Withania somnifera (Ashwagandha)

Analytical Method Development

Sanni Raju, Ph.D., R.Ph.Natreon, Inc.2D - Janine Place, New Brunswick, NJ 08901Tel: 732-296-1080; Fax: 732-777-5129E-mail: info@natreoninc.comWebsite: www.natreoninc.com

1

What is Ashwagandha?

Ashwagandha is an Ayurvedic medicine derived from the plant Withania somnifera. A ti t Anti-stress

Improves endothelial function Cognition enhancing Anti-inflammatory Lipid management

2

CONFIDENTIAL

Fitness for Purpose Statement

Method should be able to quantitatively analyze withanolide glycosides, W h f A d l h d f l h d Withaferin-A and oligosaccharides from nutraceutical matrices, such as powders, tablets, capsules and liquids.

Method is confirmatory and will be used to pass or fail a batch of the raw material (powder extract) or a finished product, such as a tablet or a capsule.

There is no regulatory requirement The method will be used in a laboratory set up. Chemists trained in HPLC/HPTLC techniques will be required to do the analysis.

3

Fitness for Purpose Statement

Points for discussion by SPDS

How does this method address Ashwagandha extracts made by different manufacturers

What should be the targeted analytes Limits of quantification Availability of reference standards

4

Extraction Methodology

Traditionally only roots have been usedNatreon developed a better extract using roots plus leaves Natreon developed a better extract using roots plus leaves

(patented)Most companies use either alcoholic or hydro-alcoholic

solvents for extraction.One company has used supercritical extraction using carbon

dioxide.Natreon’s method is completely aqueous. Depending on the extraction method used, chemical

constitution of the extract varies.

5

CONFIDENTIAL

Chemical Composition

Depending on the extraction solvent and themethod used, the Ashwagandha extract may contain any

f th f ll i tit tof the following constituents:

o Withanolide glycosides o Withaferin A o Oligosaccharides o Alkaloids o Polysaccharides yo Withanostramnolideo 27-hydroxy withanone o 27-hydroxy withanolide B

6

CONFIDENTIAL

Intellectual Property - Sensoril®

US 7,318,938: Withania somnifera composition, method for obtaining same and pharmaceutical, nutritional and personal care formulations thereofformulations thereof

US 6,713,092 B1: Withania somnifera composition, method for obtaining same and pharmaceutical, nutritional and personal care formulations thereof

US 6,153,198: Withania somnifera composition EP1569669 A2:Withania somnifera composition, method for

obtaining same and pharmaceutical, nutritional and personal care formulations thereof

Canadian Patent No. 2,508,478 – Withania somnifera composition, , , p ,method for obtaining same and pharmaceutical, nutritional and personal care formulations thereof

US 8,206,757: Combination of Ashwagandha and Indian Gooseberry

Other pending US and PCT applications

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CONFIDENTIAL

Human clinical study of Sensoril® for stress management

o Number of patients

o Randomized placebo-controlled double blind studyo Randomized, placebo controlled, double blind studyo 130 chronically stressed subjects enrolled; 98 completed

o Treatment schedule• Group 1: Sensoril

®(250 mg capsule; 2/day)

• Enrolled 35; Completed 34• Group 2: Sensoril

®(125 mg capsule; 2/day)

• Enrolled 35; Completed 30• Group 3: Sensoril® (125 mg capsule; 1/day)

• Enrolled 30; Completed 19• Group 4: Placebo (2/day)

• Enrolled 30; Completed 15• Comprehensive assessments done at

0 day, after 30 and 60 days treatment

Published: JANA, Vol. II, No.1, 208, Pages 50-56.

