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The Story of ProtectinTM: A New Class of Nutriceutical
Douglas J. Pousma, MD18 June 2003
The Story of ProtectinTM
•Background•Uses of NSAIDs •Mechanism of action of ProtectinTM and NSAIDs
•Discovery of ProtectinTM
•Efficacy and Safety in Cells, Animals, and Humans•Discussion
Uses of NSAIDs•Pain, especially pain secondary to inflammation, but also due to menstrual cramps and migraine headaches•Osteoarthritis•Rheumatoid Arthritis•Systemic Lupus Erythematosus•Scleroderma•Psoriasis•Prevention of coronary artery disease, stroke, TIAs•Prevention of FAP and colorectal CA
•Giovannucci et al., NEJM 1995; 333:609-14•Smalley, et al., AIM 1999; 159:161-66
•Alzheimer’s Disease (up-regulated COX2 expression)•Ho, et al., Arch Neurol 1998;58:487-92
40M in 1995, 60M in 2020
Inflammation
Mast cell activation, non-immune host defense, stretch/pressure receptors
Arachidonic Acid (ARA)
COX 1,2 Thromboxane and Prostaglandins *
HETEs and Leukotrienes *
5-LO
* These eicosanoids are highly pro-inflammatory, pro-bronchospastic, and pro-vasodilatory. TXA2 is a potent platelet aggregator. Leukotriene B4 (LTB4) has potent chemotactic (PMNs, eos) and chemokinetic (vasodilation, vasopermeability) effects.
Inflammation
Arachidonic Acid (ARA)
Cox 1, 2
Thromboxane and Prostaglandins
HETEs and Leukotrienes
5-LO
X NSAIDS
NSAIDS generally only inhibit COX 1 and 2 enzymes
Biochemical Model: IC50 Values (mcg/ml/unit)
Molecule COX 1 COX 2 COX2 Selective
Free-B-Ring Flavanoid (pure) 0.44 0.28 YesFree-B-Ring Flavanoid 83% 0.32 0.42 NoFlavans (pure) 0.11 0.42 NoFlavans (50%) 0.17 0.41 NoProtectinTM 0.20 0.40 NoIbuprofen 0.42 3.60 NoNexrutineTM 0.30 >500 Nocelecoxib 0.43 0.004 Yes
What is ProtectinTM? How was it Discovered?
ProtectinTM is a patented, proprietary blend of free B-ring flavanoids & flavans that inhibits COX1, COX2,
AND 5LO. 1230 plant extracts 22 COX2 inhibitors (1.8%)
In vitro assay COX1, COX2, PLA2, and 5LO challenges
Genomics testing
ProtectinTM
The “Flavan Factor”
Q: If early biochemical tests showed the flavan extract inhibited 5LO, would ProtectinTM also inhibit this enzyme?
A: Yes, the IC50= 1.38 mcg/ml/unit enzyme.
The “Flavan Factor”
Q: If ProtectinTM inhibits 5LO, does it inhibit leukotriene B4?
A: Production of newly-induced LTB4 was completely inhibited by ProtectinTM in a monocyte line that expressed COX 1, COX 2, and 5LO (IC50 = 1.5 mcg/ml).
Inflammation
StimuliArachidonic Acid (ARA)
Cox 1, 2
Thromboxane and Prostaglandins
HETEs and Leukotrienes
5-LO
X NSAIDS and ProtectinTM
X ProtectinTM
ProtectinTM effectively inhibits both COX 1 and 2 and 5-lipo-oxygenase.
The Effect of ProtectinTM on Intracellular LTB4
0
20
40
60
80
100
No Protectin Protectin 3mcg/ml
Ibuprofen 3mcg/ml
Day 0
Day 2
Percent LTB4 remaining within monocytes per ELISA. Note the nearly 80% and 60% decreases in LTB4 content with ProtectinTM compared to controls and ibuprofen (another known 5-LO inhibitor), respectively.
Cellular Toxicity
•The percent LDH release induced by ProtectinTM was negligible and similar to ASA and ibuprofen
•The percent LDH release induced by celecoxib (CelebrexTM) and indomethacin exceeded 100% at inhibitor concentrations of 100 mcg/ml. The percent LDH released induced by ProtectinTM at 100 mcg/ml = 0%.
