WO2017137767A1

WO2017137767A1 - Anti-neurodegenerative disease formulation containing apocynin and paeonol

Info

Publication number: WO2017137767A1
Application number: PCT/GB2017/050352
Authority: WO
Inventors: Nicholas John Larkins
Original assignee: Akl Research & Development Ltd
Priority date: 2016-02-11
Filing date: 2017-02-10
Publication date: 2017-08-17
Also published as: GB2547241A; GB201602486D0

Abstract

Compositions comprising 4-hydroxy-3-methoxyacetophenone or metabolite thereof and 2-hydroxy-4-methoxyacetophenone or metabolite thereof for use in the treatment of neurodegenerative diseases, for example for the treatment of neurodegenerative diseases such as dementia, Alzheimer's disease[AD] or Parkinson's disease [PD].

Description

ANTI-NEURODEGENERATIVE DISEASE FORMULATION CONTAINING

APOCYNIN AND PAEONOL

The present invention relates to formulations for the treatment of neurodegenerative diseases [for example dementia, Alzheimer's disease (AD), Parkinson's disease (PD), frontal lobar dementia or amyotrophic lateral sclerosis (ALS)] in humans or animals. Neurodegenerative diseases are among the most pressing problems of developed societies with ageing populations.

Neurodegeneration by definition disturbs the properties of the CNS and therefore affects neuronal function, as well as the structure or survival of neurons. Because the complexity of the human CNS is so great, neurodegenerative disorders that derange its function have been challenging to understand and treat. No therapeutics are known to ameliorate the natural course of neurodegenerative diseases.

Neurodegenerative diseases such as AD, PD and ALS are typified by a reactive morphology of glial cells including both astrocytes and microglia. This reaction, both cellular and molecular, is not distinguishable between one disease and another or from other conditions such as stroke or traumatic injury.

Dementia is a syndrome m which there is a deterioration in memory, thought processes, behaviour and the ability to perform everyday activities. Dementia is usually of a chronic and progressive nature. Although Dementia most commonly affects older people, it is not a normal part of aging. Dementia is one of the major causes of disability and dependency amongst older people worldwide. Dementia also has a physical, psychological, social and economic impact on caregivers, families and society at large. Worldwide, 47.5 million people have dementia and there are 7.7 million new cases every day.

Dementia can be caused by a variety of diseases and injuries that primarily or secondarily affect the brain, such as AD or stroke. AD is the most common cause of dementia and may contribute to 60-70% of Dementia cases. AD is a multifaceted neurodegenerative disorder characterized by memory loss, language impairment, personality changes and gradual loss of intellectual ability. Currently, over 35 million people worldwide are affected with AD, and this has increased with aging of the global population. If no effective treatment of AD is discovered, the number of sufferers is anticipated to increase three-fold by 2025 compared the number of sufferers in 2005.

A treatment that delays disease onset and/or progression by 5 years could halve the number of people requiring institutionalization and/or dying from AD.

To date, therapeutic options for symptomatic treatment of AD, include three acetylcholinesterase (AChE) inhibitors (donepezil, rivastigmine and galatamine) and an N-methyl- d-aspartate receptor ( MDAR) antagonist (memantine). These therapeutic options only provide a modest and temporary benefit for memory and cognitive function and do not delay or stop the progression of neurodegeneration .

Within the scientific and medical community there now exists a census of opinion that one-compound, one-target approach to combatting neurodegenerative diseases such as AD has failed.

This could be due to the multiple pathogenic mechanisms involved in neurodegenerative diseases such as AD, including amyloid β (A β) aggregation to form plaques, hyperphosphorylation to disrupt microtubule to form neurofibrillary tangles, calcium imbalance, enhanced oxidative stress, impaired mitochondrial function, apoptotic neuronal death, and deterioration of synaptic transmission, particularly at cholinergic neurons.

In view of these complex pathogenic mechanisms, and the successful treatment of chronic diseases such as HIV or cancer, with multiple drugs having complementary mechanisms of action, the inventor has developed multi- (i.e. two or more) target agents for simultaneously modulating multiple (i.e. two or more) pathogenic mechanisms leading to neuronal death.

According to the present invention there is provided a composition, (e.g. a pharmaceutical preparation), comprising 4 -hydroxy-3-methoxyacetophenone [hereafter apocynin] or metabolite thereof and 2-hydroxy-4- methoxyacetophenone [hereafter paeonol] or metabolite thereof for use in the treatment of dementia (for example in the treatment of AD or PD) . 4 -hydroxy-3-methoxyacetophenone [Apocynin] is a plant g structural formula:

Apocynin is found in plant substances and plant extracts, for example in extracts of the plants picrorrhiza kurroa, apocynum cannabinium, apocynum venatum, apocynum androsaemifolium and vanilla species such as Vanilla planifolia. Apocynin may also be synthetically produced by methods known in the art.