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CONFIDENTIAL

Clinical study results – Stress & Anxiety parameters

Hamilton Anxiety Scale Pulse rate*

Scoring system (subjective) Severe – 4 Pulse rate

Blood pressure* Sleep deprivation* Palpitation* Dry mouth* Perspiration* Appetite Fatigue* Flushes* Headache & Muscular pain*

Severe 4 Moderate – 3 Mild – 2 Occasional – 1 None – 0

Statistical significance ANOVA P <0.05 (*significant)

Conclusion Statistically significant

Forgetfulness* Irritability* Inability to concentrate* Feeling of impending

doom*

Statistically significant reduction seen in all the 12 stress & anxiety and 2 quantitative parameters

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CONFIDENTIAL

Clinical study results – % Change in gross stress & anxiety parameters

% Reduction in gross stress condition in 30 & 60-day treatment

30 60 30 day30 60

30 60

30 60

p‐values <0.001

10

p

CONFIDENTIAL

Clinical study results: Stress markers% Reduction in serum cortisol % Reduction in C-reactive protein

% Improvement in serum DHEAS

Statistical significance

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• Day 0 vs 60 day; ANOVA• 250 mg & 125 mg BID: p <0.001• 125 mg OD: p <0.01

CONFIDENTIAL

Clinical study results: Lipid profile% Reduction in cholesterol % Reduction in triglycerides

% Reduction in LDL % Improvement in HDL

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CONFIDENTIAL

Clinical study results: Effects on VLDL, hemoglobin & glucose

% Reduction in VLDL % Improvement in Hemoglobin

% Reduction in glucose

Statistical significance

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Statistical significance• Statistical significance only with 250 mg BID • One way ANOVA; p <0.001

Non-significant

CONFIDENTIAL

Comparison of Sensoril® data to Lexapro® and Effexor® data from literature - HAM-A/mHAM-A Day 0 – Day 60

Hamilton parameter: Initial vs 60-day

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CONFIDENTIAL

Sensoril® - Cardiovascular/Endothelial Dysfunction Clinical Study

Study Design Prospective, randomized, double blind study with placebo and positive

(Atorvastatin/Lipitor®) controls with 20 subjects in each group. Patients with Type 2 diabetes stabilized on Metformin treatment

Inclusion Criteria Patients of either sex, aged 18-75 years Fasting plasma glucose of ≥110 mg/dL Glycosylated haemoglobin (HbA1c) between 7 % and 9% Taking stable dose of anti-diabetic treatment for the past 8 weeks prior to the

screening visit Endothelial dysfunction defined as ≤ 6% change in reflection index (RI) on post

salbutamol challenge test

Exclusion Criteria Patients with severe uncontrolled hyperglyceamia, uncontrolled hypertension,

cardiac arrhythmia, impaired hepatic or renal function, history of malignancy or stroke, smoking, chronic alcoholism, any other serious disease requiring active treatment and treatment with any other herbal supplements and pregnant and lactating women.

15CONFIDENTIAL

Sensoril® - Cardiovascular/Endothelial Dysfunction Clinical Study – Demographics & Dosage Regimen

N

Parameter PlaceboSensoril250 mg

Sensoril500 mg Atorvastatin

N 20 20 20 20

Age57.45±8.85 55.40±8.07 57.30±9.40 56.95±8.04

Sex (M/F)12/8 14/6 13/7 13/7

Weight (Kg)66.09±5.56 68.07±6.51 67.30±6.16 68.56±8.47

BMI (Kg/m2)24.82±1.86 24.89±2.03 25.01±2.92 26.02±3.12

Duration: 12 weeks

Sensoril®: 250mg and 500 mg twice daily

Atorvastatin: One capsule of Atorvastatin 10mg (night) and one capsule of Placebo (morning)

Placebo: One capsule of placebo twice daily

16CONFIDENTIAL

Sensoril® treatment improves endothelial function

17

Refelection Index: A salbutamol challenge test employing digital volume plethysmography was used to assess endothelial function as reported by Chowienczyk et al (J Am Coll Cardiol, 34(7):2007-2014, 1999) and Naidu et al (Indian J Pharmacol,39:168-169, 2007)

