Safety, Tolerability, and Pharmacokinetics of an Oral Withania Somnifera Product in Older Adults
Phase: EARLY_PHASE1
Conditions: Healthy, Aging
Sponsor: Oregon Health and Science University

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 1 of 28 
 
 
 
 
 
 
Safety, tolerability, and 
pharmacokinetics of an oral Withania 
somnifera product in older adults 
 
NCT06171724 
 
Version Date: 10.10.2024 
 
 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 2 of 28 
 
Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in 
older adults 
 
Principal Investigator: 
Alex Speers, ND, MS  
503-418-5896
 
 
 
 
 
Co-Investigators/staff: 
Joseph Quinn, MD (Clinical Investigator) 
503-494-7231 
 
Amala Soumyanath, PhD (Co-Investigator) 
Jesus Martinez (Study staff) 
Katlin Wozniak (Study staff) 
 
503-494-6878 
A. OBJECTIVES  
A.1. Purpose 
The purpose of this pilot study is to measure the oral absorption and pharmacokinetics (PK) of 
compounds from two doses of a standardized Withania somnifera extract product (Shoden®) in 
healthy older adults. Compound levels will be measured in human plasma and urine over 48 
hours after acute oral administration and after four weeks’ use of Shoden®. Peak exposure and 
time, total exposure, half-life, and urinary excretion of compounds will be calculated. These data 
will be used to inform decisions on the dosage and dosing frequency for future phase 1 and 
phase 2 clinical trials.  
 
A.2. Primary goal 
To determine the oral bioavailability and PK of withanolides from two doses of a Withania 
somnifera product (Shoden®) in healthy older adults following single administration and after 
four weeks’ use.  
 
A.3. Endpoints  
• 
Primary endpoints:  
o Peak exposure and time (Cmax, Tmax) and total exposure (AUC0-t, AUC0-∞) of 
withanolides from Shoden® in human plasma after a single administration. 
• 
Secondary endpoints:  
o Half-life (t1/2) of withanolides from Shoden® to help determine dosage intervals. 
o Urinary excretion of withanolides from Shoden®  
o Steady-state levels of withanolides after four weeks’ use. 
o Tolerability and safety of Shoden® following single administration and after four 
weeks’ use. 
o Feasibility of assessing sleep- and stress-related outcomes remotely using REDCap 
surveys. 
B. SPECIFIC AIMS 
Aim 1: Evaluate the oral absorption and PK of selected withanolides from Shoden® in 
older adults. 
• 
Hypothesis: Selected withanolides will be detectable in the plasma and urine at both doses 
of Shoden®, with significantly greater plasma levels achieved at the 480 mg dose. 
 
Aim 2: Assess the safety and tolerability of Shoden® in older adults. 
• 
Hypothesis: A single administration and four weeks’ use of Shoden® at 240 and 480 mg 
per day will be well tolerated and produce no severe adverse events. 
 
Aim 3: Assess the feasibility of remotely measuring sleep- and stress-related outcomes.  

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
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• 
Hypothesis: Remotely measuring sleep- and stress-related outcomes using REDCap 
surveys will be feasible, as defined by at least 80% of all questionnaires completed by 
the study population. 
C. BACKGROUND 
C.1. Botanical dietary supplements 
The use of botanical dietary supplements in the US has exploded in recent years, with sales 
exceeding $10 billion for the first time in 2020,1 and further increasing to $12 billion in 2021.2 
The popularity of these products has increased despite a relative lack of evidence to support 
their widespread use,3 along with significant questions regarding product quality and variability4 
and safety.5 Rigorous efficacy trials would support the evidence-based use of botanical dietary 
supplements, but key preliminary data from early-phase clinical research are required first in 
order to optimize these efficacy trials. The National Center for Complementary and Integrative 
Health (NCCIH) has established a stepwise approach for conducting natural products research, 
which includes collecting human PK data of putative active compounds from a natural product 
and demonstrating mechanism-based biological signatures in humans prior to efficacy trials.3,6  
 
C.2. Withania somnifera (WS) 
C.2.1. Overview 
Withania somnifera (L.) Dunal (WS; common name: ashwagandha) is an example of a botanical 
for which key preliminary data from early-phase clinical studies are lacking, limiting the impact of 
efficacy trials using WS products. WS enjoys a formidable reputation in Ayurvedic medicine as a 
rasayana herb, i.e., one that can rejuvenate the body and promote the health of all the tissues.7 
WS is also classified as an adaptogen, an agent that promotes homeostasis of the whole body 
not by one specific pharmacological mechanism, but by eliciting complex responses.8 
Traditionally, WS preparations have been used for conditions such as arthritis, asthma, goiter, 
and ulcers, as well as anxiety, insomnia, and neurological disorders. These uses are linked to 
the botanical’s reputed adaptogenic, anti-stress, and anti-inflammatory properties.9,10  
Numerous WS commercial products are now readily available to the public. In the United States 
(US), WS products are classified as “botanical dietary supplements” by the Food and Drug 
Administration. According to the National Institutes of Health Office of Dietary Supplements, 
there are currently more than 1,300 products on the US market that contain WS.11 From 2018 to 
2021, yearly sales of WS botanical dietary supplements increased from $7.5 million to over $92 
million, making it the 7th best-selling botanical dietary supplement in the US.2 This dramatic rise 
in WS’s popularity, which has caught the attention of mainstream news outlets,12,13 has been 
driven in part by its status as an adaptogen.14 Clinical trials of varying rigor have investigated 
WS for conditions including stress15-20 and WS is actively being studied at OHSU’s NIH-funded 
BENFRA Center.15,21-23 Despite significant research interest in WS, human PK data are scant, 
and yet they are needed to support rigorous biological signature studies and efficacy trials of 
this popular botanical. 
 
C.2.2. Phytochemistry 
Earlier studies have examined the PK of WS compounds in silico24,25 and in mice26-28 or rats.29-35 
All these studies have focused on withanolides, a group of steroidal lactones relatively unique to 
WS,36,37 which many pre-clinical studies support as the main bioactive compounds in WS.38,39 
Several withanolides have been investigated for their therapeutic potential, with withaferin A 
being most well-studied.40,41 Figure 1 shows the chemical structures for commonly studied 
withanolides. To our knowledge, only two human PK studies of WS products have been 
published. A PK study in osteosarcoma patients42 found that a WS root extract did not produce 
detectable plasma levels of withaferin A as measured by liquid chromatography (LC) coupled to 
UV detection, a less sensitive method than LC coupled to multiple reaction monitoring mass 
spectrometry (LC-MRM-MS). Another PK study comparing two WS root extracts detected the 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
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withanolides 12-
deoxywithastramonolide and 
withanolide A (using LC-MRM-MS) from 
both products in the plasma of healthy 
men aged 18-45.43 These studies are 
helpful, but limited in relevance due to 
their use of root only extracts (WS 
leaves are also used commercially, and 
contain higher levels of withanolides15), 
the small number of withanolides 
targeted, the specificity of the data to 
the products tested, and the lack of 
female or older participants. The 
present study will be the most robust 
PK study to-date for WS, generating PK 
data, including steady state levels after 
four weeks’ use, for a wide variety of 
withanolides at two doses of a 
commercially available WS product using a randomized crossover design.  
 