A preferred composition for use in the treatment of dementia (for example in the treatment of AD or PD) comprises apocynin and paeonol wherein the ratio (by weight) of apocynin to paeonol is between about 1 to 100 and about 100 to 1, preferably between about 1 to 50 and about 20 to 1, more preferably between about 1 to 30 and about 10 to 1, more preferably between about 1 to 20 and about 5 to 1, more preferably between about 1 to 10 and about 2 to 1, more preferably between about 1 to 10 and about 1 to 1, more preferably between about 1 to 10 and 1 to 2.

The metabolite of apocynin may be selected from the group consisting of, 3' , 4 ' -dihydroxyacetophenone, acetoisovanillone and 1- ( 4 ' -hydroxy-3 ' -methoxyphenyl ) - ethanol formed by ketone reduction of apocynin, 3,4- dihydroxyacetophenone , and diapocynin. The diapocynin may be formed by oxidation of apocynin, for example in vivo oxidation during neutrophil activation in the presence of myeloperoxidase and hydrogen peroxide. The composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) may further comprise L-glutamine.

The composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) may further comprises a filler or excipient. The excipient or filler may comprise

Spirulina, or an extract thereof. Preferably the composition for use in the treatment of neurodegenerative diseases is for use in the treatment of dementia, AD or PD, most preferably AD.

The compositions and preparations for use in treatment neurodegenerative diseases (for example in the treatment of dementia, AD or PD) of the invention may include "isolated" apocynin. Isolated apocynin is apocynin which has been synthesised, or which has been extracted from plants and purified.

Alternatively or additionally, apocynin may be present in compositions or preparations according to the invention as direct extracts from plants such as those mentioned above (for example as part of an unresolved mixture of compounds in the form of an unpurified plant or root extract) . These will be referred to as apocynin "in the natural form" or "natural apocynin". For example, apocynin present in preparations for use in the treatment of dementia (for example in the treatment of AD or PD) according to the invention in the form of Picrorrhiza kurroa will be referred to as "natural apocynin". The term "natural apocynin" or apocynin "in the natural form" also includes glycosides of apocynin such as those found in the plant species in which apocynin is found. Such glycosides include androsin and other iridoid glycosides, for example.

Preferably, the composition includes apocynin in a purified or synthetic form: "isolated" apocynin.

The composition for use in the treatment of dementia (for example in the treatment of AD or PD) may include apocynin as part of an unresolved mixture of compounds in the form of an unpurified plant or root extract: "natural" apocynin. The use of active entity in the natural form in combination with the isolated active apocynin may lead to a synergistic effect between the isolated form (e.g. purified or synthetic apocynin) and the natural form (e.g. apocynin included in Picrorrhiza kurroa) . Picrorrhiza kurroa is a standardised form based on standardised iridoid glucoside fraction, such forms are well known. A preferred Picrorrhiza kurroa in standardised form comprises Picrorrhiza kurroa standardised to "apocynin min 4%". Standardised iridoid glucoside fractions between apocynin min 2% and apocynin min 8% are also preferred.

Preferably, the composition includes apocynin in a purified or synthetic form: "isolated" apocynin. Alternatively, the composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) may include apocynin which is in the natural form only, for example, Picrorrhiza kurroa. However, if this is the case it may be necessary to limit the amount of Picrorrhiza kurroa to prevent side effects (such as stomach upset which may occur due to other phytochemical species in the Picrorrhiza kurroa) . However, it is noted that most human subjects can take up to 2, 000 mg of standardised Picrorrhiza kurroa per day without any discomfort.

If natural apocynin is included in the composition for use in the treatment of dementia, (for example in the treatment of AD or PD) , then preferably the natural apocynin is one or more of Picrorrhiza kurroa, Apocynin cannabinium, Apocynin venatum, Apocynin androsaemifolium, or vanilla species such as Vanilla planifolia, or extract (s) thereof, or is a glycoside of 4-hydroxy-3- methoxyacetophenone .

2-hydroxy-4-methoxy-acetophenone [Paeonol] is a phenol having the following structural formula: 3

It may be found in plant substances and plant extracts. Paeonol may be synthetically produced by methods known in the art. For example, paeonol may be found in Paeonia suffruticosa Paeonia lactiflora, Paeonia veitchii, Paeonia obovata, Cynanchum panniculatum, Rheum palmatum (rhizome) and Scutellaria baicalensis (root) .

The Paeonol metabolite may be selected from the group consisting of resacetophenone , resacetophenone-2-O- sulfate, 2-hydroxy-4-methoxyacetophenone-5-0-sulfate, 2- hydroxy-4 -methoxyacetophenone-5-O-glucopyranuronoside, 2- hydroxyacetophenone-4-O-glucopyranuronoside and paeonol- 2-O-sulfate .

The compositions and preparations for use in the

treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) of the invention may include "isolated" paeonol, that is paeonol which has been synthesised or paeonol which has been extracted from plants and purified.

