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The Use of a Complex Flavonoid as a Treatment for ALSElijah W. Stommel*, Brent T. Harris∞, David J. Graber ∞, Jeffrey A. Cohen*, Department of Medicine (Section of Neurology) *

Department of Pathology∞ Dartmouth-Hitchcock Medical Center (DHMC), Lebanon, NH 03756Tel: 603-650-8615 Fax: 603-650-0458 Email: elijah.w.stommel@hitchcock.org

AbstractWe propose using sublingual LutiMax®, a molecular complex of the bioflavonoids (luteolin and rutin) that target many of the pathogenic mechanisms thought to be important in ALS. LutiMax® was introduced into commerce as a dietary supplement worldwide in 2002 by SYNORx, Inc. There is substantial evidence to show that bioflavonoids are neuroprotective by reducing neuroinflammation, reducing pro-inflamatory cytokines and chemokines and reducing neuronal apoptosis, all of which suggests an important role for flavonoids in counteracting neurodegeneration. LutiMax® has no known major side effects. We have followed a relatively small number of ALS patients who appear to have stabilized once they started taking LutiMax®. This unexpected observation needs to be verified with a formal clinical trial. Patients will be evaluated using the ALS Functional Rating Scale (ALSFRS) as a primary outcome with secondary outcomes being, survival, safety and FVC measured every 3 months. With this phase II trial, 60 ALS patients will be entered in a double-blind, placebo-controlled, cross-over study. We plan to use LutiMax® for 12 months vs. placebo in a cross-over with a latin square technique where the patient will be switched from real medication to placebo or visa-versa at six months. A wash out period of a week between treatments will occur. Assuming 80% complete both periods (i.e., receive both placebo and treatment) the study will have sufficient power (80%) at a type I error rate of 5% to detect an effect of treatment such that 75% of patients have a better ALSFRS score following treatment, than following placebo. The patients will have to meet strict inclusion and exclusion criteria for the study including being able to ingest the pills sublingually and having been diagnosed with the disease for less than 1 year. There will be no need to follow any specific blood labs as LutiMax® TM has not been found to have any major toxicities. Because the safety profile of LutiMax® is excellent, the expenses of the trial should be relatively low. We envision involving 5 NEALS associated centers in this trial.

Introduction

LutiMax® is a molecular complex of the bioflavonoids Luteolin(L) and Rutin(R). LutiMax® was introduced into commerce as a dietary supplement worldwide in 2002 by SYNORx, Inc. LutiMax® has not been shown to have any serious side effects. LutiMax® has many targets for neurodegenerative disease. The response appears to be dose dependent, and there have been no demonstrable biological effects to either pure L or R1. We, at DHMC, have personally seen several ALS patients who appear to have clinically stabilized while taking LutiMax®.

There is substantial evidence to show that flavonoids are neuroprotective by reducing neuroinflammation, reducing pro-inflamatory cytokines and chemokines and reducing neuronal apoptosis all of which suggests an important role for flavinoids in counteracting neurodegeneration2-5. As no rigorous clinical trial has been undertaken, we propose to look at this potentially promising compound in the setting of ALS.

Three recent publications confirm the poor bioavailability of oral L, two of which describe the dramatic increased oral bioavailability of L when combined with R. Orally ingested LutiMax® shows 1.9 to over 10 times greater plasma levels of L than the same oral dose of L6. R is hydrolyzed to Quercetin (Q) 4-6 hours after ingestion7 and plays the role of Trojan Horse for L as well as having complimentary pharmacokinetic effects on L6,8.

L and Q are some of the best inhibitors of superoxide generation by xanthine oxidase9 (XO) and nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase in nature10-11, and they block nitric oxide (NO) synthesis12-13 as well as quench the hydroxyl radical14-15. L and Q are both active in the mitochondria, effect apoptosis in cancer cells by blocking glycolysis16-17, and they both are neuroprotective to excitotoxins18-19, chemical oxidants20, neurotoxins21, cytokines5, and inflammatory molecules22. R also increase the synthesis of glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) 23-26 and Q also promotes redox homeostasis24.

L, R, and Q block the reactive oxygen species (ROS)15,24, cytokine27-28, and LPS induced expression of inflammatory genes27, protein signaling molecules2,29, kinases3,30, and inflammatory mediating enzymes including phopholipase A2

31, phospholipase C32, protein kinase C (PKC)33, cyclooxygenase-2 enzyme (COX-2)34, inducible nitric oxide synthase (iNOS) enzyme expression35-36, lipoxygenase37-38, TNF-alpha39-41,and TNF-alpha induced nuclear factor-kappa B (NF-KB) activation via I kappa B kinase beta (IKKB)42-43. The interleukins IL-1B5,28, IL-244-45, IL-446, IL-547, IL-65,48, IL-849-50, IL-1044, IL-1242, and IL-1351-52 as well as IFN-gamma28,53 all show inhibition or modulation by L or Q or both. Both L and Q are also strong inhibitors of IgE mediated allergic reactions54-55.