C.2.3. Safety 
In animals, WS extracts have also been shown to be safe in toxicity studies. Acute and sub-
acute toxicity of a hydroalcoholic root extract of WS was investigated in female Wistar rats.44 No 
behavioral signs of toxicity or gross pathological changes were observed with acute doses up to 
2000 mg/kg. Similarly, no signs of subacute toxicity were observed in rats given 500, 1000 or 
2000 mg/kg of the extract daily for 28 days. In a separate study, a hydroalcoholic root extract of 
WS was used to assess prenatal developmental toxicity in rats at doses of 500, 1000 and 2000 
mg/kg daily for 28 days.45 No signs of toxicity, gross pathology changes, or mortality were 
observed in the pregnant rats or fetuses. Further studies are required to examine the safety of 
WS extracts prepared using other extraction methods and plant parts. 
In humans, the widespread use of WS as a traditional medicine and dietary supplement, as well 
as current scientific literature, support its general safety. In a review of clinical trials that used 
root preparations of WS for a wide variety of conditions, the authors noted that reasonable 
safety outcomes were seen with no serious adverse events or changes in vital signs or 
hematological and biochemical parameters.46 Mild to moderate transient adverse events 
including somnolence, giddiness, vertigo, drowsiness, and gastrointestinal effects (e.g., nausea, 
epigastric pain, loose stools, constipation, decreased appetite) were reported in some studies, 
while other studies involving adults and children did not report any adverse events.46 Similarly, 
an earlier review reported that WS has been used in all age groups and in both sexes, even 
during pregnancy, without any reported side effects.10 One study formally measured the 
tolerability of a WS root extract using the Patient's Global Assessment of Tolerability to Therapy 
and found it to have a high tolerability score.47 Rare, serious adverse events have been 
reported, however. Isolated cases of mild to severe liver toxicity have occurred, with three 
deaths in patients who had underlying liver disease.48-50 A case report has also been published 
detailing a kidney transplant rejection due to WS.51 Liver and renal abnormalities will be 
assessed at the beginning and end of each four-week study period. 
There are also reported effects of WS for which safety implications are unclear. Several studies 
have reported increased testosterone levels in males taking WS,52-55 but not in females.16,53 
Elevated DHEA-S levels have been observed In a study of 50 aging, overweight males, with no 
related side effects,52 and a case report of a female patient with X-linked heterozygous 
adrenoleukodystrophy taking WS. However, other clinical trials that included female participants 
have found either decreased levels of DHEA-S53 or no effect on DHEA-S.56 DHEA is a precursor 
Figure 1. Chemical structures of commonly studied withanolides. 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 5 of 28 
to testosterone. Testosterone will be assessed at the beginning and end of each four-week 
study period. Potentially related side effects will be assessed with an adverse events 
questionnaire every two weeks. The risk of taking WS while pregnant or breast-feeding is 
unknown and therefore, it is recommended to avoid WS during pregnancy and breast-feeding. 
The intervention used in this study, Shoden®, is commercially available and has been studied in 
randomized, double-blind, placebo-controlled clinical trials, where it has been well-tolerated with 
no significant adverse events reported.52,53,57 The doses of Shoden® being used in this study 
are 240 and 480 mg. In a previous clinical trial of 60 participants, 240 mg Shoden® once daily 
for 60 days was not associated with any significant adverse events or laboratory 
abnormalities.53 The higher dose of 480 mg was studied in a recently completed study of 
Shoden®, where it was well-tolerated after a single administration in 16 participants.58 We 
anticipate that 480 mg of Shoden® will also be safe and well-tolerated over four weeks, but will 
closely monitor participants for potential adverse events, as described in Section N.  
While these studies suggest that WS is generally safe, the possibility of adverse events arising 
from herb-drug interactions must be considered.59 A study of 50 participants with subclinical 
hypothyroidism found that a WS root extract modulated thyroid indices,20 suggesting that 
caution is advised in patients with thyroid disease. Shoden® has been found to increase 
testosterone levels in men in two separate trials, by 11%53 and 14.7%52, indicating a need to 
avoid WS in patients with a history of prostate cancer. A small human study (n=6) found that a 
root extract of WS had hypoglycemic effects “comparable to that of an oral hypoglycemic 
drug60,” suggesting caution in diabetic patients.56,57,61,62 Pharmacodynamic interactions between 
WS and other drugs may exist as well, as evidenced by additive effects seen in rodent studies 
between WS and the drugs imipramine,63-65 diazepam,66 and fluoxetine.67 The GABA-mimetic 
and serotonergic activities seen with some WS extracts in rodents would suggest caution when 
co-administering WS with conventional drugs that work by similar mechanisms.  
Pharmacokinetic interactions may also occur when botanicals alter the activity of drug 
transporters or drug metabolizing enzymes.68 It has been suggested that an IC50 of less than 
100 μg/mL for extracts or 100 μM for active constituents should be classified as potent inhibition 
that could lead to undesirable herb-drug interactions.69,70 Various root extracts of WS had IC50 
values greater than 100 μg/mL for the cytochrome P450 isoenzymes CYP3A4, CYP2D6, 
CYP1A2, and CYP2C9 in human liver microsomes, while withaferin A and withanolide A did not 
inhibit these enzymes at doses up to 50 µM.71,72 An aqueous extract of WS did not inhibit human 
recombinant CYP3A4 at doses as high as 1000 µg/mL.73 In a detailed biopharmaceutical study 
of withanone, the compound was found to have IC50 values of >100 µM for CYP 2C9/11 in rat 
and human liver microsomes and IC50 values between 28.5 and 80 µM for other CYP 
isoenzymes, the lowest being 28.5 µM for CYP3A4 in human liver microsomes.74 The clinical 
relevance of these findings will depend on the systemic concentrations of this compound 
achieved during regular use of WS. Further studies are required to fully evaluate this important 
aspect of the use of WS. 
D. SIGNIFICANCE 
This study will be the most thorough investigation of the oral absorption and PK of withanolides 
to date, which is needed given the widespread use of WS. These PK data will support biological 
signature research and provide a rationale for mechanistic studies focusing on orally absorbed 
withanolides. PK studies of WS are important to guide clinical herb drug interaction (HDI) 
studies, per the recommendations of the Natural Products-Drug Interaction (NAPDI) Center.75,76 
This is particularly important given the high rate of polypharmacy in older adults.77  
E. PRELIMINARY DATA 
E.1. Rationale for the choice of Shoden® 
Shoden®  is a commercially available 70% ethanolic extract of WS root and leaves, 
standardized to 35% withanolide glycosides. This product was chosen for use in the current PK 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 6 of 28 
study due to its high content of withanolides compared to other products on the market and its 
use in multiple earlier studies. The effects of Shoden® have been investigated in one pre-
clinical study and three randomized controlled trials. In vitro, Shoden® upregulated gene 
expression levels of GABAAρ1 receptors and histamine H3 receptors compared to control in 
Rattus norvegicus glioblastoma (C6) cells, suggesting a potential role of Shoden® for sleep.78 In 
a mouse model of pentobarbital-induced sleep, Shoden® decreased time to sleep onset and 
increased total sleep duration.78 Shoden® also increased nonrapid eye movement (NREM) 
sleep in Wistar rats.78 In clinical trials, Shoden® has been studied for its effects on sleep-, 
stress-, and vitality-related outcomes. In a randomized, double-blind, placebo-controlled, 
crossover study of 50 aging, overweight males, a dose of Shoden® equivalent to 21 mg 
withanolide glycosides per day for eight weeks significantly increased levels of DHEA-S and 
testosterone compared to placebo, but did not affect cortisol, estradiol, fatigue, vigor, or sexual 
well-being.52 In a randomized, double-blind, placebo-controlled study of 60 adults (37 males, 23 
females) reporting high stress however, 240 mg Shoden® daily for 60 days significantly reduced 
morning cortisol and increased DHEA-S compared to placebo.53 Shoden® was also associated 
with a significant reduction in anxiety scores, as measured by the Hamilton Anxiety Rating 
Scale.53 Finally, in a randomized, 
double-blind, placebo-controlled 
study of 150 healthy adults (72 
males, 78 females), 120 mg 
Shoden® once daily for six 
weeks significantly improved 
subjective sleep quality and 
objective measures of sleep as 
measured by a wrist actigraphy 
watch (e.g., sleep efficiency, 
total sleep time, sleep latency, 
and wake after sleep onset) 
compared to placebo.57 Based 
on these studies suggesting 
likely biological efficacy, Shoden® 
is an ideal product for which to 
examine withanolide plasma levels 
and biological signatures. 
  
E.2. PK of withanolides in Shoden®: 
An unpublished clinical trial conducted in India of Shoden® (CTRI/2020/10/028397) evaluated 
the PK of three withanolides (withanolide A, withaferin A, and withanoside IV) in 16 healthy 
adults, aged 18-45 years. All three withanolides were detected by LC-MRM-MS in plasma (data 
provided by Arjuna LTD under a NDA).79 This study was limited in scope due to the dose (480 
mg), which was higher than that used in clinical trials involving Shoden®,52,53,57 the lack of older 
participants, and the limited range of withanolides targeted. 
 
E.3. LC-MS detection of withanolides 
As part of ongoing research at the BENFRA Center, Dr. Soumyanath and the BSR/PKCore at 
OHSU have been developing LC-MS methods of detecting withanolides in mouse plasma, 
which will be optimized for human plasma and urine as part of the study. Figure 2 demonstrates 
good separation for several of the withanolides that will be analyzed in this study.  
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Figure 2. LC-MRM-MS chromatogram of selected withanolides. 
Separation was achieved using a Kinetex 2.6um Phenyl-Hexyl 100A 
50x2.1mm column and a gradient of (A) water with 10 mM aqueous 
ammonium and (B) acetonitrile both containing 0.1% formic acid. 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 7 of 28 
F. RESEARCH DESIGN AND METHODS 
F.1. Study design 
The proposed study is a randomized, double-blind, crossover trial evaluating (a) the PK of 
withanolides, from two doses of a commercially available WS root and leaf extract (Shoden®), 
(b) safety and tolerability of these doses and (c) the feasibility of remotely measuring sleep- and 
stress-related outcomes in older adults. Participants will be randomized to one of two dose 
sequence groups. There will be two 4-week study periods separated by a 2- to 4-week washout 
period. During each study period, participants will attend a 13-hour baseline PK study visit and 
return for 24- and 48-hour blood and urine collections. After the 48-hour visit, they will continue 
taking Shoden® at the administered dose (240 or 480 mg) for four weeks, at which time they will 
return for a follow-up visit. A flow chart of the study is provided in Figure 3. 
 