ProtectinTM Efficacy in Animals
0
5
10
15
20
25
No Tx
Indomethacin
Protectin 50mg/kgProtectin 100mg/kgProtectin 200mg/kg
Ear swelling (mm change) after ProtectinTM 50, 100, and 200 mg/kg in olive oil or olive oil alone PO x 1 day after an ARA (or EtOH) “ear rub” in mice
ProtectinTM Efficacy in Animals
0
2
4
6
8
10
12
14
16
18
Negative ControlVehicle ControlNo Tx + ARAProtectin + ARA
Ankle swelling (mm change) after ProtectinTM 50, 100, and 200 mg/kg in olive oil or olive oil alone PO x 1 day after an intra-articular injection of ARA
ProtectinTM Safety in Animals•Chronic Administration of ProtectinTM 90 mg/kg (equivalent to 500 mg HDD), 450 mg/kg (5X HDD), and 900 mg/kg (10X HDD) PO x 28 days OR acute administration of ProtectinTM 2 grams/kg (20X HDD of 500 mg) PO x 14 days in mice resulted in no significant changes in CBC, ALT (SGPT), AST (SGOT), total bilirubin, alkaline phosphatase, GGT, total protein, albumin, globulin, AG ratio, cholesterol, Na+, K+, Na+/K+ ratio, BUN/Cr, phosphorus, Ca++, Ca++/Phos, glucose, osmolality, Cl-, CPK, and pre/post-prandial bile.
•No significant changes in weight gain, appearance, behavior.
•Gross necropsy of stomachs, livers, and duodenums revealed no histological abnormalities or changes compared with controls.
Drug Interaction Data (P450)
Types of drug interactionsCytochrome P450 (CYP450)
constitutes a group of hepatic enzymes that cause oxidative metabolism of endogenous and exogenous molecules.
CYP450 profiling is now standardCYP450 is comprised of 5 major
isoenzymes; CYP3A4 does half of all the detox “work”.
CYP450 Inhibition by ProtectinTM
CYP Isoform Reference Compound
% Inhibition by ProtectinTM
1A2 Furafylline 23
2C9 Sulfaphenazole 11
2C19 Tranylcypromine 16
2D6 Quinidine 15
3A4 Ketoconazole 11
Human Clinical TrialMulti-center, 3-month, prospective,
randomized, double-blind, placebo-controlled study in which 60 human subjects were given either placebo, CelebrexTM 200 mg PO q day, or ProtectinTM 250 mg or 500 mg PO q d (15 per group).
Exclusions included stroke, TIA, MI, USA, angina pectoris, CABG, h/o GI perf/bleed, symptomatic PUD, kidney dz, brittle DM, anyone taking NSAIDs or COX2 inhibitors, and anyone taking H2 blockers or proton pump inhibitors.
MetricsTest Baseline 30 days 90 days
Platelet aggregation
+ + +
24H urine Cr + + +
ESR + + +
RA + +
C-reactive protein
+ + +
Chemistry + + +
More Metrics
Test Baseline 30 days 90 days
HCT + + +
TNF-alpha + +
IL-1B + +
Glucose + + +
Fructosamine + +
Pregnancy + + +
Fecal Hemoccult
+ + +
ResultsEfficacy per SF-36 short form and
Western Ontario & McMaster Universities Osteoarthritis (WOMAC)
index:
Protectin 500 mg +19% P < 0.03 vs. placebo
P < 0.05 vs. Celebrex
Protectin 250 mg +18% P < 0.04 vs. placebo
Celebrex 200 mg +13%
Placebo +8%
Safety in Human Clinical Trial
No adverse events after physical examination and no significant laboratory changes after 12 weeks.
The Differentiator
Arachidonic Acid (ARA)
Cox 1, 2
Thromboxane and Prostaglandins
HETEs and Leukotrienes
5-LO
X NSAIDS and ProtectinTM
X ProtectinTM
What is the importance of “dual pathway” inhibition?
Stimuli
The Differentiator
A true “dual pathway inhibitor” prevents the most
common side effects of single pathway, COX 1 OR
COX 2 inhibitors.
The DifferentiatorLeukotrienes represent the primary ARA metabolites within the gastric mucosa following prostanoid inhibition.
Kricher, et al., “Prostaglandins, leukotrienes and essential fatty acids”, 1997;56:417-23
Celotti and Laufer, Pharmacol Research 2001;43:429-36
Near complete inhibition of the COX 2 pathway (eg with Rx COX-2 inhibitors) has resulted in shunting ARA down the 5-LO pathway, leading to increased production of leukotrienes and serious upper airway and other side effects.
Conclusions
•COX 2 inhibitors do NOT spare the kidney and GI tract, as marketed.
•An oral anti-inflammatory that significantly inhibits 5-LO represents a significant breakthrough.
Conclusions•Through novel, plant-based biochemical, proteomic, and genomic modeling, Oasis has discovered and patented the only dual-pathway anti-inflammatory molecule.
•Cell, animal, and human clinical data reveal acceptable and compelling efficacy, safety and drug interaction data.
•Currently available data make commercial release of ProtectinTM acceptable.
Conclusion
ProtectinTM represents a new class of nutraceutical for the treatment of
inflammatory states.