WO2012059920A1

WO2012059920A1 - Ladostigil dosage regime

Info

Publication number: WO2012059920A1
Application number: PCT/IL2011/000851
Authority: WO
Inventors: Marta Weinstock-Rosin
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Priority date: 2010-11-02
Filing date: 2011-11-02
Publication date: 2012-05-10

Abstract

Methods of treating individuals who have been identified as having Alzheimer's disease comprising administration of ladostigil or a pharmaceutically active salt thereof in a dosage in the range of 60-200 mg ladostigil per day are disclosed. The ladostigil or a pharmaceutically active salt thereof may be administered in a single or multiple doses per day. Unit dosage forms of ladostigil or a pharmaceutically active salt thereof are also disclosed.

Description

LADOSTIGIL DOSAGE REGIME

FIELD OF THE INVENTION

The invention relates to pharmaceutical compositions and dosage units comprising ladostigil and methods of using the same to treat Alzheimer's disease, Parkinson's disease and other neurological/neurodegenerative disorders, and the deterioration of cognitive function and depression associated therewith. BACKGROUND OF THE INVENTION

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is the most common form of dementia in the elderly, affecting up to 30% of the population 85 years of age.

Cognitive impairment, expressed as amnesic type of memory impairment, visuo- spatial deficits and deterioration of language, is the core clinical feature of AD. It correlates well with the degree of degeneration of cholinergic transmission in the temporal lobe and other cortical brain regions innervated by neurons arising in the nucleus basalis of Meynert.

Noncognitive symptoms are also frequent features of AD. Depression and behavioral problems have been reported to be highly prevalent, probably associated with the decrease in noradrenergic and serotonergic transmission in the cortex and the limbic system.

Acetylcholinesterase (AChE) inhibitors: donepezil, galantamine and rivastigmine, are currently used for the treatment of cognitive symptoms of AD. AChE inhibitors increase acetylcholine levels in the synapse and enhance cholinergic transmission in the brain, decreasing the rate of deterioration of cognitive function. However, prospective trials in subjects with only mild cognitive impairment failed to show disease modifying activity of these compounds.

The non-cognitive symptoms of AD have been partially addressed by AChE inhibitors. Some researchers have pointed to an improvement in Behavioural and Psychological Symptoms of Dementia (BPSD); however, this effect is controversial and partial. Thus, a significant proportion of AD patients receive multiple drug therapy composed of AChE inhibitors, antidepressants and antipsychotic agents. Noradrenaline (NA) and serotonin (5HT), the main neurotransmitters associated with depression, also play a fundamental role in cognitive processes linked to memory and learning. These neurotransmitter systems have been shown to be impaired in AD. Thus, enhancement of the cholinergic deficit alone is but a partial compensation of the complex neurotransmitter defect.

Monoamine oxidase (MAO) is an enzyme responsible for oxidative deamination of monoamines NA, 5HT and dopamine (DA). There are two major MAO isoforms: MAO-A, which acts preferentially on 5HT and NA, and MAO-B, which preferentially deaminates phenylethylamine (PEA). Dopamine and tyramine are substrates for both forms of MAO. Increased levels of 5HT and NA in the brain by MAO-A inhibition may also lead to beneficial effects on depression and/or anxiety, which are prevalent in AD patients but not addressed with currently available AChE inhibitors.

Thus, combining AChE and MAO inhibition in a single compound has the potential to treat both cognitive and behavioural symptoms of AD. Such a compound may alleviate cognitive impairment by affecting both the cholinergic and the monoaminergic neurotransmission systems and ameliorate depressive and other behavioural symptoms. In addition, MAO-B inhibitors have been shown to have neuroprotective effects in vitro and in vivo, which could reduce disease progression.

Ladostigil, also referred to as R(+)-6-(N-methyl, N-ethyl-carbamoyloxy)-N'- propargyl-l-aminoindan and (3R)-3-(prop-2-ynylamino)-2,3,-dihydro-lH-indan-5-yl ethyl methyl carbamate, is a propargyl-aminoindan with a carbamate moiety. Designed to combine the MAO-B inhibitory activity of rasagiline with AChE inhibitory activity of rivastigmine, ladostigil inhibits AChE and both MAO-A and B selectively in the brain. At much lower doses than those that inhibit either enzyme in vivo ladostigil has neuroprotective activity associated with a reduction of oxidative stress and microglial activation, neither of which is related to the ability to inhibit MAO or AChE.

Salts of ladostigil include the 1/2 L-tartrate salt of ladostigil. This tartrate salt of ladostigil, ladostigil tartrate-6-(N-ethyl, N-methyl carbamyloxy)-N-propargyl-l(R)- aminoindan, tartaric acid (2: 1) abbreviated as [(R)-CPAI] tartrate and also referred to as ladostigil tartrate, has CAS registry number 209394-46-7 and may be used as the active ingredient of ladostigil tablets. U.S. Patent Nos. 6,251 ,938, 6,303,650, and 6,538,025, incorporated herein by reference, disclose ladostigil and other compounds that inhibit AChE and MAO selectively in the brain. These compounds may be useful to treat Alzheimer's disease and other dementias such as senile dementia, dementia of the Parkinson's type, vascular dementia and Lewy body dementia, in addition to depression.