Study Design Diagram 
 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 8 of 28 
F.2. Study Population 
F.2.1. Number of participants 
Twelve non-demented older volunteers (6 female, 6 male) 65 years of age or older will be 
screened and enrolled in the study. We estimate that we will need to screen approximately 
twenty potential participants to meet the twelve volunteers needed to complete the study. We 
expect two dropouts, for a total of 10 participants completing the study.  
  
F.2.2. Inclusion and exclusion criteria 
Inclusion criteria: 
1. Age 65 and older, male and female 
2. Body Mass Index (BMI) greater than 17 and less than 35 at screening 
3. Sufficient vision and hearing to complete all tests 
4. Willingness to discontinue all botanical supplementation for one week prior to and 
throughout study  
5. No known sensitivity to Withania somnifera or any of its derivatives 
6. Normal or clinically not significant 12-lead electrocardiogram (ECG) recording 
7. Hepatic (ALT, AST, bilirubin), renal (creatinine, estimated GFR), and TSH parameters 
within normal range 
8. Hemoglobin ≥13.0 g/dL or hematocrit ≥39% (males) OR hemoglobin ≥12.5 g/dL or 
hematocrit ≥38% (females), per FDA recommendations on blood donation 
9. General health status that will not interfere with the ability to complete the study 
10. Willingness to attend all study visits 
11. Willingness to avoid caffeine and xanthine-containing foods or beverages (e.g., coffee, 
tea, chocolate, caffeine-containing sodas, colas, etc.), as well as grapefruit juice and 
poppy-containing foods for 48 hours prior to baseline visits 
12. Willingness to adhere to special diet (no dairy, grapefruit products, poppy-containing 
foods, high-fat meals, caffeine, or xanthine-containing foods or beverages) during 
baseline visits and until after 24-hour visit 
13. Mini-Mental State Exam (MMSE) score ≥27 
Exclusion criteria: 
1. Current smoking, alcohol, or substance abuse according to DSM-V criteria 
2. Participants who are currently pregnant, actively trying to conceive a child, or planning to 
within three months of study completion 
3. Severe aversion to venipuncture 
4. Donation of blood within 90 days of screening 
5. Participation in drug research study within 90 days of screening 
6. Serious health condition (i.e., illness, injury, impairment, or physical or mental condition 
which requires a) overnight hospitalization or b) continuing treatment that may cause 
episodic periods of incapacity of more than 3 consecutive days) within 30 days of 
screening 
7. Allergy to nightshade plants (Solanaceae family) 
8. Abnormal labs indicating symptomatic and untreated urinary tract infection  
9. History of prostate cancer 
10. History of kidney transplant 
11. Cancer within the last five years, with the exception of non-metastatic skin cancers 
12. Comorbid conditions requiring medication such as diabetes, kidney failure, liver failure, 
hepatitis, blood disorders, hypotension, thyroid disease, respiratory disorders, or 
cardiovascular disease 
13. Presence of sleep apnea, moderate to severe restless leg syndrome, major circadian 
rhythm changes, or narcolepsy 
14. Significant disease of the Central Nervous System (CNS) such as brain tumor, seizure 
disorder, subdural hematoma, cranial arteritis, or clinically significant stroke 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 9 of 28 
15. Diagnosis of major depression, schizophrenia, bipolar disorder, or other major 
psychiatric disorder as defined by DSM-V criteria 
16. Diseases associated with dementia such as Alzheimer’s disease, vascular dementia, 
normal pressure hydrocephalus or Parkinson’s disease  
 
F.2.2.1. Allowable exceptions to inclusion/exclusion criteria: 
1. Depression: Participants who report a diagnosis of depression, but (a) have a Geriatric 
Depression Scale score <5 at the screening visit (b) do not exhibit suicidal ideation, and 
(c) have been on a stable anti-depressant medication dose for at least two months prior 
to enrollment may be enrolled at the discretion of the clinical investigator.  
 
F.2.2.2. Concomitant interventions 
 
Allowed interventions: probiotic supplements, non-plant-based laxatives such as magnesium 
citrate or milk of magnesia, gastroesophageal reflux disorder medications such as antacids, 
dietary supplements that do not contain plant-based extracts, and non-narcotic analgesics 
 
Prohibited interventions: thyroid medications, anticoagulants (e.g., Warfarin), anticonvulsants, 
neuroleptics, anti-Parkinsonian agents, hypoglycemics, narcotic analgesics, sedatives 
(barbiturates, benzodiazepines, opioids, sleep-inducing drugs), nicotine (tobacco, patches, gum, 
lozenges, etc.), glucocorticoids, immunosuppressants, cannabis products (herb or edibles), 
investigational drugs used within five half-lives of baseline PK visit, systemic corticosteroids 
used within five half-lives of baseline PK visit, and any over the counter or prescribed products 
containing plant extracts such as herbal supplements, plant-based over-the-counter vitamin 
supplements, or plant-based over-the-counter protein powder supplements 
 
F.2.3. Vulnerable populations 
Based upon the age range and cognitive status needed in our study population, children and 
decisionally impaired adults will be excluded. We will exclude women who are pregnant, 
planning to become pregnant or breast-feeding. We will exclude men who are trying to father a 
child. We will exclude prisoners. We will not exclude any ethnic group. 
 
F.3. Setting 
All participants will be enrolled at Oregon Health and Science University (OHSU) and the OHSU 
Institutional Review Board (IRB) will oversee the study. There will be no additional sites for this 
study. All screening and study visits will be conducted at the Oregon Clinical and Translational 
Research Institute’s Clinical and Translational Research Center (CTRC) or the Center for Health 
& Healing, Building 1 (CHH1). Screening visits, baseline visits, 24- and 48-hour visits, and 4-
week follow-up visits will occur in the CTRC. All plasma and urine storage will occur in a -80°C 
freezer in Dr. Soumyanath’s laboratory in Richard Jones Hall at OHSU. All sample analysis will 
occur at the Bioanalytical Shared Resource/Pharmacokinetic Core (BSR/PK) in Richard Jones 
Hall at OHSU.  
G. RECRUITMENT 
Potential participants will be identified through multiple sources, including the Oregon Clinical 
and Translational Research Institute’s volunteer repository and the Oregon Center for Aging and 
Technology (ORCATECH) Life Lab. We will also query the electronic health records system 
using OHSU approved tools, such as Cohort discovery, Research Data Warehouse (RDW), and  
 
ACTNOW! Research Contact and Health Information Registry (IRB#: 11606). We will request 
patient lists that meet the eligibility criteria and medical record numbers, to perform subsequent 
chart review. Potential participants will be mailed or securely emailed an advertisement 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 10 of 28 
describing the study and requesting they call if they are interested in participation. IRB-approved 
study flyers will be posted with permission in OHSU clinics and local retirement 
communities/elder care facilities. The study will be advertised online through the March 
Wellness “March Stride” e-mail newsletter, OHSU’s StudyPages platform, and the NIH Clinical 
Trials website. All study advertisements will be submitted to the OHSU IRB before recruitment 
begins and all study materials will emphasize that participation is completely voluntary.  
 
