Human Safety and Pharmacokinetics Study of Orally Administered Icariin: Randomized,Double-Blind,Placebo-Controlled Trial

Trial Description

This pilot study was a randomized, double-blind, placebo-controlled, parallel-group clinical trial (NCT02112123) to evaluate the pharmacokinetics and safety of icariin, an over-the-counter supplement, in healthy individuals (N = 24) at UT Southwestern Medical Center, Dallas, TX. The trial was conducted between 7/2/2014 and 9/6/2016, with all participants recruited from the community using free and paid advertising. The study protocol was reviewed and approved by the UT Southwestern Institutional Review Board, and all participants signed an informed consent form undergoing any study procedures.

Sample Size and Randomization

The study used a sample size of 24 (N = 24), a group of 6 participants per icariin dose. The Wilcoxon-Mann-Whitney power analysis was conducted using median difference between icariin and placebo groups with Poisson distribution (mean of 3) of scores of cognition, mood, and side effects. A power of 76.2% was obtained from the proposed sample size of 24 (2:1 ratio icariin and placebo groups) at α = 0.05. Randomization was performed by a statistician using a computer-generated random number sequence, with participants randomized in a 2:1 icariin-to-placebo ratio in each dose group of 6 participants.

Inclusion and Exclusion Criteria

The included participants were healthy men and women (18 - 50 years old) with ≥12 years of education (or equivalent), a body mass index between 18.5 and 30, no medication changes within 30 days of baseline, and baseline RAVLT T-score ≥35 on trials A1 to A5 indicating normal baseline memory performance. The exclusion criteria were as follows: history of any major psychiatric illness, as assessed by Structured Clinical Interview for DSM (SCID-IV); current tobacco use; history of neurological disorders (eg, recurrent seizures or any seizure in the past 12 months, stroke, brain surgery, traumatic brain injury, and loss of consciousness >1 minute, multiple sclerosis, Parkinson’s disease); use of CNS-acting medications; hypertension (ie, systolic blood pressure >140 or diastolic blood pressure >90); medication therapy for erectile dysfunction or opioid therapy within 30 days of the baseline or initiation of new medications within 30 days of baseline; clinically significant vital signs values (body temperature ≥100.5°F [38°C], baseline heart rate <50 bpm or >100 bpm); clinically significant laboratory values (eg, hypokalemia, hypernatremia) or ECG results at baseline; medical contraindications to icariin; history of myocardial infarction, cancer, or diabetes; vulnerable populations (ie, pregnant or nursing women, incarcerated, those with severe cognitive disorders); baseline QIDS-C score >7; history of suicide attempt or current suicidal ideation; history of special education or intellectual disability; current participation in any other pharmacotherapy studies.

Intervention Description

Following the baseline visit, participants completed a 24-hour stay at the UT Southwestern Clinical Research Unit (CRU, an inpatient research facility), where the study intervention was initiated and blood samples drawn. Five dose levels were evaluated (100, 200, 400, 840, and 1,680 mg or placebo) over a 5-day administration period. The administration was in blocks of 6 participants who received either icariin or identical placebo and only moved to a higher dose if the previous dose was well tolerated. For example, if 100 mg was well tolerated, based on side effect and adverse event assessment in 6 participants, then the next 6 participants received 200 mg doses. Different healthy controls were used for each dose level, with the total treatment duration for each participant lasting 5 days. The first dose was administered orally by a nurse at the UT Southwestern CRU on an empty stomach (i.e. not within 1 hour prior or within 2 hours following meals). The second dose was also administered by a nurse following the last blood drawn at 24 hours. The participants were supplied with icariin doses for days 3–5 at the time of discharge.

Icariin, as a 20% icariin concentration in a 500 mg Horny Goat Weed capsule (Barlowe’s Herbal Elixirs, West Palm Beach, FL), was used in the study (Lot #C20121115). This product was selected because it was commercially available and Barlowe’s Herbal Elixirs provided a Certificate of Analysis (CoA). The CoA was a document issued by the manufacturer that confirmed that the product met product specifications, and included results from quality control testing of an individual batch of icariin. The preparation was changed to 600 mg (Lot #CYYH-A-01948) during the last 2 dose phases of the study due to commercial availability of the product; however, the dosing was adjusted to deliver the correct amount of icariin. Each of the Barlowe’s Herbal Elixirs icariin capsules had a label claim of 100 mg of icariin, and this concentration was confirmed through liquid chromatography mass spectrometry (LC-MS)/MS determination. The matching placebo, identical in appearance to the active drug, was prepared at Abrams Royal Pharmacy (Dallas, TX), and was encapsulated in clear and tasteless gelatin capsules. The placebo capsules contained the following ingredients: food color powder blue, food color powder yellow, food color powder brown, microcrystalline cellulose, silicon dioxide, and calcium carbonate. Both participants and research personnel remained blinded to the treatment assignments.