U.S. Patent No. 7,335,685 and U.S. Application Publication Nos. 20060189819, 20070088082 and 20070093549, incorporated herein by reference, disclose crystalline forms of ladostigil tartrate and methods of producing the same.

U.S. Patent Nos. 7,375,249 and 7,476,757, and U.S. Application Publication No. 20060199974, incorporated herein by reference, disclose synthesis of enantiomeric indanylamine derivatives including ladostigil.

U.S. Patent No. 7,491,847 and U.S. Application Publication No. 200701 12217, incorporated herein by reference, disclose methods for isolating propargylated aminoindans.

U.S. Application Publication No. 20060189685, incorporated herein by reference, discloses formulations comprising ladostigil.

U.S. Application Publication Nos. 20070135518 and 20070293583, incorporated herein by reference, discloses the use of low doses of ladostigil for neuroprotection.

U.S. Application Publication No. 20070203232, incorporated herein by reference, discloses methods for isolating propargylated aminoindans and discloses their use in the treatment of Alzheimer's disease.

U.S. Application Publication No. 20070232691, incorporated herein by reference, discloses the use of ladostigil to treat schizophrenia.

Ladostigil has two pharmacological activities. One is its ability to inhibit AChE. The other is its ability to inhibit MAO. In vivo, ladostigil inhibits both MAO-A and MAO-B selectively in the brain. These activities make ladostigil particularly useful in the treatment of Alzheimer's disease.

One problem associated with the use of AChE inhibitors is the high degree of side effects which develop upon oral administration. These side effects include nausea, vomiting and gastrointestinal discomfort. Minimizing these side effects simply by limiting the administered dose may not always be applicable, because the efficacy of the drug may be impaired at lower doses. There remains a need for methods and formulations of ladostigil and pharmaceutically acceptable salts thereof which can effectively treat Alzheimer's disease while minimizing side effects associated with therapies having cholinesterase inhibitory activity. SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions and dosage units comprising ladostigil and methods of using the same to treat subjects diagnosed with Alzheimer's disease, Parkinson's disease and other neurodegenerative disorders.

The present invention is based in part on the unexpected finding that ladostigil' s activity coincides with its level of absorption only until it reaches about 40% AChE inhibition after which activity falls off relative to its absorption level. Furthermore, it was found that approximately 30-40% of AChE inhibition in the plasma and cerebral cortex is sufficient in order to exert a therapeutic effect on cognitive function. The unexpected pharmacokinetic profile of ladostigil indicates the advantage of administrating doses of ladostigil in the range of 60-250 mg ladostigil per day for improving cognitive function while minimizing side effects associated with the inhibition of AChE activity.

In another embodiment, the present invention is based, in part on the unexpected finding that ladostigil should be administered in a daily dose of up to 200 mg.

The present invention further provides methods for improving cognitive function in a subject afflicted with Alzheimer's disease, comprising administering to the subject for at least 28 continuous days, a daily dose of 60 to 200 mg or 60-250 mg ladostigil or a pharmaceutically active salt thereof.

The present invention further provides methods for improving cognitive function while minimizing side effects associated with the inhibition of AChE activity in a subject in need thereof, comprising administering to the subject for at least 28 continuous days, a daily dose of 60 to 250 mg ladostigil or a pharmaceutically active salt thereof.

The present invention further provides methods for improving cognitive function while minimizing side effects associated with the inhibition of AChE activity in a subject afflicted with Alzheimer's disease, comprising administering to the subject for at least 28 continuous days, a daily dose of 60 to 250 mg ladostigil or a pharmaceutically active salt thereof. The present invention further provides methods for improving cognitive function while minimizing side effects associated with the inhibition of AChE activity in a subject in need thereof, comprising daily administration to the subject of a first dose and a second dose of 30 to 40 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administration to the subject of a first dose and a second dose of 40 to 60 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administration to the subject a first dose and a second dose of 50 to 70 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administering to the subject a first dose and a second dose of 70 to 95 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, wherein the second dose is administered at least 8 hours after the first dose, thereby improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof.

One aspect of the present invention relates to methods of treating individuals who have been identified as having Alzheimer's disease comprising the step of chronic single daily dose administration of 60-125 mg ladostigil or a pharmaceutical salt thereof formulated for immediate release.

Another aspect of the present invention relates to methods of treating individuals who have been identified as having Alzheimer's disease comprising the step of chronic administration twice daily of 60-65 mg ladostigil or a pharmaceutical salt thereof per dose (120-130 mg/day) formulated for immediate release.

