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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: [email protected]: 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
2CONFIDENTIAL
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.
5CONFIDENTIAL
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
6CONFIDENTIAL
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
7CONFIDENTIAL
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.
8CONFIDENTIAL
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
Clinical study results: Stress markers% Reduction in serum cortisol % Reduction in C-reactive protein
% Improvement in serum DHEAS
Statistical significance
11
• Day 0 vs 60 day; ANOVA• 250 mg & 125 mg BID: p
Clinical study results: Effects on VLDL, hemoglobin & glucose
% Reduction in VLDL % Improvement in Hemoglobin
% Reduction in glucose
Statistical significance
13
Statistical significance• Statistical significance only with 250 mg BID • One way ANOVA; p
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 weeksSensoril®: 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
****
**
*
**
18
**
CONFIDENTIAL
Sensoril® treatment reduces cardiovascular risk factors
****
**
*
**
19
**
CONFIDENTIAL
Sensoril® treatment reduces cardiovascular risk factors
****
**
*
**
20
**
CONFIDENTIAL
Sensoril® treatment reduces cardiovascular risk factors
****
**
*
**
21
**P=
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
25
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
27
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
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
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
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
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
40
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 s2 .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 -
3.58
1
4.85
4
5.66
8 7.5
59
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 084038Column
TemperatureAmbient
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 3015.0 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
51
WS1311176 12.61 0.2 16.73 0.72