Paeonol may be present in preparations for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) according to the invention as direct extracts from plants (i.e. as part of an unresolved mixture of compounds in the form of an unpurified plant or root extract) . These will be

referred to a paeonol "in the natural form" or "natural paeonol". For example, paeonol present in preparations for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) according to the invention in the form of Paeonia

suffruticosa will be referred to as "natural paeonol". The terms paeonol "in the natural form" or "natural paeonol" include glycosides of paeonol such as those found in the plant species in which paeonol is found.

Such glycosides include paeonin, paeonolide, paeonoside, and paeoniflorin for example. Preferably the composition for use in the treatment of dementia (for example in the treatment of AD or PD) includes paeonol in a purified or synthetic form: "isolated" paeonol. Alternatively or additionally the paeonol may be present in natural form. If the paeonol is present in natural form, preferably the paeonol is present in one or more of Paeonia suffruticosa, Paeonia lactiflora, Paeonia veitchii, Paeonia obovata, Cynanchum panniculatum, Rheum palmatum or Scutellaria baicalensis, or extract thereof, or is a 2-hydroxy-4-methoxyacetophenone glycoside.

A preferred composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) includes isolated apocynin and isolated paeonol and the ratio (by weight) of isolated apocynin to isolated paeonol is between 10:1 and 1:100, more preferably between 5:1 and 1:50, more preferably between 2:1 and 1:20, more preferably between 1:1 and 1:10, more preferably between 1:2 and 1:6 more preferably between 1:3 and 1:6, more preferably between 1:3 and 1:5, more preferably between 1:3 and 1:4. The composition may also include natural apocynin and/or natural paeonol. In the ratios above the weights of apocynin and paeonol refer to the weight of the components in the isolated form (e.g. isolated apocynin and isolated paeonol) . A composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) comprises apocynin and paeonol wherein apocynin is present in isolated form and in the natural form (e.g. Picrorrhiza kurroa) . Preferably the ratio (by weight) of isolated apocynin to apocynin in the natural form is between about 10 to 1 and 1 to 1 (based on a calculation where the natural apocynin is in the form of Picrorrhiza kurroa standardised to Apocynin min 2%) .

A further composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) comprises apocynin and paeonol wherein apocynin is present in isolated form and paeonol is present in the isolated form and in the natural form (e.g. Paeonia suffruticosa) . Preferably the ratio (by weight) of isolated paeonol to paeonol in the natural form is between about 10 to 1 and 1 to 1, more preferably between 4:1 and 2:1 (based on a calculation where the natural paeonol is in the form of Paeonia suffruticosa .

The composition may comprise apocynin and paeonol wherein apocynin is present in isolated form and in the natural form (e.g. Picrorrhiza kurroa) ; and paeonol is present in isolated form and in the natural form (e.g. Paeonia suffruticosa .

Preferably the composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) includes paeonol and apocynin in isolated form. If the composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) includes apocynin which is only in the natural form (that is no isolated apocynin) and paeonol which is only in the natural form (that is no isolated paeonol), it is preferred that the % by weight of the total composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) which is apocynin in the natural form (e.g. Picrorrhiza kurroa) is at least 2.5% (more preferably at least 5%) ; and/or the % by weight of the total composition which is paeonol in the natural form (e.g. Paeonia suffruticosa) is at least 2.5% (more preferably at least 5%) .

The compositions for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) may be pharmaceutical preparations, for the treatment of disease in humans, or veterinary preparations, for the treatment of non-human animals .

Compositions for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) may further comprise L-glutamine at a ratio (by weight of apocynin to L-glutamine) of between 1 to 10 and 1 to 40, more preferably between about 1 to 15 and 1 to 25. Preferably the composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) further includes a binder. The preparations (or compositions) for use m the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) may further comprise additional components such as pharmaceutically conventional carriers, diluents, solvents, flavourings, emulsifiers and stabilisers. They may comprise additional components (for example carriers or diluents) which are "conventional" in herbal remedies. Preferably the pharmaceutical or veterinary preparation (or composition) for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) further comprises one or more of the following:

Taste masking agents, for example yoghurt, fruit juice, honey, spirulina or extract thereof, chlorella and syrup.

The preparations (or compositions) for use in the

treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) preferably includes an excipient, spirulina. Spirulina is a cyanobacterium that is a source of amino acids, minerals,

phycocyanobilin (PCB) , trace elements and vitamins, and which may act as a taste masking agent.

The compositions and pharmaceutical/veterinary preparations for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) are suitable for oral administration. The methods of formulation of the compositions for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) for oral administration are well known in the art. For example, the composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) for administration may be prepared using a pharmaceutically acceptable carrier in a form suitable for administration. Such a carrier can be prepared as a tablet, a pill, a sugar-coated agent, a capsule, a liquid, a gel, a syrup, a solvent, a slurry, a suspension, etc. The carrier may be a herbal binder or one or more pharmaceutically acceptable carriers such as liposomes, lactose, trehalose, sucrose, mannitol, xylitol, crystalline cellulose, chitosan, calcium carbonate, talc, titanium oxide, or silica (silicon oxide) or the like.

The composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) may be obtained, for example, by combining the active ingredients with a solid excipient, pulverizing the mixture (i.e. reducing the size of the particles in the mixture) (if necessary) and inserting into a capsule, for example, a soft sealed capsule consisting of a gelatin capsule, gelatin and coating (e.g., glycerol or sorbitol) or a capsule composition suitable for vegetarians. Conventional methods of reducing the size of particles in a mixture for inclusion in a pharmaceutical composition are known in the art. One such method known in the art is mechanical micronization . Mechanical micronization increases the surface area of particles and thus enhance the solubility and dissolution of the particles, particularly when the particles are particles comprising poorly soluble drug molecules. In the soft capsule, the composition for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) may be dissolved or suspended in an appropriate liquid, such as a fatty oil, liquid paraffin or liquid polyethylene glycol, with or without a stabilizer. Polymers, cyclodextrins and liposomes may be used to improve the solubility as well as stability of the formulations. The method of including polymers, cyclodextrins and liposomes in pharmaceutical compositions is well known in the art.

The formulation (composition or preparation) for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) may also be in the form of a standardised liquid extract. Standardised liquid extracts may in some circumstances have advantages when compared to the solid dose forms (tablets and hard shell capsules). They may involve minimal processing during manufacture during manufacture and may reflect the true spectrum of the original herb (or plant etc.), in a compact and convenient form. There is also the possibility of superior bioavailability as the preparation is already in the liquid form. The prescribed dose may then be easily diluted (water, fruit juice, adding ice etc.) so as to minimise the experience of any unpleasant taste thus increasing the likelihood of patient compliance. It will be appreciated that the preparations for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) are suitable for other means of administration known in the art, for example mucosal delivery routes (for example rectal, nasal, vaginal), topical administration, and administration by injection. The methods of formulation of the compositions for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) for administration by these methods are well known in the art.

Preferably the preparation (or composition) for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) is administered to a subject (human or animal) at a concentration, per daily dose, of apocynin, of from 0.1 mg/kg body weight to 100 mg/kg body weight, more preferably 0.5 to 50 mg/kg body weight, more preferably 1 to 30 mg/kg body weight, more preferably 2 to 20 mg/kg body weight. Preferably, the preparation is administered to a human or animal subject at a concentration, per daily dose, of paeonol of lmg/kg body weight to 100 mg/kg body weight, more preferably between about 2 mg/kg and 50 mg/kg body weight, more preferably between about 2 mg/kg and 20 mg/kg body weight. These daily dose concentrations are based on isolated apocynin and isolated paeonol. In a preferred embodiment the preparation (or composition) for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) is administered to a subject (e.g. human) at a concentration, per daily dose, of isolated apocynin, of between 0.1 mg/kg body weight and 100 mg/kg body weight, more preferably 0.5 to 50 mg/kg body weight, more preferably between about 1 to 30 mg/kg body weight, more preferably 2 to 20 mg/kg body weight. Preferably, the preparation for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) is administered to a subject at a concentration, per daily dose, of isolated paeonol of lmg/kg body weight to 100 mg/kg body weight, more preferably between about 2 mg/kg and 50mg/kg body weight, more preferably between about 2 mg/kg and 20 mg/kg body weight. More preferably the preparation for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) further comprises natural paeonol (e.g. Paeonia suffruticosa) is administered to a subject (e.g. human) at a concentration, per daily dose, of natural paeonol, of between 1 mg/kg body weight and 100 mg/kg body weight, more preferably 2 to 50 mg/kg body weight (for example 10 mg/kg body weight to 40 mg/kg body weight), more preferably between about 2 and 20 mg/kg body weight.

The preparation (or composition) may further comprise apocynin, in the natural form, wherein the apocynin in the natural form is administered to the subject at a concentration, per daily dose, of natural apocynin, of between 0.25 mg/kg and 15 mg/kg body weight, more preferably 1 to 3 mg/kg body weight. The doses may be increased (e.g. doubled) at the beginning of the treatment, for example for the first 1 to 3 days, as a "loading dose". Thus, as a loading dose, the preparation (or composition) may be administered to a subject (human or animal) at a concentration, per daily dose, of apocynin, of between 0.2 mg/kg body weight and 200 mg/kg body weight, more preferably 1 to 100 mg/kg body weight, more preferably 2 to 60 mg/kg body weight, more preferably 4 to 40 mg/kg body weight. Preferably, the preparation is administered to a human or animal subject at a loading dose concentration, per daily dose, of paeonol of 2 mg/kg body weight to 200 mg/kg body weight, more preferably between about 4 mg/kg and 100 mg/kg body weight, more preferably between about 4 mg/kg and 40 mg/kg body weight. These daily dose concentrations are based on isolated apocynin and isolated paeonol.