A further aspect of the present invention relates to methods of treating individuals who have been identified as having Alzheimer's disease comprising the step of chronic administration twice daily of 75-100 mg ladostigil or a pharmaceutical salt thereof per dose (150-200 mg/day) formulated for immediate release. According to some embodiments, the present invention relates to methods of treating individuals who have been identified as having Alzheimer's disease comprising the step of chronic administration twice daily of 80 mg ladostigil or a pharmaceutical salt thereof per dose (160 mg/day) formulated for immediate release.

According to other embodiments, the present invention is directed to the use of ladostigil or a pharmaceutical salt thereof in the preparation of a medicament for treating individuals who have Alzheimer's disease, wherein the use is selected from the group consisting of: chronic single daily dose administration of 60-125 mg ladostigil or a pharmaceutical salt thereof;

chronic administration twice daily of 60-65 mg ladostigil or a pharmaceutical salt thereof per dose (120-130 mg/day) ;

chronic administration twice daily of 75-100 mg ladostigil or a pharmaceutical salt thereof per dose (150-200 mg/day); and

In some embodiments, the ladostigil or a pharmaceutically active salt thereof is administered in two equal divided doses per day.

In other embodiments, the ladostigil or a pharmaceutically active salt thereof is administered in two equal doses of 80-120 mg each per day.

According to further embodiments, the ladostigil or a pharmaceutically active salt thereof is administered in two equal doses of 80 mg each per day.

In yet further embodiments, the ladostigil or a pharmaceutically active salt thereof is administered in a single dose per day.

In other embodiments, the ladostigil or a pharmaceutically active salt thereof is formulated for immediate release.

In yet other embodiments, the ladostigil or a pharmaceutically active salt thereof is ladostigil tartrate.

Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the chemical structure of ladostigil [(R)-CPAI] (lA); and a graph indicating the mean concentration-time curves for (R)-CPAI in the three cohorts (IB).

Figure 2 shows the chemical structure of (R)-MCPAI (2A); and a graph indicating the mean concentration-time curves for (R)-MCPAI in the three cohorts (2B).

Figure 3 shows the chemical structure of (R)-ECPAI (3A); and a graph indicating the mean concentration-time curves for (R)-ECPAI in the three cohorts (3B).

Figure 4 shows the chemical structure of (R)-CAI (4A); and a graph indicating the mean concentration-time curves for (R)-CAI in the three cohorts (4B). Figure 5 shows the chemical structure of (R)-HPAI (5A); and a graph indicating the mean concentration-time curves for (R)-CPAI in the three cohorts (5B).

Figure 6 shows the chemical structure of (R)-HCPAl (6A); and a graph indicating the mean concentration-time curves for (R)-CPAI in the three cohorts (6B).

Figure 7 shows the chemical structure of (R)-MCAI (7A); and a graph indicating the mean concentration-time curves for (R)-CPAI in the three cohorts (7B).

Figure 8 shows the chemical structure of (R)-ECAI (8A); and a graph indicating the mean concentration-time curves for (R)-CPAI in the three cohorts (8B).

Figure 9 is a graph showing the kinetics of AChE and BuChE inhibition at termination.

Figure 10 is a bar graph showing MAO-B inhibition and reduction in plasma DHPG levels in the three cohorts at termination.

Figure 11 The impact of metabolic alterations: the chemical structure of 1 1A shows the metabolic susceptible positions on (R)-CPAI. Position #1 represents the site of hydrolysis of the carbamate group by ChE forming the metabolite (R)-HPAI; Position #2 represents the site of dealkylation by CYP2C19 forming the metabolite (R)-MCPAI and Position #3 is the cleavage site of the propargylamine group by CYP1A2 upon which the secondary metabolite (R)-MCAI is formed from (R)-MCPAI; the table of 1 IB shows the in vitro enzymatic inhibitory activity of ladostigil and its major metabolites.

Figure 12 is a scheme summarizing the Open-label phase study-titration.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention relates to pharmaceutical compositions and dosage units comprising ladostigil or active salts thereof and methods of using the same for treating subjects suffering from neurodegenerative disease such as but not limited to Alzheimer's disease and Parkinson's disease. In one embodiment, the present invention provides that unexpectedly ladostigil's activity coincides with its level of absorption only until it reaches about 40% cholinesterase (AChE) inhibition after which activity falls off relative to its absorption level. In another embodiment, approximately 30-40% of AChE inhibition in the plasma and cerebral cortex is sufficient in order to exert a therapeutic effect on cognitive function. In another embodiment, the unexpected pharmacokinetic profile of ladostigil indicates that administrating doses in the range of 60-200 mg ladostigil per day provides improvement of cognitive function coupled to reduction of side effects associated with the inhibition of AChE activity. In another embodiment, the present invention provides that 60- 200 mg ladostigil per day minimizes undesired inhibition of AChE activity by ladostigil therapy.

