Safety, Tolerability and Behavioural Effects of Souroubea-Platanus in Healthy Volunteers
Safety, Tolerability and Behavioural Effects of Souroubea-Platanus in Healthy Volunteers
Souroubea sympetala extracts have shown anxiolytic properties in animal models. Souroubea and
 its active principle betulinic acid appear to exert these effects by acting as an agonist for
 the benzodiazepine (BZD) binding site of the GABAA receptor with no withdrawal effects on
 food intake, locomotor activity, or other symptoms typically associated with BZD agonism. As
 such, this may offer a valuable source for an alternative anti-anxiety treatment.

 The primary objective of this study is to (1) to evaluate the safety and tolerability of a
 single daily dose of an extract of a mixture of Souroubea spp. leaf and small branch material
 and Platanus spp. bark when administered orally over two weeks in healthy volunteers. Based
 on its safety in canine trials, we hypothesize that Souroubea-Platanus (SP) preparation will
 be well tolerated with adverse event profile similar to placebo.

 The secondary objective is (2) to establish whether some of the anxiolytic properties of
 Souroubea-platanus seen in animal models will translate to human participants. We hypothesize
 that Souroubea-Platanus preparation will demonstrate anxiolytic and/or stress-reduction
 properties as indicated by salivary cortisol levels and self-report measures of anxiety.
Background

 Within Latin American indigenous communities tea brewed from fresh or dried crushed Souroubea
 leaves is used to treat Susto, a culture-bound syndrome resembling manifestations of Anxiety
 Disorders. Souroubea sympetala (Ss) leaf extract has shown to be effective as an anxiolytic
 in animal models, and betulinic acid (BA) is one of the active molecules responsible for
 mediating this effect.

 Plant-based natural products are sought by many individuals as alternatives or supplements to
 standard medical care. As a result, more research is required in order to develop botanical
 treatments that are evidence-based, safe and guided by health practitioners.
 Souroubea-Platanus may offer such alternative.

 Safety

 Evidence suggests that Souroubea-Platanus was well tolerated in Beagles in a 28-day pilot
 study at 8x recommended dose, producing significant reductions in plasma cortisol 1h
 post-administration without any adverse reactions, except slightly reduced platelet count in
 2 animals attributed to infection with Ehrlichia canis, likely contracted prior to the study
 onset. In a follow-up 16 beagles and up to 5x the recommended does for 28 days such decrease
 in platelet count was not observed, no there were any other adverse events. No adverse events
 have been reported in animals for the animal health product version of the drug (Zentrol)
 that has been on the market for 2 years. The naturalistic observations of Souroubea
 consumption suggest that it is safe for human use, however, this has not been established in
 a formal study. In addition, drug interactions have occurred with some herbal products,
 particularly those that strongly inhibit or induce CYP enzymes. As Souroubea may act on the
 BDZ binding site of the GABAA receptor, possible side-effects and symptoms similar to those
 associated with benzodiazepine anxiolytics are possible.

 Clinical Relevance

 The study has significant clinical relevance for anxiety and stress-related disorders and may
 lead to developing a new anxiolytic agent or supplement with more selective effects and
 reduced risks for the patients.

 Overview of study design

 Forty-five healthy adult volunteers will be enrolled in the single-center, randomized,
 double-blind, parallel group study. Power analysis revealed that to achieve a power of 0.80,
 with an estimated effect size of 0.40 (estimate based on rodent studies), a total sample size
 of 42 would be required. Accounting for approximately 10% predicted participant dropout rate
 per group, it is expected that 45 participants will be enrolled in the study.

 Participants will be randomly assigned to one of the 3 treatment groups with 15 individuals
 in each for detecting a dose-response effect. Treatment groups will be either (a) 190mg
 extract (b) 380mg extract, or (c) inert placebo, all in identical vegicaps.