CONFIDENTIAL

Sensoril® treatment improves endothelial function

****

**

*

**

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**

CONFIDENTIAL

Sensoril® treatment reduces cardiovascular risk factors

****

**

*

**

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**

CONFIDENTIAL

Sensoril® treatment reduces cardiovascular risk factors

****

**

*

**

20

**

CONFIDENTIAL

Sensoril® treatment reduces cardiovascular risk factors

****

**

*

**

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**P=<0.05

CONFIDENTIAL

Sensoril® treatment reduces cardiovascular risk factors

****

**

*

**

1% C

hang

e

22

** 3

1 p<0.01 when compared to Placebo2 p<0.001 when compared to Placebo3 p<0.001 when compared to Placebo

CONFIDENTIAL

Sensoril® - Clinical Study - Mental Cognition in Stable Bipolar Patients – University of Pittsburgh

Study Design: As per IND with US FDA Double blind, randomized, parallel group, adjunctive treatment, 30

subjects in each group.j g p Sensoril® vs. Placebo, added to ongoing maintenance bipolar

medications

Dosage Regimen 250 mg first week, once a day, increased to 250 mg twice daily, for rest

of the study Total Duration: 8 weeks

Funded by NARSAD (NY based sponsor of studies in bipolar and schizophrenic NARSAD (NY based sponsor of studies in bipolar and schizophrenic

patients)

Accepted for publication in the Journal of Clinical Psychiatry

23CONFIDENTIAL

Sensoril® - Cognition Study - Demographics

Group 1 (n=30) Group 2 (n=30)

Age (years) Mean SD Mean SD

46.9 10.38 45.93 10.40

Gender Male Female Male Female

13 17 10 20

Race White

AfricanAmerican

Other WhiteAfricanAmerican

Other

21 9 0 18 11 1

EducationHigh School or Less

University OtherHigh School or Less

University Other

7 22 1 8 20 27 22 1 8 20 2

Marital StatusSingleMarried Divorced/Separated

13610

13710

24CONFIDENTIAL

Cognition study results

Effect Size - Sensoril 500 mg/day

Cohen’s “d” Effect Size

a b

a b

0.2 Small

0.5 Medium

0.8 Large

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Working memory – Digit Span Backwards: (a) number correct backward; (b) span backward Social Cognition - Penn Emotional Acuity Test: mean response rating; Attention - Flankers Continuous Performance Test: (a) RT neutral mean; (b) RT neutral median

CONFIDENTIAL

Cognition study results –Adverse EventsSensoril Study- Adverse Events by Event (n=60) 19 Events in Sensoril (n=30) 26 Events in Placebo (n=30)

Nervous System Sleepiness 3 6 headache 0 2

i id d / i ht 3 3 vivid dreams/nightmares 3 3 dizziness 1 0 tiredness/fatigue 1 2 lightheadedness 0 1Gastrointestinal nausea/upset stomach 1 4 constipation 0 2 diarrhea 5 1 decrease in appetite 0 1 increase in gas 1 0Dermatological rash 1 1 itching 0 1Cardiovascular palpitations 0 1Circulatory swelling in feet 1 0 heaviness in legs 0 1 tingling in fingers 0 1Psychiatric increase in depression 1 1Genitourinary frequent urination 1 0Respiratory

26CONFIDENTIAL

Effect size of Latuda® and Lexapro® from Literature:

Latuda(L id )(Lurasidone)

Lexapro(Escitalopram)

10 mg/day

40 mg/day

120 mg/day 20 mg/day

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NOTE: The data shown here is not cognition data and presented only to give an idea about the effect sizes with psychiatry drugs

CONFIDENTIAL

Cognition Study - Summary

The effect sizes for working memory (digit span test), social cognition (using the PENN emotional acuity test), or attention (using the Continous Performance Test – Flankers version) were in favor of Sensoril over placebo.