In one embodiment, the present invention provides a method of improving cognitive function while minimizing side effects associated with the inhibition of AChE activity in a subject in need thereof, comprising administering to the subject 60 to 250 mg ladostigil or a pharmaceutically active salt thereof per day, thereby improving cognitive function while minimizing side effects associated with the inhibition of AChE activity in a subject in need thereof. In another embodiment, a range of 60 to 200 mg ladostigil or a pharmaceutically active salt thereof per day ensures efficacy while minimizing side effects associated with the inhibition of AChE activity. In another embodiment, a dose comprising from 40 to 100 mg ladostigil or a pharmaceutically active salt thereof ensures efficacy while minimizing side effects associated with the inhibition of AChE activity . In another embodiment, up to 2 doses of the invention per day ensure efficacy while minimizing side effects associated with the inhibition of AChE activity. In another embodiment, a higher daily dose of about 250 mg ladostigil is administered to patients with a slow metabolic rate, in order to provide sufficient levels of MCPAI.

In one embodiment, cognitive function refers to a person's ability to process thoughts. In another embodiment, cognitive function includes, inter-alia, memory, the ability to learn new information, speech, and reading comprehension. In another embodiment, cognitive function is impaired in an aging subject. In another embodiment, an aging subject is a subject in need thereof according to the invention. In another embodiment, a subject in need thereof is a subject suffering from a decline in cognitive function. In another embodiment, a subject in need thereof is a subject suffering from a certain loss in cognitive function. In another embodiment, a subject in need thereof is a subject suffering from multiple sclerosis (MS). In another embodiment, a subject in need thereof is a subject suffering from a neurodegenerative disease. In another embodiment, the neurodegenerative disease is Parkinson's, Alzheimer's, and Huntington's disease. In another embodiment, the neurodegenerative disease is Amyotrophic lateral sclerosis (ALS). In another embodiment, the neurodegenerative disease is ataxia telangiectasia, Batten disease, corticobasal degeneration, dementia, Amnesia, aphasia, Creutzfeldt-Jakob disease, Fatal familial insomnia, infantile refsum disease, Lyme disease, Machado-Joseph disease, Multiple system atrophy, Neuroacanthocytosis, Niemann-Pick disease, associated with Protein aggregation, Refsum disease, Sandhoff disease, Shy-Drager syndrome, Spinocerebellar ataxia, Subacute combined degeneration of spinal cord, Tabes dorsalis, Tay-Sachs disease, Toxic encephalopathy, Wobbly hedgehog syndrome, In another embodiment, the neurodegenerative disease occurs as a result of neurodegenerative processes.

In another embodiment, improving cognitive function includes enhancement of cognitive function and/or prevention of a decline in memory and thought. Doing activities such as word problems, memory problems, and mathematics may "exercise" the brain so that fewer cells die or become inactive over time. In another embodiment, improving cognitive function is increase in cognitive skills. In another embodiment, improving cognitive function includes restoration of cognitive function. In another embodiment, improving cognitive function includes improving the concentration ability. In another embodiment, improving cognitive function includes maintaining or improving the IQ score. In another embodiment, improving cognitive function includes minimizing episodic memory loss. In another embodiment, improving cognitive function includes minimizing episodic memory gaps. In another embodiment, improving cognitive function includes ameliorating amnesia. In another embodiment, improving cognitive function includes ameliorating spells of aphasia. In another embodiment, improving cognitive function includes ameliorating episodic problems with confusion. In another embodiment, improving cognitive function includes ameliorating problems with fine/gross motor coordination. In another embodiment, improving cognitive function includes ameliorating brief dramatic changes in emotional status. In another embodiment, minimizing side effects associated with the inhibition of ChE is minimizing the risk of SLUDGE syndrome. In another embodiment, side effects include but are not limited to: anorexia, nausea, vomiting, diarrhoea, insomnia, bradycardia, hypotension, hypersecretion, bronchoconstriction, GI tract hypermotility, prolonged muscle contraction, and decrease in intraocular pressure. In another embodiment, minimizing is abolishing. In another embodiment, minimizing is reducing the risk of any side effect or a combination of side effects within a treated population by at least 10%, 20%, 40%, 50%, 60%, 75%, 80%, or 90%. In another embodiment, minimizing is reducing the frequency of any side effect or a combination of side effects in a patient by at least 10%, 20%, 40%, 50%, 60%, 75%, 80%, or 90%.