Bioflavonoids

Luteolin

Quercetin

Rutin

L enhances proteosomal degradation of misfolded proteins33, and both L and Q increase the redox sensitive transcription factor (Nrf-2) mRNA and stabilize Nrf-2 to proteosomal degradation56. L also activates the cell regulating protein, Sirtuin57. L and Q block the transcription factor AP-127,51, and immune activation in experimental autoimmune encephalitis (EAE)41,58. L and Q block extracellular signal-regulated kinases ERK1/2 and the protein kinase Akt59, modulate intracellular calcium60-61 and increase mitochondrial efficiency and inhibit glycogen synthase kinase 3 (GSK-3)30,62. Recent research on L and Q show these bioflavonoids target autophagy63 MPTP related Parkinson’s disease*56, astrocytic mediated apoptosis, management of blood brain barrier (BBB) to T Cells and macrophages*41,58, and IL-6 production in microglia by inhibiting the mitogen-activated protein kinase JNK phosphorylation and the activator protein (AP-1)*61. (*studies sponsored by SYNORx)

The recent research on L and the synergistic mixtures with R provide a body of evidence to support the clinical evaluation of LutiMax® in ALS. ALS is a multifactorial disease with multiple pharmacologic targets, and the multifactorial pharmacodynamics of L, R, and Q match many of the same targets at the transcriptional to the enzymatic and metabolic level. Overall, there are compelling arguments to try LutiMax® in a clinical trial with ALS patients.

Proposed Trial We feel that there is potential for LutiMax® as a treatment for ALS after observing some patients who appear to have stabilized on this compound. We propose treating, through a multi-center trial, 60 ALS patients with LutiMax®. With this phase II trial, patients will be entered in a double-blind, placebo-controlled, cross-over study. We plan to use LutiMax® for 12 months vs. placebo in a cross-over with a latin square technique where the patient will be switched form real medication to placebo or visa-versa at six months. Assuming 80% complete both periods (i.e., receive both placebo and treatment) the study will have sufficient power (80%) at a type I error rate of 5% to detect an effect of treatment such that 75% of patients have a better ALSFRS score following treatment, then following placebo. The patients will have to meet strict inclusion and exclusion criteria for the study including being able to ingest the pills sublingually. Patients would be evaluated using the ALSFRS as a primary outcome with secondary outcomes being survival and FVC, measured every 3 months. A neurological screening exam will be done on initial visit and then every three months. There will be no need to follow any specific blood labs as LutiMax® has not been found to be toxic in rats nor in humans64-66. No acute oral toxicity was found with testing in mice using LutiMax® at 5.0 grams/kg of body weight and 7.5 grams/kg of body weight as performed by the Chinese Academy of Sciences, Institute of Materia Medica, National Institute of Pharmacological Screening in Beijing in 2007 (SYNORx, Inc.,unpublished data). The lack of toxicity should make compliance better and will reduce the budget for the trial.

A dose of LutiMax® (each lozenge contains 100mg of luteolin and 100mg of rutin) will ultimately be 8 lozenges sublingually 4 times a day. We expect patients to be able to get to this dose within a week of starting. The sublingual delivery for LutiMax® is a convenient reliable method which avoids first-pass metabolism in the liver and pre-systemic elimination in the gastrointestinal tract67. A washout period of a week will be initiated between treatments. LutiMax® has made a placebo lozenge which is indistinguishable by taste, texture and color to the standard lozenge. The placebo lozenge has been tested by chromatography.

High intakes of carbohydrate and low intakes of fat and some kinds of fatty acids may, when combined appear to increase the risk of ALS68. A ketogenic diet has been proposed as a therapy for ALS69-70. LutiMax® works best in a low-carbohydrate diet, especially low in sugars (Thomas Lahey: personal observations). We will recommend a high protein, high unsaturated fat, low carbohydrate diet with a target of 3000 calories per day to maintain weight and muscle mass. To accomplish this, a nutritionist will provide an integrated shopping list and meal recommendations and patients will keep a dietary record.

Funding for this project is still being pursued. The budget for this trial should be relatively small.

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LutiMax® vs. Controls (MGH study)