Medium to close to large effect sizes for cognition studies in psychiatry are not at all common; thus for a 2 month study with Sensoril® at a 500 mg/day the results are encouraging.

When add on drugs are administered along with CNS drugs, there are often more side effects; however when Sensoril® was given are often more side effects; however, when Sensoril® was given along with other CNS drugs in this 2 month study, the side effect profile was quite benign.

Study was done as per IND filed with US FDA and not funded by Natreon.

28CONFIDENTIAL

Effect of Sensoril® on Cold Pressor-inducedCardiovascular Changes in Healthy Volunteers

Study Design: Randomized, double-blind, placebo-controlled, crossover

Subjects: 20 healthy male subjects

Dosage: 2 x Sensoril® 250mg BID2 x Matching placebo capsules BID

Duration of treatment: 2 weeks

29CONFIDENTIAL

Effect of Sensoril® on Cold Pressor-induced Change in Aortic Pressure

Mean % Change in Aortic Pressure Induced by Cold Pressor Test with Sensoril® & Placebo

ress

ure,

mm

Hg

n %

Cha

nge

30CONFIDENTIAL

Aor

tic P

Mea

n

Data Expressed as Mean ± SD

Effect of Sensoril® on Cold Pressor-induced Change in Augmentation Index

Mean % Change in Augmentation Index Induced by Cold Pressor Test with Sensoril® & Placebo

enta

tion

Inde

x n

% C

hang

e

31CONFIDENTIAL

Aug

me

Mea

n

Data Expressed as Mean ± SD***p<0.001 Compared to Pretreatment

***

Effect of Sensoril® on Mental Stress-inducedCardiovascular Changes in Healthy Volunteers

Study Design: Randomized, double-blind, placebo-controlled, crossover

Subjects: 20 healthy male subjects

Dosage: 2 x Sensoril® 250mg BID2 x Matching placebo capsules BID

Duration of treatment: 2 weeks

32CONFIDENTIAL

Effect of Sensoril® on Mental Stress-induced Change in Aortic Pressure

Mean % Change in Aortic Pressure Induced by Mental Stress Test with Sensoril® & Placebo

c Pr

essu

re,

mm

Hg

ean

% C

hang

e

33CONFIDENTIAL

Aor

tic Me

Data Expressed as Mean ± SD*p<0.05 Compared to Pretreatment

*

Effect of Sensoril® on Mental Stress-induced Change in Augmentation Index

Mean % Change in Augmentation Index Induced by Mental Stress Test with Sensoril® & Placebo

men

tatio

n In

dex

an %

Cha

nge

**

34CONFIDENTIAL

Aug

mM

e

Data Expressed as Mean ± SD**p<0.01 Compared to Pretreatment

Effect of Sensoril® on Psychomotor Performance in Healthy Volunteers

Study Design: Randomized, double-blind, placebo-ll d controlled, crossover

Subjects: 20 healthy male subjects

Dosage: 2 x Sensoril® 250mg BID2 x Matching placebo capsules BID

35CONFIDENTIAL

Duration of treatment: 2 weeks

Effect of Sensoril® on Psychomotor Performance in Healthy Volunteers

Test  Reaction Time with Sensoril®, ms Reaction Time with Placebo, ms 

Pre Treatment Post Treatment Pre Treatment Post Treatment 

Finger tapping  187±16 183±17 (ns) 192±14 194±14 (ns) 

Simple reaction test  306±39  287±43 **  313±45  316±26 (ns) 

Choice discrimination test  471±34  455±51 *  476±34  477±30 (ns) 

Digit Symbol Substitution Test  1695±263  1557±233 **  1690±279  1739±313 (ns) 

Digit vigilance task  1360±57 1317±88 ** 1371±79 1395±82 (ns) 

Card sorting test  96±19  84±13 *  98±17  98±20 (ns) 

         