In another embodiment, the present invention relates to methods of treating individuals afflicted with a neurodegenerative disease such as Alzheimer's disease, comprising the step of administering a single daily dose of 60-100 mg ladostigil or a pharmaceutical salt thereof. In another embodiment, the present invention relates to methods for treating individuals afflicted with a neurodegenerative disease such as Alzheimer's disease, comprising the step of administering twice daily 60-65 mg ladostigil or a pharmaceutical salt thereof per dose (daily dose of 120-130 mg/day). In another embodiment, the present invention relates to methods for treating individuals afflicted with a neurodegenerative disease such as Alzheimer's disease, comprising the step of administering twice daily 75-100 mg ladostigil or a pharmaceutical salt thereof per dose (daily dose of 150-200 mg/day). In another embodiment, the present invention relates to methods for treating individuals afflicted with a neurodegenerative disease such as Alzheimer's disease, comprising the step of administering twice daily 80 mg ladostigil or a pharmaceutical salt thereof per dose (daily dose of 160 mg/day). In another embodiment, the present invention relates to methods for treating individuals afflicted with a neurodegenerative disease such as Alzheimer's disease, comprising the step of administering twice daily 100 mg ladostigil or a pharmaceutical salt thereof per dose (200 mg/day). In another embodiment, the administration is a chronic administration. In another embodiment, the phrases "neurodegenerative disease" and "neurodegenerative disorder" are used interchangeably.

In another embodiment, the present invention provides that the phrase "daily dose" is the total dose of ladostigil or a salt thereof per day (24 hours). In another embodiment, the present invention provides that a "daily dose" is divided into 2 or more doses which are administered concomitantly or sequentially. In another embodiment, the present invention provides that a "daily dose" is divided into 2-4 doses which are administered concomitantly or sequentially. In another embodiment, the present invention provides that a "daily dose" is administered in one dose or once a day. In another embodiment, the present invention provides that a "daily dose" is administered in two doses or twice a day. In another embodiment, the present invention provides that a "daily dose" is divided into two doses. In another embodiment, the present invention provides that a dose is administered every 6, 7, 8, 9, 10, 11, or 12 hours.

In another embodiment, ladostigil or a pharmaceutical salt thereof is/are formulated for immediate release. In another embodiment, ladostigil or a pharmaceutical salt thereof is/are formulated for sustained release.

In another embodiment, the present invention is directed to the use of ladostigil or a pharmaceutical salt thereof in the preparation of a medicament for treating a subject afflicted with a neurodegenerative disease such as Alzheimer's disease, wherein the use is selected from the group consisting of: total daily administration of 60 to 200 mg ladostigil or a pharmaceutical salt thereof once a day or twice a day; total daily dose administration of 60- 100 mg ladostigil or a pharmaceutical salt thereof once a day or twice a day; chronic administration twice daily of 60-65 mg ladostigil or a pharmaceutical salt thereof per dose (120-130 mg/day); chronic administration twice daily of 75-100 mg ladostigil or a pharmaceutical salt thereof per dose (150-200 mg/day);). In some embodiments, ladostigil or a pharmaceutically active salt thereof is administered in two equal divided doses per day. In other embodiments, ladostigil or a pharmaceutically active salt thereof is administered in two equal doses of 80-120 mg each per day. According to further embodiments, ladostigil or a pharmaceutically active salt thereof is administered in two equal doses of 80 mg each per day. In yet further embodiments, ladostigil or a pharmaceutically active salt thereof is administered in a single dose per day. In yet other embodiments, a pharmaceutically active salt thereof is ladostigil tartrate. In another embodiment, the present invention is based on the unexpected finding that ladostigil should be administered in a daily dose of up to 200 mg.

In another embodiment, the present invention provides a method characterized by periodically increasing the amount of ladostigil or a pharmaceutically active salt thereof. In another embodiment, periodically increasing the amount of ladostigil or a pharmaceutically active salt thereof minimizes the risk of adverse events associated with AChE inhibition. In another embodiment, periodically is 4-10 days. In another embodiment, periodically is 6-8 days. In another embodiment, periodically is 7 days. In another embodiment, the present invention provides a method for improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof, comprising administering to the subject 30 to 40 mg ladostigil or a pharmaceutically active salt thereof twice a day for at least 4 days (equals to daily dose of 60-80 mg of ladostigil or a pharmaceutically active salt thereof), followed by administering to the subject 40 to 60 mg ladostigil or a pharmaceutically active salt thereof twice a day for at least 4 days (equals to daily dose of 80-120 mg of ladostigil or a pharmaceutically active salt thereof), followed by administering to the subject 50 to 70 mg ladostigil or a pharmaceutically active salt thereof twice a day for at least 4 days (equals to daily dose of 100-140 mg of ladostigil or a pharmaceutically active salt thereof), followed by administering to the subject 70 to 95 mg ladostigil or a pharmaceutically active salt thereof twice a day for at least 4 days (equals to daily dose of 140-190 mg of ladostigil or a pharmaceutically active salt thereof), thereby improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof.

