI–Ls-AChBP complex. b CTD–Ls-AChBP complex. The binding site is located at the interface between two adjacent subunits, as drawn in green for (+)-chains and in cyan for (-)-chain (see text). The subunits EA and CD interfaces are shown for the binding sites for IMI (a) and CTD (b), respectively. Orange broken lines depict hydrogen bonds. In each ligand and amino acid, chlorine, nitrogen, oxygen and sulfur atoms are colored purple, blue, red and yellow, respectively
Another difference between CTD and IMI is that the former exposes a hydrogen bond donor NH at position 1, whereas the latter lacks it. We suggested earlier that this NH may form a hydrogen bond with the backbone carbonyl of Trp143 in loop B (Ihara et al. 2007b). As suggested, the NH of CTD made a hydrogen bond with the backbone carbonyl of Trp143 from the (+)-chain in the crystal structures of the Ls-AChBP (Fig. 4b), although it was observed at only 2 of 5 interfaces. Such hydrogen bond formation may assist CTD in keeping contact with nAChRs when large conformational changes occur in LBD to open the central channel (Miyazawa et al. 2003; Unwin 2005), accounting, at least in part, for higher agonist efficacy of CTD compared to IMI and ACh in certain native and recombinant nAChRs (Brown et al. 2006; Ihara et al. 2004).
Interactions with loop D and selectivity of neonicotinoids
We have previously shown that substitution of a glutamate for the glutamine corresponding to Gln55 of Ls-AChBP (Fig. 1c) markedly reduced the maximum response to IMI and nitenpyram of the chicken α7 nAChR, whereas mutation of the same glutamine to arginine enhanced it, slightly shifting the concentration–response curve to the left (lower concentrations). In complete contrast, the same mutation reduced the (-)-nicotine sensitivity of the α7, whereas substitution with a glutamate enhanced it (Shimomura et al. 2002). Therefore, it was postulated that this glutamine residue is likely to be located in close proximity to the nitro group of neonicotinoids, and that marked changes in the α7 response to neonicotinoids as well as (-)-nicotine are the results of electrostatic interactions between the added residues and the nitro group. Consistent with this hypothesis, Gln55 faced the nitro groups of IMI and CTD in the crystal structure of the Ls-AChBP within a hydrogen bondable distance in the AChBP–IMI complex (<3.3 Å in 2 of 5 interfaces in the crystal structure of the Ls-AChBP) (Fig. 4). For CTD, however, the distance between the Gln55 and the nitro group was longer than 3.5 Å, excluding or minimizing a contribution of hydrogen bonding to the binding potency of CTD.
All vertebrate nAChRs, perhaps apart from those containing either human or rat β4 subunit, are likely to show reduced sensitivity to neonicotinoids due to the lack of the basic residues in loop D. The human β4 subunit (Fig. 1c), as well as rat β4 subunit (sequence not shown), possesses a lysine as residue corresponding to Gln55 of Ls-AChBP. However, the vertebrate β4 subunits possess a glutamate residue as that corresponding to Thr57 in Ls-AChBP which is located in the vicinity of Gln55 (Fig. 5). This glutamate residue is postulated to interfere electrostatically with the neighboring basic residue–neonicotinoid interactions, thereby reducing the neonicotinoid sensitivity of nAChRs containing the β4 subunits.
Fig. 5Interactions between loops C, D and F. IMI-binding site of Ls-AChBP is presented by especially focusing on loops C, D and F. The binding site at the interface of the subunits C and D is drawn in green for (+)-chains and in cyan for (-)-chain (see text). In each ligand and amino acid, chlorine, nitrogen, oxygen and sulfur atoms are colored purple, blue, red and yellow, respectively. Orange broken lines depict hydrogen bonds
Role of loops C and F in their interactions with neonicotinoids
We have found that replacement by glutamate of the proline corresponding to Ser186 in the YXCC motif in loop C of the Drosophila Dα2 subunit reduces the IMI sensitivity of the Dα2β2 hybrid nAChR, whereas its reverse mutation in the α4β2 nAChR enhanced sensitivity (Shimomura et al. 2004), suggesting a contribution of the loop C residue to the neonicotinoid sensitivity of nAChRs. However, another view has been proposed in which the serine or threonine residue corresponding to Ser186 from (+)-chain of Ls-AChBP in loop C of insect nAChRs (see Fig. 1c) forms a hydrogen bond with the nitro group of neonicotinoids, thereby enhancing the binding potency of neonicotinoids (Tomizawa et al. 2007). In the crystal structures of Ls-AChBP, Ser186 made hydrogen bonds with Glu163 and Tyr164 in loop F of (-)-chain but not with the nitro group of IMI (Fig. 5). This hydrogen bond web, as well as the basic residues in loop D, seems to play an important role in determining the neonicotinoid sensitivity of nAChRs. In most heteromeric vertebrate nAChRs, α subunits possess acidic residues as those corresponding to Ser186 in loop C, resulting in electrostatic repulsion of the acidic residues in loop F of β subunits corresponding to Glu163 in Ls-AChBP (Fig. 1c). On the other hand, insect nAChR α subunits do not possess such an acidic residue in loop C (Fig. 1c). Hence, it is conceivable that amino acids not only from α subunits, but also from non-α subunits of insect nAChRs, contact more effectively with neonicotinoids than those of vertebrate nAChRs. In this context, Tyr164 may also contribute indirectly to the neonicotinoid sensitivity of insect nAChRs because vertebrate non-α subunits possess phenylalanine residue incapable of assisting the formation of the hydrogen-bond-network at this position.
In conclusion, we have for the first time elucidated the crystal structures of the Ls-AChBP in complex with two commercial neonicotinoids IMI and CTD. Met114 in loop E and Gln55 in Loop D closest to the nitro group in neonicotinoids, suggesting that corresponding residues in insect nAChRs may play important roles in determining neonicotinoid sensitivity. Ser186 in loop C was found to form hydrogen bonds with Gln163 as well as Tyr164 in loop F, suggesting that these hydrogen bondable residues can also indirectly influence the selective interactions with neonicotinoids. CTD forms hydrogen bond with the backbone carbonyl of Trp143 in loop B and shows weaker hydrophobic contacts with Trp143 compared with other neonicotinoids tested, which may altogether lead to its unique actions on nAChRs. Although there are sequence and structural similarities between AChBPs and nAChRs, there are also important differences. Future structural studies on AChBP mutants engineered to resemble more closely insect or human nAChRs could help to enhance even further our understanding of neonicotinoid actions and selectivity. Nevertheless, we have provided new insights into the molecular basis of differential actions of neonicotinoid molecules. These results may be of value in the design of even safer new crop protection agents.

acetylcholine binding protein (lymnaea stagnalis)