      P value compared to baseline in Reaction Time

36CONFIDENTIAL

p

      ns – Not significant;   * p<0.05;    ** p<0.001 

 

Effect of Sensoril® on Pain Threshold Force and Threshold Time in Healthy Volunteers

Mechanical Pain Model

Placebo Sensoril® p value

Baseline threshold force (grams)

443.06 ±65.02 450.3 ± 75.49 ns

Post Drug threshold force (grams)

446.53 ±65.95 538.5 ± 72.27 <0.05

Baseline threshold Time (seconds) 5.54 ± 0.81 5.62 ± 0.94 ns

Post Drug threshold Time (seconds) 5.58 ± 0.82 6.73 ± 0.90 <0.05

All values - Mean ± Standard deviationp values represent the students paired t test between the placebo and Sensoril® group

Effect of Sensoril® on Pain Threshold Time Healthy Volunteers Hot Air Pain Model

80

100

20

40

60

Tim

e in

sec

ond

s

baseline

postdrug

p<0.001 $

p-ns*

43.79 ± 6.79 43.99 ± 6.9344.28 ± 9.19 49.89 ± 7.07

0

20

Placebo Sensoril

Values given as Mean ± SD* compared to baseline$ compared to baseline and placebo

Sensoril® - Summary Reduces stress, fatigue and sleeplessness and increases energy

Improves endothelial function and lipid profile in Type 2 diabetics

Improves mental cognition without significant adverse events

Decreases hsCRP, a biomarker for inflammation.

Backed by 7 clinical studies, self-affirmed GRAS, Pre-DSHEA

No complaints in 9 years of world-wide market history

39CONFIDENTIAL

Ashwagnadha

Analytical Methodology

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Current Specifications of Bioactives in Sensoril® brand Ashwagandha

Withanolide glycosides (combined Withanoside Iv, With id V With lid A t ) ≥ 10 0%Withanoside V, Withanolide A, etc.) : ≥ 10.0%

Withanolide aglycones as Withaferin-A : ≤ 0.5%

Oligosaccharides : ≥ 32.0%

41CONFIDENTIAL

Current HPLC Method - Quantification of Withanolide Glycosides and Withaferin-A

HPLC Condtions:Merck-Hiber® Pre-Packed column RT 250-4, LiChrosorb® RP-18, Particle

size 5μM, 4 x 250 mm cartridge column, Ord No.1.50333.0001 with a

reverse phase guard column.A bi tAmbient

Acetonitrile: Water-1: 1 (v/v)

0.6 ml/min

20 min

Isocratic

UV 225 nm

20μl (with a loop injector)

Waters HPLC 515 PDA Detector (Waters™ 2996, Photodiode Array

Detector), evaluation with Empower Software

Pdt. No. UN-1648 Acetonitrile (Merck), Pdt No. 93956 Water for HPLC

(Merck)

42CONFIDENTIAL

Method with external standard; evaluation of area of peaks.

Withaferin A isolated from Withania somnifera by multiple column

chromatography was used as external standard. Withaferin A, 0.5 mg was

dissolved in 1 ml of methanol and four different concentrations were

applied to HPLC. Calibration curve was plotted between area and different

concentration. The linear regression equation of the calibration curve was

obtained as follows:

Y = 2217082.726 X+ 200001.715 with a correlation coefficient of 0.9998.

Where Y is the area and X is the concentration in g.

Current HPLC Method - Quantification of Withanolide Glycosides and Withaferin-A

Withaferin-A reference standard, developed in-house, is used for both aglycone as well as glycoside estimation

The peak appearing at tR 7.5 min is considered that of With f i AWithaferin-A

Peaks appearing at tR 2.0-7.4 min are considered those of withanolide glycosides.