The preparation (or composition) for use in the treatment of neurodegenerative diseases (for example in the treatment of dementia, AD or PD) may further comprise L- glutamine administered to the subject at a concentration, per daily dose, of L-glutamine of 1 mg/kg body weight to 280 mg/kg body weight, more preferably 20 mg/kg body weight to 280 mg/kg body weight, more preferably between 100 mg and 180 mg/kg body weight.

The daily dose may be provided as a single capsule, tablet or other solid or liquid form known to those skilled in the art, or may be provided in divided doses (for example 1 to 3 doses) to make up the full daily dose. The doses of apocynin and paeonol may be provided together in the capsule, tablet, etc. or the two may be provided as separate capsules or tablets for sequential administration.

The composition may be administered for a period of 14 days or more, for example a period of 28 days or more, for example a period of 42 days or more.

The composition may be administered daily for a period of 14 days or more, for example daily for a period of 28 days or more, for example daily for a period of 42 days or more.

The inventor has observed that that there may be a time- lag between 14 days up to 42 days (or more) - before any obvious clinical benefits can be observed. Once such clinical benefits are observed, depending on the chronicity and severity, clinical improvements continue to accrue.

According to the present invention in a further aspect, there is provided kit of parts for a preparation for treatment or prevention of neurodegenerative diseases (for example dementia, AD or PD) comprising at least one dose of apocynin or metabolite thereof; and at least one dose of paeonol or metabolite thereof. It is envisaged that the kit of parts may be provided as, for example, a blister pack containing capsules containing doses or partial doses, for example, apocynin, and separate capsules containing doses or partial doses of paeonol. The pack may be provided with instructions for sequential administration of the doses. When administered either together or separately the compounds should be administered such as to maintain a suitable blood level of each of the components. When administered separately the compounds (apocynin and paeonol) should be given within 4 hours of each other, preferably within 2 hours, and most preferably substantially simultaneously.

Pharmacokinetic studies in rats have shown that both apocynin and paeonol are traceable in serum and cross the blood brain barrier.

Paeonol is lipophilic and apocynin is hydrophilic. The hydrophilic apocynin is rapidly metabolised in comparison to paeonol which being lipophilic undergoes a slower metabolism.

Following systemic absorption, two early studies in rats indicated that the majority of apocynin is rapidly excreted in the urine unchanged; for example, 80% of the acetovanillone administered via intraperitoneal inj ection was excreted unchanged.

An APPA (Apocynin and Paeonol combination) 28 day oral (gavage) administration study was undertaken in the rat and which was followed by a 2 week treatment-free period. The assay was fully validated for apocynin and paeonol in compliance with regulatory standards and exhibited high levels of selectivity, accuracy and precision.

Exposure of paeonol was significantly higher than apocynin (and both absent in control animals) . Apocynin underwent significant metabolism as evidenced by drug related peaks in the MRM chromatograms which affected the apocynin chromatography at Week 4 and also affected the paeonol ISR (Incurred Sample Reanalysis).

No half-lives could be calculated at any dose level for Apocynin .

Half-lives of Paeonol were between 1.91 and 6.98 hours on Day 1 and Week 4, with no notable trend relating to gender, dose level or number of doses. Where the apocynin half-life could not be determined, the corresponding Area under the Curve, AUC (0-∞), value was also not reported.

In view of these observations these data supported the proposition that the two compounds, apocynin and paeonol, when administered together (APPA) , affect the metabolism of each other. In view of these observations, it was determined that apocynin fraction of APPA enhances the biological exposure of paeonol.

Accumulation of apocynin appeared following daily dosing for 4 weeks at all dose levels. There did not appear to be any trend in accumulation ratios relating to gender or dose level.

Little or no accumulation of paeonol appeared following daily dosing for 4 weeks at all dose levels. There did not appear to be any trend in accumulation ratios relating to gender or dose level . These unique and unexpected outcomes could not have been predicted from the metabolisms of the two compounds in isolation and this only becomes apparent when both apocynin and paeonol (APPA) are administered in combination .

The different exposure levels between apocynin and paeonol, when combined together as APPA, in conjunction with the evidence of extensive apocynin metabolism provided evidence that apocynin enhances the biological exposure of paeonol.

The hydrophilic apocynin, when and only when administered in conjunction with the lipophilic paeonol fraction in APPA, binds in an exceedingly short time-period to serum albumin. Serum albumin is the most abundant protein in the circulatory system and the main protein responsible for the transport of endogenous and exogenous compounds by reversible binding. A reversible apocynin- serum albumin combination would leave apocynin in a non-detectable state such that, as been observed, the apocynin half-life in not detectable at any dose levels. Such mega-rapid serum binding thus allows the apocynin to be biologically available over a more prolonged time. This observation can be confirmed by the findings that accumulation of apocynin appeared following daily dosing for 4 weeks at all dose levels whereas little or no accumulation of Paeonol appeared following daily dosing for 4 weeks at all dose levels.