 Capsules containing the placebo and the extracts will be produced under GMP conditions. In
 accordance with Health Canada guidelines, extract will conform to the acceptable limits for
 heavy metal and biological contamination. The placebo will consist of microcrystalline
 cellulose suitable for use in foods.

 The capsules will be taken once a day in the morning with water for 2 consecutive weeks. The
 safety evaluations will be conducted at screening and repeated at the end of the 2-week study
 period. Adverse events will be evaluated after 1 week and 2 weeks of treatment and 1 week
 after discontinuation. The participants will be asked to maintain their regular lifestyle and
 diet during the study period so long as those habits do not violate the study exclusion
 criteria.

 Participants will be asked to take the capsules at the same time every morning. Compliance
 will be facilitated by daily reminders by e-mail, text, or phone call and a review of the
 returned medication vials.

 The participants will have 24-hour phone access to the study physician for emergencies.

 Participant Compensation

 Participants will be reimbursed for their time in participation, as well as for
 transportation and parking costs on prorated basis corresponding to minimal hourly wage.

 Randomization and blinding

 Blinded treatment will be used to reduce potential bias during data collection and evaluation
 of clinical endpoints. Participant numbers will be assigned in sequential order as
 participants enroll in the study. All participants will be asked to come in at the same time
 for all testing periods after screening (Days 1, 7, 14, and 21) in order to account for
 time-of-day effects on cortisol.

 Participants will be assigned to one of the treatment groups according to a computer
 generated randomization schedule prepared by contracted pharmacy personnel. The randomization
 will be balanced by using permuted blocks. Pharmacy personnel will package and label study
 drug for each participant.

 The study investigator or his/her designate and study participant will remain blind to the
 participants' group until the completion of the study. The blind should be broken only if in
 emergency requiring knowing the treatment status of the participant.

 Study procedures

 1. Baseline screening will include:

 1. Medical history, physical examination, and vital signs, including temperature,
 heart rate, respiratory rate and blood pressure;

 2. Mental status examination, including completion of the DSM-5 Self-Rated Level 1
 Cross-Cutting Symptom Measure - Adult with follow-up on any items rated as present
 in at least mild degree;

 3. Laboratory tests and urine drug screen as described below;

 4. Serum pregnancy test for females of childbearing potential; must not be pregnant

 2. Safety evaluations:

 1. Each volunteer will provide a blood and urine sample in the morning, within 14 days
 before the start of the study (baseline), 7 days after the first dose, and on the
 last day of the study (day 14). Body weight will be measured at baseline and day
 14. Physical examination including vital signs and inquiry of adverse events will
 be performed on days 7 and 14. Additional evaluation of adverse events will be done
 one week after discontinuation of the study drug. Vital signs will be monitored
 until 1 hour after administration of the first dose.

 The following laboratory parameters will be measured:

 -  complete blood count: white blood cell (WBC) count with differential, red blood cell
 (RBC) count, hemoglobin, hematocrit and indices, platelet count;

 -  chemistry panel: glucose, calcium, electrolytes (sodium, potassium, chloride), BUN,
 creatinine, albumin, total protein, alkaline phosphatase, gamma glutamyl transferase,
 aspartate aminotransferase, alanine aminotransferase, bilirubin, creatine kinase;

 -  urinalysis will be standard, microscopic if indicated;

 -  salivary cortisol will be measured as a stress indicator.

 Pregnancy testing in females will consist of a serum test (for convenience, since other blood
 samples are already being collected during screening; this should not normally require
 additional venipuncture).

 Saliva samples will be collected using sterile saliva sample tubes. Blood and urine samples
 will be collected at the clinical laboratory. Blood samples will be drawn by venipuncture.

 Records of health data identifying participants will be kept on record for 25 years before
 being destroyed; all records will be kept confidential during that time.