U 1 2 0

1 .4 0

1 .6 0

1 .8 0

2 .0 0

2 .2 0

2 .4 0

2.86

5

43

AU

0 .0 0

0 .2 0

0 .4 0

0 .6 0

0 .8 0

1 .0 0

1 .2 0

Min u te s

2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0

Pea

k2 -

3.5

81

4.85

4

5.66

8 7.55

9

8.67

2

10.3

39

Current HPLC Method - Quantification of Oligosaccharides

HPLC Conditions:

Equipment Waters HPLC system consisting RI detector Waters 2414, pump Waters 515,

evaluation with Empower software.

C l C b h d t A l i C l [ t ] 300 3 9 P t N WAT 084038Column Carbohydrate Analysis Column [waters] 300 x 3.9 mm, Part No.WAT 084038

Column Temperature

Ambient

Eluent Acetonitrile: Water in the ratio of 80:20 (v/v)Run Time 10 minFlow rate 2 ml/minGradient

ProgrammeIsocratic

Injection volume 20μl (with a loop injector)Sensitivity 16

Reagents Pdt. No. UN-1648 Acetonitrile (Merck)Pdt. No. 93956 Water for HPLC (Merck)

Evaluation Method with external standard

44

Evaluation Method with external standard.

External

Standard

Oligosaccharide isolated from Withania somnifera by multiple column chromatographies was used as external standard. Oligosaccharide 10 mg was weighed and dissolved in 1 ml of water. Four different concentrations (10g, 50g, 100g and 200g in 20l) were applied to HPLC. Calibration curve was plotted between concentration and area. The linear regression equation was obtained as follows.Y = 6620.5 X+ 72099 with a co-relation coefficient of 0.996where Y is the area and X is the concentration in g.The area of peaks appearing between 4-10 minutes is addedand contents of oligosaccharides are calculated using linearregression equation

New HPLC Method

Reference standards of Glycosides (Withanoside-IV, Reference standards of Glycosides (Withanoside IV, Withanoside-V, Withanolide-A, Withanoside-UV active) and Aglycones (Withaferin-A, Withastramonolide, Withanone) were obtained from ChromaDex.

Withanoside IV is used to calculate the total withanolideglycoside content

Withaferin-A is used to calculate the total aglycone content

45CONFIDENTIAL

New HPLC Method – HPLC ConditionsHPLC conditions:

System : Alliance-Water (PDA 2996)

System number : NAT/HPLC/03

Method : Sensoril Gradient systemy

Column : Lichrocart (250 × 4 mm, 5 μm Merck) [NI/LC/RPC18/005[250×4]]

Solvent system : Gradient; (A) Acetonitrile (B) Water

Detection : 225 nm (PDA detector)

Flow rate : 0.5 ml/min

Run time : 30 min

HPLC programming for mobile phase is given below:

Table 1: Gradient system of solvent-A and solvent-B

Time (Min.) Solvent A (%) Solvent B (%)0 10 90

4.0 20 80

46

7.0 80 2010.0 70 30

15.0 60 4017.0 100 022.0 100 025.0 20 8030.0 10 90

 

New HPLC Method - Sensoril® HPLC Chromatogram

47

New HPLC Method – Retention Times of Standards

Sample Retention Time (Min.)

Withanoside-IV 11.20

Withanoside-V 11.96

Withanoside-UV-active 11.23

Withaferin-A 13.08

Withanone 14.42

48

Withastramonolide 13.64

 

New HPLC Method – Calibration Curve for Withanoside IV

49

New HPLC Method – Calibration Curve for Withaferin-A

50

Current HPLC Method vs New HPLC Method

Batches

Sensoril Monograph Method New HPLC Method

Glycoside

(% w/w)

Aglycone

(% w/w)

Glycoside

(% w/w)

Aglycone

(% w/w)

WS1307165 10.49 0.22 13.77 0.53

WS1311173 9.46 0.214 15.13 0.73

WS1311174 10.42 0.218 21.90 1.01

WS1311175 12.54 0.29 17.53 0.79

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WS1311176 12.61 0.2 16.73 0.72