Such unexpected outcomes supported repeated clinical findings that when absent from the synergism of the complementary molecule apocynin, that a significantly higher dosage of paeonol, as a stand-alone compound, is required .

It is preferred that the preparation is administered orally, for example, in pill or capsule form, although it is possible to use other known conventional administration techniques, such as injection.

According to the present invention in a still further aspect there is provided the use, in the manufacture of a preparation for the treatment of neurodegenerative diseases (for example dementia, AD or PD) , of and 4- hydroxy-3-methoxyacetophenone (apocynin) or metabolite thereof and 2-hydroxy-4-methoxyacetophenone (paeonol) or metabolite thereof. According to the present invention in a still further aspect there is provided a method of treatment of neurodegenerative diseases (for example dementia, AD or PD) comprising a step of administration to a human or non-human animal subject in need thereof of a composition comprising 4 -hydroxy-3-methoxyacetophenone (apocynin) or metabolite thereof and 2-hydroxy-4- methoxyacetophenone (paeonol) or metabolite thereof.

In the method of treatment of neurodegenerative diseases (for example dementia, AD or PD) , the 4-hydroxy-3- methoxyacetophenone is preferably administered to the subject at a daily dose equivalent to between 1 mg/kg body weight and 100 mg/kg body weight isolated 4-hydroxy-

3-methoxyacetophenone , and the 2-hydroxy-4- methoxyacetophenone is preferably administered to the subject at a daily dose equivalent to between 5 mg/kg body weight and 150 mg/kg body weight isolated 2-hydroxy-

4 -methoxyacetophenone . Preferably the composition/preparation includes apocynin in isolated form.

Preferably the composition/preparation includes paeonol in isolated form.

The compositions, preparations and doses for use in treating neurodegenerative diseases (for example dementia, AD or PD) according to the invention may include other components (e.g. an excipient, Spirulina) which are suitable for treating dementia (for example AD or PD) . However, it is preferred that the paeonol and the apocynin (in isolated form and/or in natural form) represent (or lead to) substantially all of neurodegenerative disease (for example dementia, AD or PD) activity of the compositions, preparations and doses.

The compositions, preparations, methods and uses for treating neurodegenerative diseases (for example dementia, AD or PD) may be used in combination with other (conventional) medicaments or preparations. The other (conventional) medicaments and preparations may include (but are not limited to) donepezil and/or rivastigmine and/or galantamine and/or memantine.

The present invention will now be described in detail with reference to illustrative examples.

CASE STUDY EXAMPLES

The inventor has observed that there may be a time-lag, for example of between 14 days up to 42 days (or more) before any obvious clinical benefits can be observed. Once such clinical benefits are observed, depending on the chronicity and severity, clinical improvements continue to accrue.

EXAMPLE 1

An 8 year old male dog was diagnosed with Osteoarthritis in its left shoulder, was 3/5 lame at walk and showing early signs of dementia. The dog showed a diminishing degree of interest in sport activity over a period of several months. It was decided to treat the dog with a composition comprising apocynin and paeonol.

The following components were mixed in a conventional manner to provide the formulation used to treat the dog.

(isolated)

Total lOOOg

The mixture (APPA) was divided and prepared in a form suitable for dosing, in a capsule form for oral dose.

Formulation 1 was given to the dog in its feed at a rate of 0.53 grams per day per 25-kilogram body weight.

After a period of several weeks following treatment with Formulation 1, the dog was more mentally alert and active and very active in all forms of sport activity. The above is the usual daily dose. The animal was started (1-7 days) on a loading dose of 1.05 grams per day per 25- kilogram body weight and once a clinical benefit was determined the dose was reduced to those noted above. EXAMPLE 2

An 80 year old male human had been diagnosed with early- to-mid stage dementia, Alzheimer's disease, and

osteoarthritis [OA] . It was decided to treat the

individual's OA with a composition comprising apocynin and paeonol. The individual was not receiving any other medication to treat his Alzheimer's disease.

The individual took two 500 milligram capsules of the composition of formulation 1 per day for the first 30 days of treatment.

Thereafter, the individual took one 500 milligram capsule of the composition of formulation 1. After three weeks the patient's wife reported that her husband' s OA was much improved and was interested to report that his mental attitude was much improved. At 8 weeks his daughter said (unprompted) : "It was like his eyes had switched back on, he was in the moment, and more able to grasp what was going on than in the previous 18 months. He is happier, sharper, more with it, more fluid in conversation." The patent's wife reported that

independently the patient's granddaughter noted a

significant change too which she mentioned to her mother. Other friends have independently noticed a marked

improvement in his ability to hold a conversation too.

The effect in this patient is evidence that the

formulation has a beneficial effect in the treatment of Alzheimer's disease.