Pharmacokinetic Profile Determination

Icariin (98+%), purchased from INDOFINE Chemical Company (Hillsborough, NJ), was used as the analytical standard as received. Biochanin A, purchased from Sigma-Aldrich (St Louis, MO), was used as the internal standard and used as received. All other reagents were purchased and used as received from Fisher Scientific (Hampton, NH).

Bioanalytical Sample Preparation

The bioavailability and pharmacokinetics of orally administered icariin were determined by plasma analysis following the initial oral dose of icariin. After dosing, 15 blood samples (approximately 2 mL each) were obtained over 24 hours at 0, 30, 60, 90, 120, 150, 180, 210, 240, 300, 360, 420, 480, 1080 and 1440 minutes. These samples were turned into plasma and stored at −80 ± 10°C until analysis.

Bioanalytical Method

An LC-MS/MS method for the determination of icariin in human plasma was developed and qualified with a lower limit of quantitation of 1 ng/mL. The plasma from subjects receiving doses of 100 mg (n = 6), 200 mg (n = 5), 400 mg (n = 6), 840 mg (n = 3), and 1,680 mg (n = 3) was analyzed. Essentially all plasma samples contained levels of icariin below the lower limit of quantitation with the exception of a few samples.

Specifically:

LC-MS/MS analysis of the capsules demonstrated that the capsules contained 82% of the label claim; therefore, the lack of bioavailability was not due to improperly formulated capsules. The bioanalytical method was qualified through analysis of 3 calibration curves prepared on 3 separate days. The calibration curve was determined to be linear (r > 0.99) over the concentration range of 1.0 to 1000 ng/mL on all 3 days. Accuracy and precision were assessed by determination of the percent relative error (accuracy) and percent standard deviation (precision) and the results of this determination are presented in Supplemental Table S2. The accuracy varied between 93.9% and 114%. The precision varied between 2.87% and 10.2%.

Bioanalytical Conditions

The icariin plasma concentrations were determined using an in-house developed liquid chromatography tandem mass spectrometry method. Standards and samples (100 µL) were prepared by protein precipitation after addition of the internal standard biochanin A. Chromatographic separation was achieved using gradient elution with a Kinetex XB C18 30 × 2.1 mm, 2.6 µm, column, which was maintained at 60°C, and an injection volume of 1 µL. Mass spectrometric quantitation of icariin and biochanin A was carried out in multiple-reaction monitoring mode with positive polarity, by monitoring the precursor >product transitions of m/z 677.4 > 531.2 for icariin and m/z 284.9 > 152.0 for IS. Unit resolution was used for both precursor and product ions. The analytical data obtained were processed by Analyst software (version 1.6.2). The same methodology was utilized for analysis of the commercially available icariin capsules.

Study Assessments

Various assessments were administered to participants to examine safety, tolerability, and effect of icariin on cognition and mood. The SCID-CV ²² was used to rule out exclusionary psychiatric illness, while mood was assessed using clinician-rated instruments, such as Young Mania Rating Scale, ²³ Hamilton Rating Scale for Anxiety, ²⁴ and QIDS-C. ²⁵ Different cognitive domains were evaluated using RAVLT (verbal declarative memory), ²⁶ TMT (executive function, attention, sequencing, visual search, motor function), ²⁷ and the RMCPT (working memory). ²⁸ To monitor icariin safety and tolerability, vital signs were collected at each visit, an ECG was obtained at baseline, 24 hours after the first dose of study medication, and at the final study visit, and a physical examination was performed at baseline and exit. The side effects were monitored using the SAFTEE ²⁹ and A-SEX, ³⁰ as well as via a psychiatric evaluation at each study visit. Data were analyzed using SPSS 24.0, and Wilcoxon signed rank test and Mann-Whitney U-test nonparametric tests were utilized in the analysis.