In another embodiment, the present invention provides a method for improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof, comprising administering to the subject 30 mg ladostigil or a pharmaceutically active salt thereof twice a day for at least 4 days (equals to daily dose of 60 mg of ladostigil or a pharmaceutically active salt thereof), followed by administering to the subject a first dose of 40 mg ladostigil or a pharmaceutically active salt thereof and a second dose of 60 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days (equals to daily dose of 100 mg of ladostigil or a pharmaceutically active salt thereof), followed by administering to the subject 60 mg ladostigil or a pharmaceutically active salt thereof twice a day for at least 4 days (equals to daily dose of 120 mg of ladostigil or a pharmaceutically active salt thereof), followed by administering to the subject 80 mg ladostigil or a pharmaceutically active salt thereof twice a day for at least 4 days (equals to daily dose of 160 mg of ladostigil or a pharmaceutically active salt thereof), thereby improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof.

In another embodiment, the present invention provides a method for improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in afflicted with Alzheimer's disease, comprising daily administering to the subject a first dose and a second dose of 30 to 40 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administering to the subject a first dose and a second dose of 40 to 60 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administering to the subject a first dose and a second dose of 50 to 70 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administering to the subject a first dose and a second dose of 70 to 95 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, wherein the second dose is administered at least 4 hours after the first dose, thereby improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject afflicted with Alzheimer's disease.

In another embodiment, the present invention provides a method for improving cognitive function in a subject afflicted with Alzheimer's disease, comprising daily administering to the subject a first dose and a second dose of 30 to 40 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administering to the subject a first dose and a second dose of 40 to 60 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administering to the subject a first dose and a second dose of 50 to 70 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administering to the subject a first dose and a second dose of 70 to 95 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, wherein the second dose is administered at least 4 hours after the first dose, thereby improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof.

In another embodiment, the present invention provides a method for improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject afflicted with Alzheimer's disease, comprising administering to the subject 30 mg ladostigil or a pharmaceutically active salt thereof twice a day for at least 4 days, followed by administering to the subject a first dose of 40 mg ladostigil or a pharmaceutically active salt thereof and a second dose of 60 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by administering to the subject 60 mg ladostigil or a pharmaceutically active salt thereof twice a day for at least 4 days, followed by administering to the subject 80 mg ladostigil or a pharmaceutically active salt thereof twice a day for at least 4 days, thereby improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof.

In another embodiment, gradual increase in the dose of ladostigil or a pharmaceutically active salt thereof maintains the efficacy of the drug while minimizing side effects associated with AChE inhibition. In another embodiment, gradual increase in the dose of ladostigil or a pharmaceutically active salt thereof, in time periods of 4-8 days, maintains the efficacy of the drug while minimizing side effects associated with AChE inhibition. In another embodiment, the exact time period of "at least 4 days" includes determination of reduced side effects associated with AChE inhibition. In another embodiment, at least 4 days is at least 7 days. In another embodiment, at least 4 days is 7 days. In another embodiment, at least 4 days is 8 days. In another embodiment, at least 4 days is 9 days. In another embodiment, at least 4 days is 18 days. In another embodiment, gradual increase in the dose of ladostigil or a pharmaceutically active salt thereof, in time periods of at least 7 days, maintains the efficacy of the drug while minimizing side effects associated with AChE inhibition. In another embodiment, gradual increase in the dose of ladostigil or a pharmaceutically active salt thereof, in time periods of 7 days, maintains the efficacy of the drug while minimizing side effects associated with AChE inhibition. In another embodiment, the time period is designed for each patient individually according to blood measures as described herein and/or side effects associated with AChE inhibition as described herein.

In another embodiment, administering ladostigil or a pharmaceutically active salt thereof twice a day is administering equal doses of ladostigil or a pharmaceutically active salt thereof twice a day. In another embodiment, there is at least 6 hours gap between each dose. In another embodiment, there is at least 8 hours gap between each dose. In another embodiment, there is at least 10 hours gap between each dose. In another embodiment, there is at least 12 hours gap between each dose.

In another embodiment, the first dose is administered during the morning hours. In another embodiment, the first dose, the second dose or both is/are administered prior to eating and/or drinking. In another embodiment, the first dose is administered prior to eating and/or drinking in the morning hours. In another embodiment, the first dose is administered at least 15 minutes prior to eating and/or drinking in the morning hours. In another embodiment, the first dose, the second dose or both is/are administered at least 15 minutes prior to eating and/or drinking. In another embodiment, the first dose, the second dose or both is/are administered at least 30 minutes prior to eating and/or drinking. In another embodiment, the first dose, second dose or both is/are administered at least 45 minutes prior to eating and/or drinking. In another embodiment, the first dose, second dose or both is/are administered at least 60 minutes prior to eating and/or drinking. In another embodiment, the first dose, second dose or both is/are administered at least 75 minutes prior to eating and/or drinking. In another embodiment, the first dose, second dose or both is/are administered at least 90 minutes prior to eating and/or drinking. In another embodiment, daily administering is administering within 24 hours. In another embodiment, daily administering is administering from awakening to two hours before bedtime.