EXAMPLE 3

In 2014, a canine study (double blinded placebo

controlled) at the University of Vienna, the study investigators reported that:

"Although this study requires validation in a larger group of dogs, APPA (the combination of 4-hydroxy-3- methoxyacetophenone and 2-hydroxy-4-methoxyacetophenone ) seemed to be particularly effective at improving the self-directed activities (standing posture, walking, trotting) evaluated in the orthopaedic examination, which may indicate an effect beyond the merely antiinflammatory or analgesic. APPA evidently improved the dogs' comfort and consequently may have encouraged more spontaneous activity, which could have contributed to better strength and co-ordination over time. Significant improvements in general activity and in ability to rise, walk, run and climb stairs in the APPA group support this possibility, as rising to standing from lying down, running, and climbing stairs in particular require both comfort and strength. Thus, an improvement in "ability to walk" on the Pain Interference assessment may be pointing to improvements in strength and stamina as well as in comfort... One might also consider improvements in mood as factoring in to willingness to participate in daily activities such as going for walks."

EXAMPLE 4

A man now in his mid-60s had survived a ruptured cerebral aneurysm about 25 years ago, which put him at high risk for early-onset AD. Until taking early retirement, he had been a senior office in the Army. He began showing signs of AD at around age 60, and had been taking various medications to treat his AD, without success. His condition had deteriorated in the past 6 months to the point that his carer reported events such as: "tried to shave this morning with his toothbrush", and "asked him to bring a mop and eventually turned up with an ironing board . "

After receiving APPA for 6 weeks, his caretaker reported the following: "So far, the changes, which took around 4 to 5 weeks to note, are quite dramatic. I am expecting ongoing improvement, but I have no idea by how much or what the ceiling will be; however, I am super excited by this. We have run out of APPA. You know it is certainly working. The big difference is that he is much more engaged. He was becoming a bit isolated and withdrawn, but I can see a difference and friends have said he is on good form and thought he would be much worse. So, thank you for letting us use the APPA. " EXAMPLE 5

A man in his mid-70s reported that over the past 12 months or so he had become increasingly more forgetful. Although he still had a good long-term memory, he was having frequent short-term memory failures. In addition, he was experiencing a form of nominal aphasia, where he was able to recognise people's faces but unable to relate his relationship with them until reminded. After 8 weeks on APPA, he reported that his memory is much better, and asked to continue on APPA.

Example 6

A 17 year old female dog has been causing the owner concerns as to her loss of quality of life. The dog was sleeping fitfully during the night spending, sometimes hours, pacing backwards and forwards as if a 'trance' . During the day similar 'no longer with it' behaviour had become commonplace. Such symptoms are typically encountered in canine cognitive dysfunction syndrome (dementia) . The owner, a veterinarian, decided to implement the formulation as outlined in 'Example 1' (APPA) . The APPA dose rate was given at 500mg per day. After 14 days the owner reported that the dog's behaviour and mental acuity had improved. After several more week the owner reported that the dog was 'pretty well much back to her old normal self . After several months the owner decided to discontinue any further APPA. After a period of around 14 days the owner reported the dog had deteriorated with her neurological being much the same or even worse that before starting the APPA. As the dog^'s quality of life continued to deteriorate after a further 4 weeks the owner resumed dosing with APPA. The owner reported that on this occasion there was a noticeable positive clinical response within 10 days : Taking into consideration her increasing old age -she has regained once again her quality of life' . Example 7

An 8 year old male dog had always shown an anti-social behaviour with both humans and other dogs. Other than enjoying prolonged countryside walks and/or long runs he was very much a 'loner'. A trauma to various ligamentous attachments around his pelvic girdle area prevented taking any further distance exercise until sufficient healing had occurred. To assist the recovery the dog was prescribed APPA - 500mg twice per day. Over several weeks the owner observed initially subtle' positive character changes. The dog had stared to become 'more socially connected' with other the other household dogs and 'was on occasions actually playing with them' . Continuing to take APPA over the ensuing 18 months the owner reported that the social changes have been so dramatic it was if he had become 'a canine Dorian Grey and reinvented his life'

Example 8

Loss of lean mass (sarcopenia) is accelerated in early AD and is associated with brain atrophy and cognitive performance, as a direct or indirect consequence of AD pathophysiology or through shared mechanisms common to both AD and sarcopenia. A 93 yo female who had been taking APPA over several years fell over and partially de-gloved the skin over distal limb requiring hospitalization and a consequent skin graft. After 3 weeks bedridden just going to the bathroom unattended was very problematic. Back at home, after several weeks taking APPA the patient was able to walk up to 1 kilometre per day. The patient now routinely walks 1 kilometre twice per day to her workplace where she continues be fully active.

Claims

C L A I M S

1. A composition for use in the treatment of neurodegenerative diseases (for example, dementia, Alzheimer's disease (AD) or Parkinson's disease (PD) ) comprising 4 -hydroxy-3-methoxyacetophenone or metabolite thereof and 2-hydroxy-4-methoxyacetophenone or metabolite thereof .