G.1. Incentives 
Participants will be given the option of complimentary transportation by taxi to and from their 
study visits or a parking permit for each study visit. They will not be provided with transportation 
for screening visits. To compensate them for their time and effort, participants will receive $200 
for completion of the study via the ClinCard. Payments will be prorated, $100 for each 
completed baseline PK visit. While not a direct incentive, participants will be provided with three 
complete meals and snacks during each study visit that will not interfere with the PK analysis.  
H. INTERVENTION 
H.1.  Shoden® powder, a commercial, dried 70% ethanolic extract of Withania somnifera 
(ashwagandha, WS) root and leaf. 
Shoden® powder will be prepared from WS root and leaf by the company Arjuna Natural, using 
their standard procedures in GMP-compliant facilities. WS root and leaf will be purchased from 
“qualified” suppliers, i.e., suppliers who have previously supplied raw herbs that have passed 
Arjuna’s standards for identity, and pesticide and heavy metal levels. Every batch of plant 
material will be examined by the following tests: macrosocopic examination for verification of 
identity, thin layer chromatography (TLC) to verify the presence of characteristic withanolides, 
impurity testing to confirm that pesticide and heavy metal tests (lead, arsenic, cadmium, and 
mercury) remain below specified limits, and high-performance liquid chromatography (HPLC) 
analysis to confirm the presence of acceptable levels of total withanolide glycosides. WS root 
and leaf raw materials will then be subjected to two-stage extraction with 70% ethanol. After 
each extraction, the extract will be examined by thin layer chromatography (TLC) to verify the 
successful extraction of withanolides and concentration during the second stage. The extract 
will be dried by spray drying and the dried extract will undergo impurity testing, microbial testing, 
and HPLC analysis to confirm the presence of acceptable levels of total withanolide glycosides. 
A dossier with a paper trail from the raw material to finished product will be maintained by the 
company and a Certificate of Analysis will be provided for each batch of extract detailing 
analytical results. 
The PI will ensure that the Shoden® powder provided by Arjuna undergoes the following tests 
through three collaborating institutions of the NIH-funded Botanical Research Core of the 
BENFRA Botanical Dietary Supplements Research Center: 
1. High-performance TLC (HPTLC) of the Shoden® extract will be performed in Dr. 
Soumyanath’s laboratory at OHSU. Using HPTLC chromatographic methods described 
in the United States Pharmacopoeia monograph for ashwagandha extract, the presence 
of sitosterol and withanolide A (characteristic of WS root) and withaferin A (characteristic 
of WS leaf) in Shoden® will be verified. 
2. The Shoden® sample will be analyzed at Oregon State University Mass Spectrometry 
Center (OSMSC) using methods they have developed for BENFRA Center research. 
Liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-
MRM-MS) will be used for targeted quantification of several withanolides for which 
commercial reference standards are available. LC coupled to high resolution tandem 
mass spectrometry (LC-HRMS/MS) will be used to record a fingerprint of all detectable 
components in the extract. 
3. The Shoden® sample will be analyzed at or through the QC laboratory at the dietary 
supplement company, Oregon’s Wild Harvest (OWH), for pesticides, heavy metals (lead, 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 11 of 28 
arsenic, cadmium, and mercury) and microbial content (coliforms, pathogenic E.coli 
0157, Salmonella, yeast, mold, and total aerobic plate count). 
Arjuna Natural will custom prepare capsules containing two dose levels (120 mg or 240 
mg/capsule) from the same batch of Shoden® powder for which they provided samples for 
characterization studies. The capsules will also contain a standard mixture of inactive excipients 
(microcrystalline cellulose (filler) and vegetable cellulose (capsules)). The weight of excipients 
will be adjusted so that capsules of the two doses have the same final total weight to preserve 
blinding. The capsules will be provided in sufficient quantities for the entire clinical trial as well 
as QC and stability tests to be conducted. Voucher specimens of the Shoden® powder and 
capsules (120 mg and 240 mg) will be maintained in Dr. Soumyanath’s laboratory at -80 ˚C. 
Randomization and blinding will be done by the OHSU research pharmacy. A single dose of 
Shoden® will be dispensed by the research pharmacy to a member of the study team just prior 
to the participant’s baseline PK visit for each study period. Participants will receive two single 
administration doses of Shoden® (240 and 480 mg). The order in which they receive these 
doses will be random to prevent a tolerance effect. The OHSU Research Pharmacy will 
randomize participants to one of two dose sequence groups, stratified by sex. Participants will 
consume Shoden® in the presence of a study team member following baseline measurements 
in order to ensure adherence to the study protocol. Prior to the 48-hour visit for each study 
period, a 35-day supply of Shoden® (240 or 480 mg per day) will be dispensed to a study team 
member. Whenever the study intervention is dispensed, the OHSU research pharmacy will 
record the date, participant ID number, and dose for accountability.  
 
H.2. Administration 
A single dose (240 or 480 mg) of the study intervention will be administered in an outpatient 
setting at each baseline PK visit. The dose sequence will be randomized. The research 
pharmacy will dispense one dose of Shoden® to a member of the study team, who will provide it 
to the participant for oral consumption. At the 48-hour visit for each study period, participants 
will be given a 35-day supply of the study intervention. 
 
H.3. Criteria for intervention discontinuation and stopping guidelines 
Participants will be advised during the informed consent process that they have the right to 
permanently discontinue Shoden® and/or withdraw from the study at any time without negative 
repercussions. Participants will also be counseled during the consent process that they may be 
withdrawn from the study at the discretion of the Principal Investigator or designees. A 
participant may discontinue and/or withdraw and/or be withdrawn from the study for the 
following reasons: 
 
Administrative: 
1) Withdrawal of participant consent 
2) Request of site investigator or designees 
3) Request of primary care physician 
4) Non-compliance 
5) Protocol deviation 
6) Premature termination of the study 
 
Adverse event (AE)/experience: 
1) Worsening of pre-existing disease 
2) Intercurrent illness 
3) Death 
4) Major/clinically significant alteration in laboratory values or biometric assessments after 
beginning product 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 12 of 28 
5) Other AE 
6) Other reasons concerning the participant’s health or well-being 
 
The study will be terminated if two participants terminate due to intervention-related serious 
adverse events. Severe adverse events will be reviewed immediately by the PI to determine if 
severity and relatedness warrant termination of the study (Section N). 
I. SCREENING AND STUDY ACTIVITIES 
A schedule of study visits and assessments is shown in Table 1. 
 
I.1. Phone screening 
Potential participants will be screened over the phone by the study team, prior to attending an 
in-person screening visit. An IRB-approved telephone script will be used. During the phone 
screen we will ask participants to self-report that they are not are currently pregnant, breast 
feeding, or actively trying to conceive a child. The telephone screening will take approximately 
10-15 minutes. Eligibility will be documented and potential participants that meet the eligibility 
criteria will be scheduled for a screening visit.  
 
I.2. Consent process 
To ensure full comprehension of all aspects of their participation in the study, all potential 
participants will be mailed or securely emailed a copy of the IRB approved consent form prior to 
their in-person screening visit. They will be instructed to review the form ahead of time but not to 
sign it until the visit. For participants with limited English proficiency a qualified interpreter will be 
present for screening, the consent discussion, and the subsequent study visits. As this study 
does not specifically target participants with limited English proficiency, the short consent form 
in the language needed will be submitted to the IRB prior to use. All in-person screening visits 
and consenting will occur in a CTRC outpatient room. At the in-person screening visit, a 
member of the study team will review the consent form and then assess their understanding by 
asking the participant to describe key points of the study (e.g., purpose of study, number of 
visits, length of study) before signing. After the participant signs the consent, screening 
assessments and baseline measurements will start. A copy of their signed consent form will be 
provided to each participant. To ensure ongoing consent, participants will be asked if they wish 
to continue their participation in the study at the beginning of every in-person study visit and 
phone visit.  
 
I.3. Screening visit 
After the consent form is signed the participant’s health status will be evaluated using a 
biometric assessment (height, weight, and body mass index), vital signs (blood pressure, pulse 
rate, temperature, and respiration rate), a screening physical exam, a mood assessment 
(Geriatric Depression Scale-15), and cognitive assessment (Mini-Mental State Examination). A 
non-fasting plasma and urinary laboratory evaluation (complete blood count (CBC), 
comprehensive metabolic panel (CMP), thyroid-stimulating hormone (TSH), and urinalysis) will 
be performed and submitted to the OHSU clinical core laboratory to screen for abnormalities 
that could impact study results. Study staff will collect approximately three teaspoons (15 ml) of 
venous blood by peripheral venipuncture and the participant will collect 20 ml of urine via a 
clean-catch method for analysis. A screening electrocardiogram (ECG) will also be taken at the 
screening visit. The visit is expected to take 60 to 90 minutes.  
The study clinician (Dr. Quinn) will review screening and laboratory results with the candidate 
prior to participant enrollment. Participants will be contacted by phone and told whether they 
qualify for the study or not. Participants who meet all final eligibility criteria will be invited to 
participate in the intervention stage of the study. The OHSU research pharmacy will randomize 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 13 of 28 
each participant into one of two dose sequence groups. Any participant with abnormal health 
screening or laboratory evaluation will be directed to their primary care physician for treatment 
and excluded from the study. In the event that a participant does not qualify for the study, their 
data will be destroyed at the end of the study. 
 