In another embodiment, administering ladostigil or a pharmaceutically active salt thereof twice a day is administering ladostigil or a pharmaceutically active salt thereof in a first dose and a second dose. In another embodiment, the first dose comprises equal or less amount of ladostigil or a pharmaceutically active salt thereof than the second dose.

In another embodiment, twice a day includes a first dose and a second dose. In another embodiment, a first dose is administered in the morning hours. In another embodiment, a second dose is administered in the evening hours. In another embodiment, a second dose is administered 3-6 hours before bed time. In another embodiment, a second dose is administered before 20:00, 19:00, or 18:00. In another embodiment, both the first dose and the second dose are administered prior to eating and/or drinking as provided herein. In another embodiment, the first dose is administered upon awakening and the second dose is administered 3-6 hours before bed time. In another embodiment, the first dose is administered at least 6 hours before the second dose. In another embodiment, the first dose is administered at least 7 hours before the second dose. In another embodiment, the first dose is administered at least 8 hours before the second dose. In another embodiment, the first dose is administered at least 5-8 hours before the second dose.

In another embodiment, 30 to 40 mg ladostigil or a pharmaceutically active salt thereof twice a day (or a first and a second dose) is 40 mg ladostigil or a pharmaceutically active salt thereof twice a day (a first and a second dose, each of 40 mg ladostigil or a pharmaceutically active salt thereof). In another embodiment, two doses (or a first and a second dose) of 40 to 60 mg ladostigil or a pharmaceutically active salt thereof is one or first dose of 40 mg ladostigil or a pharmaceutically active salt thereof and one or second dose of 60 mg ladostigil or a pharmaceutically active salt thereof. In another embodiment, 40 to 60 mg ladostigil or a pharmaceutically active salt thereof twice a day (or a first and a second dose) is 50 mg ladostigil or a pharmaceutically active salt thereof twice a day (a first and a second dose, each of 50 mg ladostigil or a pharmaceutically active salt thereof). In another embodiment, 50 to 70 mg ladostigil or a pharmaceutically active salt thereof twice a day (or a first and a second dose) is 60 mg ladostigil or a pharmaceutically active salt thereof twice a day (a first and a second dose, each of 60 mg ladostigil or a pharmaceutically active salt thereof). In another embodiment, 70 to 95 mg ladostigil or a pharmaceutically active salt thereof twice a day (or a first and a second dose) is 80 mg ladostigil or a pharmaceutically active salt thereof twice a day (a first and a second dose, each of 80 mg ladostigil or a pharmaceutically active salt thereof).

In another embodiment, administration as described herein is chronic administration. As used herein, the term "chronic administration" refers to repeated administration of pharmaceutical compositions comprising a specific amount of active ingredient for at least 12 days, at least 16 days, at least 21 days, or four continuous weeks. Preferably such administration is repeated for at least 12 weeks or more, 24 weeks or more or 52 weeks or more.

In another embodiment, Ladostigil and its pharmaceutically acceptable salts, when administered in the dosages and formulations disclosed herein provide unexpectedly minimal undesired side effect with good safety and efficacy profiles. In another embodiment, ladostigil and its pharmaceutically acceptable salts, when administered in the dosages and formulations disclosed herein minimize side effects associated with the inhibition of AChE activity. In another embodiment, ladostigil and its pharmaceutically acceptable salts are administered in multiple doses or as a single dose. In either case, the amount of drug released and available for absorption ensure that side effects are minimized while exposing the subject to sufficient drug to provide a clinically beneficial effect.. In some embodiments, the multiple dose regimen provides two equal doses per day.

Single doses are provided which may be formulated for immediate. Immediate release formulations may be administered in a single dose with sufficient drug to inhibit ChE for a sufficient amount of time, e.g. 6, 8 or 12 hours, to impart a clinically beneficial effect.

Pharmaceutically active salt of ladostigil may include for example hydrochloride, sulfate, tartrate, maleate, citrate, phosphate, acetate, lactate, fumarate, hydrobromide, mesylate, pamoate, hydroiodide, nitrate, and methylsulfate. In some embodiment, ladostigil is provided as ladostigil hemitartrate. In another embodiment, the term "Ladostigil" includes its metabolites. In another embodiment, Ladostigil metabolites include: 1) (R)-HCPAI; 2) (R)-MCPAI; 3) (R)-ECPAI; 4) (R)-CAI; 5) (R)-HPAI; 6) (R)-MCAI and 7) (R)-ECAI. In another embodiment, a metabolite of the invention is formed through the actions of CYP 450 isozymes in the liver, with the exception of (R)-HPAI, which is the result of ladostigil hydrolysis by AChE. In another embodiment, the metabolites listed above inhibit AChE at different concentrations with the exception of (R)-HPAI which does not inhibit AChE but is a potent inhibitor of MAO activity.