2. A composition for use according to claim 1 wherein the ratio (by weight) of 4-hydroxy-3-methoxyacetophenone to 2-hydroxy-4-methoxyacetophenone is between 1 to 100 and 100 to 1.

3. A composition for use according to claim 2 wherein the ratio (by weight) of 4-hydroxy-3-methoxyacetophenone to 2-hydroxy-4-methoxyacetophone is between 1 to 1 and 1 to 10.

4. A composition for use according to any preceding claim further comprising an excipient.

5. A composition for use according to claim 4 where the excipient is Spirulina.

6. A composition for use according to any preceding claim for use in the treatment of Alzheimer's disease or Parkinson's disease.

7. A composition for use according to any preceding claim including 4 -hydroxy-3-methoxyacetophenone in the form of isolated 4-hydroxy-3-methoxyacetophenone .

8. A composition for use according to any preceding claim including natural 4 -hydroxy-3-methoxyacetophenone .

9. A composition for use according to claim 8 wherein the natural 4-hydroxy-3-methoxyacetophenone is one or more of Picrorrhiza kurroa, Apocynin cannabinium, Apocynin venatum, Apocynin androsaemifolium, or vanilla species such as Vanilla planifolia, or extract (s) thereof, or is a glycoside of 4-hydroxy-3- methoxyacetophenone .

10. A composition for use according to any preceding claim including isolated 2-hydroxy-4-methoxyacetophenone .

11. A composition for use according to any preceding claim including natural 2-hydroxy-4-methoxyacetophenone .

12. A composition for use according to claim 11 wherein the natural 2-hydroxy-4-methoxyacetophenone is one or more of Paeonia suffruticosa, Paeonia lactiflora, Paeonia veitchii, Paeonia obovata, Cynanchum panniculatum, Rheum palmatum or Scutellaria baicalensis, or extract thereof, or is a 2-hydroxy-4-methoxyacetophenone glycoside.

13. A composition for use according to any previous claim where in the composition is a pharmaceutical or veterinary preparation.

14. A composition for use according to any previous claim comprising a unitary dose of isolated 4-hydroxy-3- methoxyacetophenone equivalent to between 0.1 mg/kg body weight and 100 mg/kg body weight; and a unitary dose of 2-hydroxy-4-methoxyacetophenone of 1 mg/kg body weight to 100 mg/kg body weight.

15. A composition for use according to claim 14 wherein the unitary dose of isolated 4-hydroxy-3- methoxyacetophenone is equivalent to between 2 and 20 mg/kg body weight.

16. A composition for use according to claim 14 or claim 15 wherein the unitary dose of 2-hydroxy-4- methoxyacetophenone is between about 1 and 100 mg/kg body weight.

17. A composition for use according to any of claims 14 to 16 further comprising 4 -hydroxy-3-methoxyacetophenone in the natural form; wherein the unitary dose of natural 4 -hydroxy-3-methoxyacetophenone is between 0.25 mg/kg and 15 mg/kg, more preferably 1 to 3 mg/kg.

18. A kit of parts for a preparation for use in the treatment or prevention of dementia comprising at least one dose of 4-hydroxy-3-methoxyacetophenone or metabolite thereof; and at least one dose of 2-hydroxy-4- methoxyacetophenone or metabolite thereof.

19. A kit of parts according to claim 18 for preparation for use in the treatment or prevention of dementia,

Alzheimer's disease or Parkinson's disease.

20. The use of 4-hydroxy-3-methoxyacetophenone or metabolite thereof and 2-hydroxy-4-methoxyacetophenone or metabolite thereof in (or in the manufacture of) a preparation for the treatment of neurodegenerative diseases .

21. The use of 4-hydroxy-3-methoxyacetophenone or metabolite thereof and 2-hydroxy-4-methoxyacetophenone or metabolite thereof in (or in the manufacture of) a preparation according to claim 20, for the treatment of dementia, Alzheimer' s disease or Parkinson's disease.

22. A method of treatment of neurodegenerative diseases comprising a step of administration to a human or non- human animal subject in need thereof of a composition comprising 4 -hydroxy-3-methoxyacetophenone or metabolite thereof and 2-hydroxy-4-methoxyacetophenone or metabolite thereof .

23. A method according to claim 22, for the treatment of dementia, Alzheimer's disease or Parkinson's disease.

24. A method according to claim 22 or claim 23 in which the 4-hydroxy-3-methoxyacetophenone is administered to the subject at a daily dose equivalent to between 0.1 mg/kg body weight and 100 mg/kg body weight isolated 4- hydroxy-3-methoxyacetophenone ; and the 2-hydroxy-4- methoxyacetophenone is administered to the subject at a daily dose equivalent to between 1 mg/kg body weight and 100 mg/kg body weight isolated 2-hydroxy-4- methoxyacetophenone .

25. A composition for use in treatment of neurodegenerative diseases substantially as hereinbefore described with reference to Examples 1-3.

26. A composition for use according to claim 25 in the treatment of dementia, Alzheimer's disease or Parkinson's disease .