I.4. PK Visits 1 and 2 
1.4.1. Baseline Visit (0-12hr) 
Visit Timing: For the first study period, Baseline Visit 1 will occur a minimum of three days after 
the screening visit to allow for review of laboratory results and scheduling of appropriate 
facilities. For the second study period, Baseline Visit 2 will occur a minimum of two weeks (and 
not more than three weeks) after the four-week follow-up visit for the first study period. 
Sample collection: Participants will be asked to fast for 10-12 hours, excluding water, the night 
before each baseline visit in an attempt to standardize gastrointestinal transit time and minimize 
delayed absorption of the study intervention due to the presence of food. Participants will be 
reminded during the pre-visit reminder call to arrive at the visit well-hydrated to facilitate blood 
collections. Study staff will place a peripheral intravenous catheter in the participant’s arm or 
hand to allow for serial plasma specimen collection. Prior to administration of Shoden®, a 
baseline plasma sample of two teaspoons (10 ml) will be collected through the catheter using a 
syringe and transferred immediately into a heparinized Vacutainer tube. The participant will then 
be administered two capsules of Shoden® (240 or 480 mg total) on an empty stomach in the 
presence of study staff to ensure compliance. Participants will be asked to abstain from food 
and drink (excluding water) for the first two hours following consumption of Shoden® capsules 
to allow for transition into the small intestine and absorption. If signs of hypoglycemia are 
present, participants will be allowed to consume a high glycemic food and monitored for 
improvement. Participants will be provided water at the visit and will be encouraged to drink four 
16-ounce cups of water over the 12 hours to maintain good hydration status. Serial blood 
samples (two teaspoons (10 ml) per sample) will be collected through a peripheral intravenous 
catheter, at 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300, 360, 540, and 720 minutes after 
dosing. A baseline urine sample and a 12-hour pooled urine collection will be obtained to 
measure excretion of WS metabolites.  
Tolerability and safety: Participants’ weight, height, body mass index, age, blood pressure, 
temperature, respiration rate, and pulse rate will be recorded at each study visit to monitor for 
adverse events and asymptomatic illnesses that may impact study results. Vitals will be 
collected at baseline, 7 hours post-Shoden administration, and at the end of each baseline visit. 
At the beginning of each baseline visit, participants will complete an electrocardiogram (ECG) 
and the Geriatric Depression Scale-15. Four teaspoons of blood (20 ml) will be collected for 
safety labs (CBC, CMP, urinalysis, TSH, serum total testosterone). ECG will be repeated 7 
hours post-intervention to assess for cardiac changes. One teaspoon (5 ml) will be collected for 
a repeat CMP at 10 hours post-intervention to evaluate kidney and liver function. Participants 
will be interviewed and administered a standardized multi-system adverse events questionnaire 
at the beginning and end of each baseline visit. This visit is expected to take 13 hours a total of 
175 mL of blood will be collected.  
If the baseline CBC shows hemoglobin or hematocrit levels below what is recommended by the 
FDA for blood donation, the visit will be halted immediately. Participants will be asked to stay for 
2.5 hours after the study agent has been administered to assess for acute side effects. Two and 
a half hours was chosen because common withanolides have reached their CMAX by this time 
based on a previous PK study using Shoden®.80 The end-of-visit adverse events questionnaire 
will be administered by phone and missed safety evaluations (7-hour ECG and vitals, 10-hour 
CMP, 12-hour vitals) will be collected at the 24-hour visit.  After the 24-hour visit, the participant 
will be withdrawn from the study.  

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 14 of 28 
If study staff have difficulties collecting blood samples for any reason, the decision may be 
made to halt the visit. Participants will be asked to stay for 2.5 hours after the study agent has 
been administered to assess for acute side effects. If the visit ends before the 7-hour mark, the 
end-of-visit adverse events questionnaire will be administered by phone and missed safety 
evaluations (7-hour ECG and vitals, 10-hour CMP, 12-hour vitals) will be collected at the 24-
hour visit. Participants will be allowed to remain on the study if difficulties with blood sample 
collection were the only reason for halting the study visit early.  
 
I.4.2 24- and 48-Hour Visits 
Participants will return at 24 hours and 48 hours for a single blood collection (two teaspoons (10 
ml)) and urine collection. Vitals will be collected. A CMP and ECG may be collected at the 24-
hour visit if the baseline visit was halted prior to 7 hours. At the 48-hour visit, the adverse events 
questionnaire will be administered as the baseline measure for the subsequent four-week 
period. After the 48-hour visit, participants will be issued a 35-day supply of the appropriate 
dose of Shoden® (240 or 480 mg), which they will begin taking before bed every day for four 
weeks. These visits are expected to take 20 minutes. After analyzing the plasma and urine 
samples for the first two participants, a determination will be made as to whether the 48-hour PK 
visit is needed for future participants. If no withanolides are detected in plasma or urine at 48-
hours, this time point will be eliminated to avoid undue participant burden. 
 
1.5. Two-Week Adverse Event Phone Visit 
The study team will call the participant two weeks after the baseline visit during each study 
period to administer an adverse events questionnaire and the Geriatric Depression Scale-15, 
and schedule the 4-week follow-up visit. To ensure ongoing consent, participants will be asked if 
they want to continue participation in the study. This visit is expected to take 15-30 minutes. 
 
1.6. Four-Week Follow-Up Visit 
Participants will be asked to fast for 10-12 hours, excluding water, the night before each four-
week follow-up visit in order to collect fasting glucose as part of the CMP. A single blood sample 
(two teaspoons (10 ml)) and a urine sample will be collected to assess steady state plasma 
levels and excretion of WS compounds. Returned capsules will be counted to assess for 
adherence. Participants will have an ECG and blood and urine samples will be sent for safety 
labs (CBC, CMP, urinalysis, TSH, serum total testosterone, serum DHEA-S). The Geriatric 
Depression Scale-15 will be administered. At the four-week follow-up visit for the second study 
period, participants and investigators will be queried as to their perception of the dose sequence 
administered. This visit is expected to take 45-60 minutes. 
 
1.7. Final Phone Visit 
Two weeks following the four-week follow-up visit for the second study period, participants will 
be telephoned to assess for delayed adverse events using interviews and a standardized multi-
system adverse events questionnaire. The Geriatric Depression Scale-15 will be administered. 
Participants will be instructed to contact the study team if they become pregnant or father a child 
after they have completed the study. 
 
1.8. REDCap Surveys 
Participants will be emailed a link to electronic surveys in REDCap at four time points during the 
study: during the week prior to each PK visit and during the week prior to each four-week follow-
up visit. Participants will complete two surveys at each time point: Pittsburgh Sleep Quality 
Index (PSQI) and the Perceived Stress Scale (PSS). 
 
I.9. Washout Period  

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 15 of 28 
The two study periods will be separated by a 2- to 4-week washout period where the participant 
will not take the study agent. If the washout period is longer than 21 days, participants will be 
called at two weeks to assess for delayed side effects. 
 
I.10. Subject participation duration 
Including the screening visit, a two to four-week washout period between doses, and an exit 
phone interview for adverse event monitoring, it will take participants 13 to 14 weeks to 
complete the study.   
 
Table 1. Schedule of study visits and assessments. 
 
Screen 
PK visit 
2-week 
f/u 
4-week 
f/u  
Washout Period 
PK visit 
2-week 
f/u 
4-week 
f/u 
Final 
visit +2 
weeks  
Study Period 
 
1 
1 
1 
2 
2 
2 
 
Event 
Clinic 
Visit 
0 to 
12h 
24h 
48h 
Phone 
visit 
Clinic 
visit 
0 to 
12h 
24h 
48h 
Phone 
visit 
Clinic 
visit 
Phone 
visit 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Vitals and biometrics 
X 
X✚ 
X 
X 
 
X 
X✚ 
X 
X 
 
X 
 
Health screening 
X 
 
 
 
 
 
 
 
 
 
 
 
Electrocardiogram (ECG) 
X 
X$ 
X˚ 
 
 
X 
X$ 
X˚ 
 
 
X 
 
CBC*, CMP*, TSH▲, and UA* 
X 
X^ 
X˚ 
 
 
X 
X^ 
X˚ 
 
 
X 
 
Serum testosterone  
 
X 
 
 
 
X 
X 
 
 
 
X 
 
Single dose of intervention  
 
X 
 
 
 
 
X 
 
 
 
 
 
Single-dose PK blood draws 
 
X 
X 
X 
 
 
X 
X 
X 
 
 
 
Issue 35-day supply of 
Shoden® 
 
 
 
X# 
 
 
 
 
X# 
 
 
 
Steady state PK blood draw 
 
 
 
 
 
X 
 
 
 
 
X 
 
12-hour urine collection 
 
X 
 
 
 
 
X 
 
 
 
 
 