 Behavioural evaluations

 1. State-Trait Anxiety Inventory (STAI) on Days 1 (baseline and 1 hour post-dose), 7 and
 14.

 2. DSM-5 Self-rated Level 2 anxiety adult short form on Days 1, 7, and 14

 3. Cortisol (physiological marker of stress): salivary concentration; analysis - difference
 between pre- and 1 h post dose on Day 1, max changes from baseline and group x time
 effects and interactions.

 Data analysis

 Statistical means and standard deviations will be calculated for each continuous variable.
 ANOVA followed by post hoc Tukey's test will be used to evaluate significant differences
 among dose groups and different days. A P-value of 0.05 will be considered as the level of
 significant difference in the analysis of the data. Behavioural variables will be analyzed
 using mixed effects ANOVA for repeated measures where appropriate.

 Transformations for non-normality and/or corrective procedures will be applied if data is
 found to be non-normally distributed or otherwise in violation of the assumptions of the
 statistical tests.

 STAI: the total sum scores (T-scores); analysis - max changes from baseline and group x time
 effects and interactions.

 Cortisol: salivary concentration; analysis - difference between pre- and 1 h post dose on Day
 1, max changes from baseline and group x time effects and interactions.

 Safety evaluations will be based on the baseline to treatment-period differences in
 laboratory tests, physical examinations, vital signs and the incidence of adverse events.

 Adverse events reporting

 All adverse events (AE) regardless of casual relationship with investigational treatment will
 be recorded. An AE will be considered treatment-emergent if it is new in onset or aggravated
 in severity or frequency following administration of the investigational agent. All treatment
 emergent AE will be followed until resolution or a stable clinical endpoint.

 Serious AE, e.g. any AE that is fatal, immediately life threatening, requires or prolongs
 hospitalization, causes permanent or significant disability will be reported to Health Canada
 and IRB and immediately addressed medically as appropriate.

 Participants will be encouraged to report AEs spontaneously or in response to general,
 non-directed questioning. All treatment-emergent AEs will be recorded in the source document
 using medical terminology.
Inclusion Criteria:

 1. Male and female participants aged 18-70 years

 2. Females of childbearing potential must use reliable contraception during the study
 period; acceptable methods of birth control include all hormonal methods, IUDs,
 complete abstinence, effective physical barriers (e.g. condoms), or confirmed
 vasectomy of partner (if that information is volunteered by the participant)

 3. Participants must be in good health status based on medical history and physical
 examination, including vital signs and clinical laboratory tests

 4. Participants must refrain from taking any prescription or over the counter medication
 within the 14 days prior to the beginning of the study and during the study period

 5. Participants must be non-smokers or light/occasional smokers (not more than 10
 cigarettes per day)

 6. Alcohol consumption should not exceed 3 drinks on any single day and no more than 7
 drinks per week in women and no more than 4 drinks on any single day and no more than
 14 drinks per week for men

 7. BMI of 18.5-30 kg/m2 excluding markedly underweight and obese individuals

 8. Participants must be willing to give written informed consent after the nature of the
 study has been fully explained

 Exclusion Criteria:

 1. Participants demonstrating uncontrolled clinically significant physical disease or
 abnormal laboratory test results within 14 days prior to the start of the study

 2. Participants with current or history of alcohol or drug use disorder

 3. Participants with any history of significant drug super-sensitivity, anaphylaxis, or
 anaphylactic reaction

 4. Participants taking prescription medication or OTC medication within 14 days prior to
 inclusion

 5. Participants who have a history of any significant psychiatric disorder or currently
 significant psychiatric symptoms

 6. Participants with a family history of serious mental illness in first-degree relatives

 7. Participants in a situation or having any condition which, in the opinion of the
 investigator, may interfere with optimal participation in the study

 8. Participants testing positive for amphetamine, cannabinoids, cocaine, barbiturates,
 benzodiazepines, opioids or hallucinogens

 9. Use of other non-prescription natural health products with known anti-anxiety effects

 10. Known allergy to any of the medicinal and non-medicinal ingredients in any conditions
 of the study