Learn how a poly-pharmacy of compounds found in herbal medicine influence the body's response and how in turn, the potential of synergistic activity might be understood.

1. PhytoPharmacology
An evidence-based
perspective
Dr. Michael Tims
Academic Director, Herbal Program
Maryland University of Integrative Health

2. What today will cover
 How science measures medicine in herbal products
 Pharmacokinetics
 Pharmacogenetics
 Synergy
 Pharmacodynamics
 Clinical Trial
 Standardized Reporting

4. Paper Chromatography
 Compounds dissolve in a
solvent
 Mobile & stationary phase
 Interact uniquely with the
stationary phase
 Retention characteristics
- elution order

5. Plant Identification
Sambucus canadensis
Polyphenolics Anthocyanins
Sambucus nigra
Jungmin Lee and Chad E. Finn, (2007) Anthocyanins and other polyphenolics in American elderberry (Sambucus canadensis) and European
elderberry (S. nigra) cultivars, J. Sci. Food Agric., 87:2665–2675

6. Pharmacokinetics

7. Xenobiotic Metabolism
Age
Genetic variation
Renal
Hepatic function
Disease
Smoking
Diet

8. Pharmacokinetics
Phase I - a variety of enzymes act to introduce reactive
and polar groups into their substrate
Phase II reactions
 Conjugates endogenous hydrophilic substance (acetyl,
methyl, etc…)
 Increases water solubility and thus excretion
 Reported as elimination half-life

9. Absorption
(A) plasma before consumption of
elderberry extract (1.2 μg of anthocyanins)
(B) plasma 30 min after
(C) plasma 60 min after
(D) elderberry extract containing 1.2 μg of
anthocyanins
Cao, G. and Prior, R.L. (1999) Clinical Chemistry, 45: 574-576

10. Plasma Concentration-Time
Profile

11. Two Formulations Compared
MTC = min. toxic conc.
MEC = min. effective conc.

12. Pharmacogenetics / -genomics
 > 50% of drugs don’t work in 50% of people
 Lab animal studies offer little genetic variability
 More than 20 human enzymes associated with drug
metabolism have polymorphisms.
 N-acetyltransferase
 S-methyltransferase
 Cytochrome p450 oxidative enzymes.

13. Metabolic Acetylation
 Liver inactivates metabolites by attaching 2-carbon acetyl
group donated by acetyl co-enzyme A
 The caffeine clearance diagnostic technique in saliva
 60% Caucasians, 20% Asians are slow acetylators
Galbraith, Bullock & Manias 2004

14. Cytochrome P450 Isozymes
CYP → Phase I enzymes
CYP2 → C P450’s
CYP2D → Subfamily
CYP2D6 → Single enzyme
Largely in endoplasmic reticulum of Liver

15. CYP2D6
 Over 100 drugs are metabolized
 Sparteine - alkaloid from Cystisus scoparius (scotch
broom) probe for CYP2D6 polymorphism
Metabolic activity (where extensive = ‘normal’)
 Poor
7.5% White Americans
< 2% Asian & African Americans
< 1% Polynesians

16. Example #1 Kava
Therapeutic drug monitoring required for drugs with a
narrow therapeutic window.
CYP2D6 Metabolism Effect
PM type plasma [ ] ↑ Drug Toxicity
EM type plasma [ ] ↓ Therapeutic Failure

17. Example #2
Codeine
Pro-drug
CYP2D6 PM types never achieve therapeutic plasma levels
→ o-demythylation
Phase I
biotransformation
Morphine
Active

18. Synergistic Effects

19. Williamson, E.M. 2001. Synergy and other interactions in phytomedicines. Phytomedicine 8:401-409.
Nociceptive Flexion Reflex
A physiological tool to study spine pain sensitivity

20.  Multiple receptors may be influenced by different
molecules in whole plant extract
 Multiple gene signaling pathways triggered
 Cellular receptor or transmembrane signaling altered at a
non-receptor site
 Antioxidants stabilize activity of other compounds
 Enhance bioavailability of other compounds - Piper
nigrum, Black Pepper
 Complex mixtures lead to individual variation in elimination
half-life
Synergistic Effects of Multiple
Constituents

21. Ephedra Pharmacokinetic Data
 Ephedrine – vasoconstrictor (BP ↑ and HR↑)
 Pseudoephedrine - bronchial dilator and relieve nasal
congestion
 Compared
 Powdered Ma Huang (Ephedra sinensis)
 Extracted ephedra in formula
 Ephedrine in tablet
 Ephedrine in solution
White, LM et al (1997) Pharmacokinetics and cardiovascular effects of Ma-Huang
(Ephedra sinica) in normotensive adults, J. Clin. Pharmacol. 37: 116-122.

22. Ma Huang (Ephedra sinensis)

23. Study Design
 12 normotensive patients (6 women)
 non-smokers
 age 23-40
 no medications known to ↑ either BP or HR
 caffeine consumption same for both phases
 Blood (5ml) taken 0, 0.25, 0.5, 0.75, 1.0, 2.0,
4.0, 6.0, 9.0 hours after 8:00 am.