Single urine collection 
 
X¶ 
X 
X 
 
X 
X¶ 
X 
X 
 
X 
 
Collect and count unused 
capsules 
 
 
 
 
 
X 
 
 
 
 
X 
 
Adverse events questionnaire 
 
X★ 
 
X 
X 
X 
X★ 
 
X 
X 
X 
X 
Cognitive screen (MMSE) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Depression screen (GDS) 
X 
X◆ 
 
 
X 
X 
 
X 
 
 
X 
X 
X 
REDCap surveys 
 
X▼ 
 
 
 
X▼ 
 
X▼ 
 
 
 
X▼ 
 
f/u – follow-up 
$  ECG collected prior to Shoden® administration and at 7 hours post-Shoden® administration 
* CBC = Complete Blood Count with differential; CMP = Comprehensive Metabolic Panel; UA = Urinalysis 
^  CMP collected prior to Shoden® administration and at 10-hours post-Shoden® administration 
▲ TSH = Thyroid-stimulating hormone 
# This will take place at the 24-hour visit if 48-hour visit is eliminated, as described in Section I.4. 
¶  Aliquot of baseline urine prior to administration of Shoden® 
★Adverse events questionnaire administered at beginning and end of baseline visits 
◆ If PK Visit 1 is ≥14 days since the screening visit 
▼REDCap surveys will be delivered to participants’ email during the week prior to each study visit. 
˚An ECG and CMP will be collected at the 24hr visit if the baseline visit was halted prior to the 7hr ECG and CMP being collected. 
J. STUDY DURATION 
Recruitment and data collection is expected to begin upon approval from the IRB and continue 
for nine months. We estimate a total of six months for final data analysis. Total estimated study 
duration is 21 months (Table 2). 
 
 
 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 16 of 28 
Table 2. Study timeline.  
 
Month 
                                                  
Activity 
1-3 
4-6 
7-9 
10-12 13-15 
16-18 
19-21 
22-24 
Characterization studies 
  
        
        
      
 
 
 
 
 
 
 
 
 
Product quality control 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
LC/MS Method Optimization 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Recruitment/enrollment 
  
        
        
      
 
 
 
 
 
 
 
 
 
Data collection 
  
        
        
      
 
 
 
 
 
 
 
 
 
Plasma/urine LC/MS analysis 
  
        
        
      
 
 
 
 
 
 
 
 
 
PK data analysis/summary 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Reports, manuscripts 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
K. DATA AND SPECIMENS 
The principal investigator will oversee the management of the participants, intervention, and 
analysis, which will be carried out by the clinical investigators, research statistician, and 
research assistant. Data entry, quality control and preparation, and participant management will 
be ongoing throughout the study. 
 
K.1. Data collection methods  
The case report forms for this study include a telephone screening script, a medical history 
form, a cognitive screening questionnaire (Mini Mental State Examination), a mood screening 
questionnaire (Geriatric-Depression Scale-15), a stress questionnaire (Perceived Stress Scale 
(PSS)), a sleep questionnaire (Pittsburgh Sleep Quality Index (PSQI)), an adverse events 
questionnaire, and a 2-week follow-up phone script. Other data collection methods include 
administering an electrocardiogram, urine collection, and blood collection. The PSS and PSQI 
will be delivered to participants’ emails via REDCap.  
 
K.2. Handling of data and specimens 
Upon enrollment, each participant will obtain a unique identification number. This number will be 
associated with all specimens and data collected from that participant. Following the collection 
of whole blood from the participant, a trained member of the study team will transfer the blood to 
the specified vacutainer tubes. Blood will be centrifuged in the CTRC’s laboratory to separate 
the plasma for analysis. The isolated plasma will be transferred into labeled tubes and placed 
on ice until transported by a member of the study team via a sealed Styrofoam cooler labeled 
with biohazard to the Soumyanath Lab (Richard Jones Hall, OHSU). The plasma and a 50mL 
aliquot of the urine specimens will be stored in labeled boxes in Dr. Soumyanath’s -80°C 
freezer.  
 
Electronic data from case report forms and specimen analysis will be entered in REDCap. 
Analyzed data will be stored in an OHSU approved cloud storage (OneDrive). Only study team 
members will be granted access to the REDCap database or OneDrive with access specific to 
their role in the project. Paper records including participant contact information, telephone 
screening responses, consent forms, electrocardiogram reports, lab results, and paper case 
report forms will be stored in participant-specific binders, kept in a locked cabinet at the 
Parkinson’s Center of Oregon office suite (Sam Jackson Hall), where Dr. Speers is located. 
 
K.3. Sharing of results with participants 
All screening laboratory results, performed by the CLIA-certified OHSU Core Laboratory, will be 
shared with the participant through their medical record. If a screening result is found abnormal 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 17 of 28 
(an incidental finding), the participant will be instructed to share their result with their primary 
care provider. Electrocardiograms (ECGs) will be performed at the screening visit, PK visits, and 
four-week follow-up visits. If the ECGs are found to be abnormal, the participant will be 
instructed to share their result with their primary care provider. As there will be no additional 
laboratory assessments with clinical implications beyond the screening visit, no results from the 
study will be shared with the research participant or their provider(s).  
 
K.4. Data, Specimen banking, and Repository Procedures 
All data obtained from this study will be used for research purposes only and will comply with 
HIPAA regulations. No genetic research will be performed on these specimens. The specimens 
and data collected relate specifically to this project; however, data, plasma and urine specimens 
will be stored for future analyses in a repository included in this protocol. Specimens and data 
from other studies will not be accepted in this repository. For future analyses, plasma and urine 
specimens will be stored in a locked -80˚C freezer in Dr. Soumyanath’s laboratory, access to 
which is badge-restricted. All specimens will be coded with each participant’s unique numeric ID 
code, visit number, and data of collection. The key for the coded specimens will be stored on 
OneDrive, and access to the key will be limited to the PI and the Repository Guardian. As this 
study is NIH funded, a Certificate of Confidentiality was issued.  
 
Participants will sign a consent form stating that specimens and data will be stored for future 
use. The PI will notify the Repository Guardian promptly if consent has been withdrawn for 
future use of specimens or data. If consent is withdrawn, the Repository Guardian or designee 
will remove identifiers from the samples and data, but the material will not be destroyed, and we 
will continue to use it for research. 
 
The repository guardian will be Alex Speers. For repository requests, the repository guardian or 
designee will review the requestor’s IRB approval memo, protocol, and repository sharing 
agreement before samples/data are released. Separate IRB approval/determination will be 
required for each specific human subject research activity that uses coded data/specimens from 
the repository. The Guardian or designee will check for genetic opt out status, withdrawn 
consent for data/samples, and limitations on future use of data/samples. A signed Repository 
Sharing Agreement will be collected before data or samples are released. The Repository 
Guardian will ensure that material transfer agreements for the transfer of biological materials 
and data use agreements for data shared outside of OHSU are executed as applicable. Data 
and specimen releases will be documented on the repository tracking sheet. Data will be 
released using OHSU approved cloud storage or secure email. Samples will be hand delivered 
or mailed in accordance with OHSU dangerous goods shipping policies. Specimens and data 
will be coded with the participant’s unique ID code, visit number, and date of collection. The key 
for requested specimens and data will be provided separately and with the appropriate IRB 
approval. 
L. DATA ANALYSIS 
L.1. Specimen analysis (Aim 1) 
Eleven withanolides (withanolide A, withanolide B, withaferin A, withanone, withanoside IV, 
withanoside V, 12-deoxywithastramonolide, sominone, viscosalactone B, 4-oxo withaferin A, 
and 2,3-dihydro-3β-methoxy withaferin-A) have been initially selected based on their availability 
as reference standards. Final selection of withanolides to be included in the PK analysis will be 
based on product characterization studies conducted at the Oregon State Mass Spectrometry 
Center. Only withanolides that are confirmed to be in the test product, or an acid hydrolysate of 
the test product (mimicking deglycosylation), will be included in the PK analysis.  
Targeted analysis of the selected withanolides in plasma and urine samples will be performed 
using LC-MRM-MS instrumentation at OHSU’s BSR/PKCore. The BSR/PKCore has developed 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 18 of 28 
a 15-minute LC-MRM-MS method for the BENFRA Center for the analysis of seven 
withanolides using digoxin-d3 as an internal standard (section E.3, Figure 2). The BENFRA 
center (through Dr. Soumyanath) is working with the BSR/PKCore lab to include the four 
additional withanolides in the method and optimize mouse plasma work-up methods for the 
analysis. These methods will be adapted for human plasma and urine in this study, using 
commercially available human plasma and urine spiked with reference withanolides under the 
guidance of Dr. Soumyanath and Dr. Andrea DeBarber, director of the BSR/PKCore.  
Each withanolide analyte will be identified by its retention time and characteristic mass spectral 
fragmentation pattern using selected reactions within a multiple-reaction monitoring paradigm. 
The peak area ratio to the appropriate internal standard will be identified and evaluated against 
the standard calibration curve of each analyte prepared by spiking known concentrations into 
blank plasma. Each analyte’s concentration in participant plasma will be used to generate a 
concentration-time curve for pharmacokinetic profiling. The primary endpoints are the maximum 
concentration (Cmax) and its corresponding timepoint (tmax) of each withanolide analyte. To 
determine the rate of elimination (k), linear regression of the plasma concentration against time 
will be used. This value will be used to determine the elimination half-life (t1/2) using the formula 
t1/2=0.693/k. Area under the concentration-time curve from dosing (time 0) to time t (AUC0-t) will 
be calculated using the linear-log trapezoidal method. Area under the concentration-time curve 
from dosing (time 0) extrapolated to infinite time (AUC0-∞) will be calculated using the equation 
AUC0-t + AUCt-∞, where AUCt-∞ = Clast/ke. Clast is the last observed concentration and ke is the 
elimination rate constant. Differences in in Cmax, AUC0-t, AUC0-∞, and t1/2 for the two doses of 
Shoden® will be evaluated using paired t-tests or the non-parametric equivalent Wilcoxon 
paired test, while differences in tmax will be compared between dose groups using chi-square 
tests of distribution. With numerous analytes of interest, post-hoc multiple comparison 
adjustments in p-values will be applied using the Holm-Sidak stepwise correction.  All final 
statistical tests will be two-tailed and considered significant at α=0.05. 
Peak area for each withanolide analyte and internal standard will be obtained using Analyst 
software from Sciex Technologies (version 1.7.1). The Core lab will provide and set up LC-
MRM-MS instrumentation. The applicant will prepare the plasma and urine samples for analysis 
in Dr. Soumyanath’s lab and retrieve and analyze the LC-MRM-MS data generated at the Core 
Lab to obtain concentrations of each withanolide in each sample. The applicant will work with 
Dr. Munar to obtain PK curves and parameters. 
 