24. Two Phase Study
 Phase I (control) – took BP every 15 min. 7:00 am –
8:00 pm
 Phase II (treatment) - took BP for same time period
 8:00 am treatment with light breakfast
 5:00 pm treatment with light dinner
 Investigators administered all capsules and meals
 Participants were moderately mobile, stayed on site
 Extraction and HPLC measurement of blood sera
(blind) and Ephedra capsules

25. Absorption Results
Ephedrine treatment Ka (hr-1) Tmax (hr)
Powdered herb 0.49 3.9
Extracted ephedra in formula 1.36 2.8
Ephedrine in tablet 1.73 1.69
Ephedrine in solution 2.35 1.81
Ephedrine was the only alkaloid detected
Ka = rate of absorption
Tmax = how long it stayed around

26. Pharmacodynamics
Pharmaceutical Model

27. Low cost biochemical and cellular assays
• Detect bioactive compound in crude extract
• Guide fractionation to purified bioactive compound
• Biological characterization of mechanism
• Isolated organ or intact cells

28. But….
What does one lose by throwing out the context in
which the target operates?

29. An iterative process
Low cost biochemical and cellular assays
Expensive animal models
• Identifies new targets and/or chemical novelty
• Toxicology
• Therapeutic blood levels
↓

30. Low cost biochemical and cellular assays
↓
Human clinical trials
Expensive animal models
↓

31. Clinical Trials
Efficacy vs. Effectiveness Along a Continuum
 Efficacy trials (explanatory trials) determine whether an
intervention produces the expected result under ideal
circumstances.
 Effectiveness trials (pragmatic trials) measure the degree of
beneficial effect under “real world” clinical settings.
Gartlehner G, Hansen RA, Nissman D, et al. (2006) Criteria for Distinguishing
Effectiveness From Efficacy Trials in Systematic Reviews. Technical Reviews,
No. 12. Rockville (MD): Agency for Healthcare Research and Quality (US).

32. Clinical Significance/Relevance
 Selection of study participants
 Selection of outcome measures
 Establishing an evidence picture
* What special considerations, if any, do we need
for herbs?

33. Clinical Trial Study Design
 E. angustifolia root preparations were developed from a
single lot
 Root material was extracted with either
 supercritical carbon dioxide
 60 % ethanol
 20 % ethanol
 Placebo: mixture of alcoholic beverage, denatonium
benzoate (bitter flavor) and water

34.  Treatments given 3x each day as 1.5-ml tincture
 Single dose contains equivalent of 300 mg
echinacea root
 Based on German Commission E monograph

35.  The 437 volunteers were randomly assigned in blocks to
receive one of the seven treatment regimens
 Participants and all study staff were blinded to the group
assignments until all data had been collected and
transmitted to study statistician

36. Seven Possible Treatment
Assignments
1. CO2 extract during both phases
2. 60 % extract during both phases
3. 20 % extract during both phases
4. Placebo during prophylaxis phase and CO2 extract
5. Placebo during prophylaxis phase and 60 % extract
6. Placebo during prophylaxis phase and 20 % extract
7. A control group received placebo throughout both
phases of the study period

37. Getting Sick, Experimentally
 Volunteers took assigned study medication as
outpatients on days -7 to 0
 On day 0, all asymptomatic volunteers were challenged
with rhinovirus type 39
 They were isolated in individual hotel rooms for the
remainder of the study
 Second phase of dosing regimen if effect on day 0

38.  Between virus challenge and morning of day 5, symptom
scores evaluated morning and evening
 A nasal lavage was performed each morning after
symptom scoring was completed
 Approximately three weeks after the virus challenge, all
volunteers returned to the study site for testing for
antibody to rhinovirus type 39

40. Effect of Echinacea on
Rhinovirus-induced Inflammation
 Assessed by measurement of interleukin-8 and
polymorphonuclear-leukocyte concentrations in nasal-
lavage specimens
 Neither prophylaxis nor echinacea treatment had a
significant effect
 Significant correlation between the interleukin-8 and
polymorphonuclear-leukocyte responses and symptom
severity

41. So what’s bad about this study?
Previously, Cochrane reviews and meta-analysis
demonstrated positive responses for echinacea

42. It’s All in the Dose
• Dosage administered in the trial may have been too low
to produce an appreciable benefit
• 1.5 ml extract 3 times daily equivalent to 900 mg / day of
dried root of E. angustifolia.
• Dose based on German Commission E monograph
• Monograph mentions E. pallida root, not E. angustifolia
root
• WHO and Canadian Natural Health Products directorate
recommend a dosage of 3000 mg / day of dried E.
angustifolia root

43.  When asked to comment , trial co-author David
Gangemi wrote, “I would like to see results from
a higher dosage group, but NIH funding
limitations didn't allow for this…”

44. Loading Dose
Recent dosing research dovetails nicely with in
traditional literature on Echinacea dosing
• 1st day 5 mL for 8 doses (8000 mg/day)
• Subsequently 5 mL 3x/day (3000 mg/day) for six days

45. Echinacea Immunologic Activity
 Cichoric acid (phenolic) – hydrophyllic
 Alkylamides (lipid) – hydrophobic
 High ethanol ratio extracts of Echinacea purpurea root and
leaf produce consistently high levels of alkylamides.
 Arabinogalactan proteins (AGPs) – Bioavailability?
 Only low alcohol (< 40%) or aqueous root extracts contain
 European products contain aerial portions, while
American products contain aerial and root parts.

46. Cannabinoid Receptors
 Alkylamides identified as cannabinoid ligands
 Not the neuronal receptors associated with
psychotrophic effects of Cannabis
 Responsible for immunomodulatory responses
at numerous immune cells
 Points to dependence of immunological “tone” of
system being treated

47. Consolidated Standards of
Reporting Trials (CONSORT)
http://www.consort-
statement.org/extensions/interventions/

48.  Latin Binomial
 Product Name
 Plant Part
 Type of product (fresh vs. dry)
 Voucher specimen
 Extract Concentration
 Authentication Method
 Dosage/Duration – rationale
 Filler
 If standardized, quantity of active/marker compounds
 In standardized product - rationale for choice of active
 Chemical fingerprint and method used
 Purity testing
 Rationale for placebo
 Practitioner training and experience