L.2. Safety analysis (Aim 2) 
Changes in laboratory outcomes from the baseline visit to the four-week follow-up visit for each 
study period will be presented using descriptive statistics (e.g., means and standard deviations, 
frequency and percent). Within group differences for laboratory outcomes at each dose will be 
evaluated for significance using paired t tests. Data will also be presented visually using 
histograms and line graphs. Treatment-emergent AEs will be organized by body system and 
summarized in table format by dose, with AE severity and possible relation to study drug 
reported.  
 
L.3. Feasibility analysis (Aim 3) 
The feasibility of administering REDCap surveys will be assessed by calculating the percentage 
of administered questionnaires that are returned and fully completed by participants, with 
feasibility defined as at least 80% of all administered questionnaires returned and completed. 
For each of the four timepoints (prior to PK Visit 1 and 2, prior to 4-Week Follow-Up Visit 1 and 
2), the percentage of administered questionnaires returned and the percentage of returned 
questionnaires that are fully completed will also be reported.  
 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 19 of 28 
L.4. Sample size 
Based on a paired sample t-test with alpha=0.05 and a sample size of 10 (anticipating two 
dropouts), we will have 95% power to detect an effect size of 1.3 (Cohen’s d). Based on plasma 
levels of withaferin A, withanoside IV, and withanolide A observed in an unpublished PK study 
of 480 mg Shoden® in 16 healthy adults,79 this effect size would be a difference in Cmax between 
the two doses of about 6.5 ng/ml for withaferin A, 2.2 ng/ml for withanoside IV, and 1.17 ng/ml 
for withanolide A. We expect to see much bigger differences between the two doses based on 
previous botanical studies where half doses resulted in Cmax values that were 35-50% lower 
than the full dose.81-84  
 
L.5. Data quality and management 
The Principal Investigator will oversee study progress including recruitment, retention, 
demographics, and study participant status. Tabulation of study data will have both physical and 
digital formats available. A member of the study team will record data from each study visit and 
phone visit on paper case report forms. Data will be input into REDCap promptly after the 
participant’s visit and double-checked for accuracy by a separate member of the study team or 
the Principal Investigator. This will include limited options for responses, protection against 
redundancy and double-data entry, and locking of the database for information entry upon study 
completion. This combination of steps will promote cohesiveness of study outcomes from study 
visit to data entry while maintaining the necessary protection of participant privacy. Study 
personnel will calculate PK outcomes using the Phoenix WinNonlin program and results will be 
double-checked by Dr. Munar to ensure accuracy.  
M. PRIVACY, CONFIENTIALITY, AND DATA SECURITY 
Confidentiality will be assured in this study through several mechanisms. Individuals interested 
in participation in the study will call the study coordinator’s personal office telephone for a phone 
screening. During screening and study visits, the investigators will ensure physical privacy by 
conducting interviews and examinations in a closed room. Participant data and specimens will 
be labeled with their participant ID. Finally, participants will not be identified by name in any 
reports or publications, nor will data be presented in such a way that the identity of individual 
participants can be inferred. All staff are trained and annually re-certified regarding these 
procedures in Responsible Conduct of Research and HIPAA.   
 
Data for this project will be stored in OCTRI's installation of REDCap, a highly secure and robust 
web-based research data collection and management system. Features of REDCap that protect 
participants' privacy and data security include: 
• 
Physical Security: OCTRI's REDCap software is housed on servers located in ITG's 
Advanced Computing Center providing locked physical security 
• 
Electronic Security: The REDCap servers are housed behind both the OHSU firewall and a 
second ACC firewall.  All web-based data transmissions are encrypted with industry-
standard SSL methods.   
• 
Controlled User Access: REDCap is employs a robust multi-level security system that 
enables researchers to easily implement "minimum necessary" data access for their 
research staff, including specification of data fields that are identifiers. This feature includes 
“single click” ability to provide completely deidentified (removing all identified data fields and 
shifting dates) for analysis or other purposes.  User activities are logged to enable auditing 
of all data access.   Access is integrated with OHSU's network such that users who are also 
OHSU employees are authenticated against their OHSU network credentials.   
• 
Data Integrity: REDCap is jointly managed in accordance with OHSU Information Security 
Directives by ACC staff and members of OCTRI's Biomedical Informatics Program, ensuring 
fidelity of database configuration and back-ups.  User activities are logged to enable auditing 
of all data changes. 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 20 of 28 
N. PROVISIONS TO MONITOR THE DATA TO ENSURE THE SAFETY OF SUBJECTS 
To ensure participant safety, we will monitor for adverse events (AE) with AE questionnaires, 
ECGs, and laboratory tests. During the baseline visit and four-week follow-up visit for each 
study period, participants will undergo an ECG and laboratory testing (CBC, CMP, TSH, 
testosterone, and urinalysis). At baseline visits, ECG will be repeated 7 hours post-Shoden® 
administration to assess for cardiac changes and CMP will be repeated 10 hours post-Shoden® 
administration to evaluate liver and kidney function. Participants whose ECGs and/or lab results 
reflect abnormalities will be further screened for potential causes of the abnormalities as 
determined by Dr. Quinn. We will notify the participant and the participants’ primary care 
physician if blood and urine safety parameters fall outside the normal range or if abnormalities 
are seen on ECG, and Dr. Quinn will undertake the necessary steps to ensure appropriate 
clinical care.  
Although assessment of AEs will also include solicitation of non-specific changes (e.g., "do you 
feel different since beginning the treatment?"), a multisystem checklist covering all major organ 
systems will be included to prevent missing any AEs. The standardized adverse event 
questionnaire will be administered to participants at the beginning and end of each baseline visit 
and four-week follow-up visit. In addition, the AE questionnaire will be administered by phone 
two weeks after the baseline visit for each study period, and two weeks after the final study visit. 
The nature of each AE, such as severity, likelihood of relationship to the study intervention, 
duration, and necessary palliative treatments will be recorded.  
Participants will be reminded and encouraged during clinic visits and telephone calls to contact 
a member of the study team if a moderate or serious AE occurs. Participants will be given a 
telephone number and email address they can contact to report potential adverse effects. The 
participant will also be advised to go to their primary care physician or emergency room as 
warranted for treatment of AEs.  
The principal investigator will do real-time review of AEs and protocol deviations to determine if 
the event meets the OHSU IRB’s definition of reportable new information. All AEs and protocol 
deviations will be reported to the IRB at the time of continuing review.  
Adverse events will be organized based on System Organ Class (SOC), based on the highest 
level of the MedDRA system (https://evs.nci.nih.gov). Within each SOC, events will be defined 
using the same criteria held by the National Cancer Institute’s Common Terminology Criteria for 
Adverse Events (CTCAE). Severity classification will similarly follow the five-point grading scale 
for each defined event as described under the CTCAE. Events will be classified as severe for 
any grade 3 (severe), 4 (life-threatening) or 5 (resultant death) and marked for immediate 
reporting to OHSU’s IRB. Severe AEs (SAEs) are defined as events that are life-threatening or 
resulted in hospitalization, permanent disability, cancer, death, or other significant outcome. All 
SAEs will be reported promptly to the PI and clinician Co-Investigator.  
To reduce pregnancy risk, participants will be instructed at the Exit Phone Call to contact the 
study team if they believe they are pregnant or have fathered a child post-study. A records 
review will be conducted by a member of the study team one month after each participant has 
completed the study to check for reports and outcome of pregnancy.  
 
N.1. Attribution to intervention 
Items identified on the Adverse Event Monitoring Form will be compared to baseline levels to 
determine if the symptom was preexisting. If the symptom was pre-existing at the same or a 
previously greater severity, the symptom will not be considered attributable to the study 
intervention. If the symptom was pre-existing at lesser severity (or was not previously present), 
the symptom may be considered attributable to the study intervention. If the intervention is 
considered attributable, the co-investigators and the PI will determine if an alternative clinical 
explanation exists for the adverse event. If an alternative explanation does not exist, the 
adverse event will be considered attributable to the study intervention. If an alternative 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 21 of 28 
explanation does exist, the adverse event will not be considered attributable to the study 
intervention. 
 
N.2. Plan for review and reporting 
The study team will submit continuing review questionnaires to the OHSU IRB per institutional 
policy. The PI will review protocol deviations, reports, and AEs in real time to determine if they 
must be reported as Reportable New information (RNI). Events that are RNI will be reported to 
the OHSU IRB per RNI policy. All other events will be summarized and reported at continuing 
review.  
O. RISKS AND BENEFITS 
O.1. Risks to subjects 
Potential harm to participants will be minimized by excluding participants with the conditions 
detailed in section F.2.2. The risks to the subjects participating in this proposed research project 
are mainly those associated with venous access, potential toxicity of the test material, and time 
commitment. 
 
Venipuncture: The potential risks to participants are minimal and reversible. Likely risks include 
pain and bruising from the intravenous catheter or venipuncture needle insertion. Occasional 
risks include feeling lightheaded or faint during or immediately after a blood sample is collected. 
Unlikely risks include swelling, infection, transient hematomas, transient thrombophlebitis, a 
fractured catheter embolus, or an allergic reaction to the adhesives or sterilization materials 
used at the venous access point. To minimize risk, catheter insertions and blood draws will be 
conducted in a seated position and using aseptic technique by trained personnel. 
 
Study Diet: The study diet requires that participants avoid any caffeine for 48 hours prior to each 
baseline visit. If participants regularly drink caffeinated beverages like coffee, they may 
experience symptoms of caffeine withdrawal, including headache, fatigue, decreased 
energy/activeness, decreased alertness, drowsiness, decreased contentedness, depressed 
mood, difficulty concentrating, irritability, and feeling foggy/not clearheaded.85 
 
Shoden® extract: Shoden® is a 70% ethanolic extract of Withania somnifera root and leaves, 
standardized to 35% withanolide glycosides and produced in a good manufacturing practice 
(GMP)-certified facility. We will also conduct independent analyses of impurities (pesticides, 
heavy metals, microbes) through Oregon’s Wild Harvest (Redmond, OR) and confirm dose 
levels through analysis at Oregon State University (Corvallis, OR).  
Shoden® is commercially available and has been studied in randomized, double-blind, placebo-
controlled clinical trials, where it has been well-tolerated with no significant adverse events 
reported.52,53,57 The doses of Shoden® being used in this study are 240 and 480 mg. In a 
previous clinical trial of 60 participants, 240 mg Shoden® once daily for 60 days was not 
associated with any significant adverse events or laboratory abnormalities.53 The higher dose of 
480 mg was studied in a recently completed study of Shoden®, where it was well-tolerated after 
a single administration in 16 participants.79 We anticipate that 480 mg of Shoden® will also be 
safe and well-tolerated over four weeks, but will closely monitor participants for potential 
adverse events, as described in Section N. The widespread use of WS as a herbal dietary 
supplement1 and reviews of clinical studies using WS46,86 supports its general safety. When side 
effects have been reported in clinical trials with other WS products, they have been mild to 
moderate transient effects including somnolence, giddiness, vertigo, drowsiness, and GI effects 
(e.g., nausea, epigastric pain, loose stools, constipation, decreased appetite).46 There have also 
been rare reports of mild to severe liver toxicity for other WS products48,49 and a case of kidney 
transplant rejection in a patient using WS.51 There is also evidence that WS may modulate 
thyroid indices20 and testosterone.52-55 Liver and renal abnormalities, thyroid function, and 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 22 of 28 
testosterone will be assessed at the beginning and end of each four-week study period. The risk 
of taking WS while pregnant or breast-feeding is unknown and therefore, it is recommended to 
avoid WS during pregnancy and breast-feeding.  
 
Treatment-emergent suicidal ideation and behavior have been identified as a concern for a 
number of drugs and drug classes. There is currently no published evidence that WS increases 
suicidal ideation and behavior.15 Suicide rates increase with age,87 however, and because 
safety information for ashwagandha in older adults is limited, a prospective assessment for 
suicidal ideation and behavior will be monitored. Heisel et al. (2011)88 have identified a “suicide 
ideation” subscale in the Geriatric Depression Scale (GDS). We are using the GDS as part of 
study procedures and will pay special attention to the identified subscale questions to assess 
potential risk of suicidal ideation or behaviors. Specifically, items 3, 7, 11, 12 and 14 are used to 
screen for suicidal ideation. We are using the GDS as part of study procedures, and if the 
participant has a total score of 2 or more on the five items listed, it is considered indicative of 
possible suicidal ideation. This participant will then be administered the Columbia-Suicide 
Severity Rating Scale (C-SSRS), which determines suicidal risk severity. If the scale reveals 
active suicidal ideation with some intent to act but without a specific plan, active suicidal ideation 
with a specific plan and intent, or suicidal behavior, a study clinician or neuropsychologist will 
call the participant and assess. Participants with high risk as determined by the clinician will be 
excluded from the study and referred for mental health intervention. 
 
Risks associated with a breach of confidentiality: There is a small risk that information about a 
study participant could be inadvertently disclosed to non-study personnel. Procedures to 
minimize this risk have been described in section M of this protocol. 
 
Other risks/discomforts associated with participation: The participants will experience a time 
burden by participation in the research study, as they will need to attend an in-person screening 
visit plus eight additional study visits, including a baseline PK visit during each study period 
where participants will remain in the Clinical and Translational Research Center for 13 hours. To 
compensate participants for their time, they will be remunerated in a prorated fashion ($100 per 
completed study period) for their participation. To offset travel costs, $1500 is set aside in the 
project budget to pay for transport to and from study visits for participants when needed. To 
mitigate the burden associated with bringing food that doesn’t interfere with the PK analysis, 
participants will be provided with three meals and snacks at each baseline PK visit at no 
expense.  
 
O.2. Potential benefits to subjects 
There is no direct benefit to participation in the study.  
P. IMPORTANCE OF KNOWLEDGE GAINED 
WS is an Ayurvedic herb commonly used for stress relief and stress-related neuropsychiatric 
disorders (e.g., anxiety, depression, and insomnia).15 Despite being one of the most popular 
herbal dietary supplements in the United States,2 there is a lack of data on the oral absorption 
and PK of WS compounds in humans. This study will assess the oral absorption and PK of 
eleven withanolides from Shoden® in healthy older adults. Compared to previous PK studies 
using WS, this study will a) include several additional withanolides, b) measure withanolide 
levels in urine as well as plasma, c) measure steady state levels after 4 weeks of consumption, 
and d) provide PK data in an important population (i.e., older adults) where dietary supplement 
use is common and risk of HDIs is higher. The data on dosing and tolerability will be directly 
applicable to future trials of Shoden® in family caregivers of people with dementia. The data 
generated from this study will also inform biological signature studies, mechanism-based 
research on WS compounds, and HDI trials. 

Study Title: Safety, tolerability, and pharmacokinetics of an oral Withania somnifera product in older adults 
Principal Investigator: Alex Speers, ND, MS  
IRB#: STUDY00026055 
Version Date: 10.10.2024 
 
Page 23 of 28 
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Version Date: 10.10.2024 
 
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