In another embodiment, studies in rodents, monkeys and humans surprisingly indicate that the concentrations of ladostigil metabolites (R)-MCPAI and (R)-MCAI found in the blood after ladostigil administration were the only ones high enough to contribute to AChE inhibition, while the concentrations of the other carbamate-containing metabolites were too low to cause AChE inhibition (see Figure 1 1A and B). It is to be further emphasized that blood concentrations of (R)-HPAI were the only ones sufficient to inhibit MAO-A and MAO- B. The increase in the blood level of (R)-MCPAI, the major metabolite responsible for AChE inhibition, is only proportional to the dose of ladostigil within the range of 40-1 10 mg; higher doses of ladostigil do not result in higher levels of AChE inhibition.

In another embodiment, the plateau reached in (R)-MCPAI levels and AChE inhibition could be due to limitation of the rate of conversion of (R)-CPAI by CYP2C19 in the liver to (R)-MCPAI and its further metabolism by CYP1A2 to (R)-MCAI. Thus, according to embodiments of the present invention, it is surprisingly disclosed that there is no advantage in giving doses of ladostigil in excess of 1 10 mg, as this does not result in increased blood levels of (R)-MCPAI. In another embodiment, each dose of a pharmaceutical composition of the invention comprises 40 to 1 10 mg of ladostigil or a salt thereof.

In addition, as MAO inhibition by (R)-HPAI is irreversible, clinically relevant inhibition of both isoenzymes (MAO-A and MAO-B) can be obtained in the dose range of ladostigil of 40-1 10 mg after 4-5 weeks of treatment, or in other embodiments in the dose range of ladostigil of 60-125 mg after 4-5 weeks of treatment.

Thus, embodiments of the invention are directed to a method of treating an individual afflicted with a neurodegenerative disease comprising chronic administration of ladostigil or a pharmaceutical salt thereof in a dosage selected from the group comprising or consisting of: single daily dose administration of 60-125 mg; twice daily administration of 60-65 mg per dose (120-130 mg/day); twice daily administration of 75-100 mg per dose (150-200 mg/day); In some embodiments, methods comprise chronic single daily dose administration of 60-125 mg ladostigil or a pharmaceutical salt thereof formulated for immediate release. In some embodiments, methods comprise chronic single daily dose administration of 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 1 15 or 120 mg ladostigil or a pharmaceutical salt thereof formulated for immediate release. In some embodiments, methods comprise chronic single daily dose administration of 60-120 mg, 60-115 mg, 60-1 10 mg, 60-105 mg, 60-100 mg, 60- 95 mg, 60-90 mg, 60-85 mg, 60-80 mg, 60-75 mg or 60-70 mg. In some embodiments, methods comprise chronic single daily dose administration of 65-125 mg, 65-120 mg, 65-1 15 mg, 65-1 10 mg, 65-105 mg, 65-100 mg, 65-95 mg, 65-90 mg, 65-85 mg, 65-80 mg, 65-75 mg or 65-70 mg; In some embodiments, methods comprise chronic single daily dose administration of 70-125 mg, 70-120 mg, 70-1 15 mg, 70-1 10 mg, 70-105 mg, 70-100 mg, 70- 95 mg, 70-90 mg, 70-85 mg, 70-80 mg or 70-75 mg. In some embodiments, methods comprise chronic single daily dose administration of 75-125 mg, 75-120 mg, 75-1 15 mg, 75- 1 10 mg, 75-105 mg, 75-100 mg, 75-95 mg, 75-90 mg, 75-85 mg or 75-80 mg. In some embodiments, methods comprise chronic single daily dose administration of 80-125 mg, 80- 120 mg, 80-115 mg, 80-1 10 mg, 80-105 mg, 80-100 mg, 80-95 mg, 80-90 mg or 80-85 mg. In some embodiments, methods comprise chronic single daily dose administration of 85-125 mg, 85-120 mg, 85-115 mg, 85-110 mg, 85-105 mg, 85-100 mg, 85-95 mg or 85-90 mg. In some embodiments, methods comprise chronic single daily dose administration of 90-125 mg, 90- 120 mg, 90-115 mg, 90-1 10 mg, 90-105 mg, 90-100 mg or 90-95 mg. In some embodiments, methods comprise chronic single daily dose administration of 95-125 mg, 95-120 mg, 95-1 15 mg, 95-1 10 mg, 95-105 mg or 95-100 mg

In some embodiments, methods comprise chronic administration twice daily of 60-65 mg ladostigil or a pharmaceutical salt thereof per dose (120-130 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 60 mg ladostigil or a pharmaceutical salt thereof per dose (120 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 65 mg ladostigil or a pharmaceutical salt thereof per dose (130 mg/day) formulated for immediate release.

In some embodiments, methods comprise chronic administration twice daily of 75-100 mg ladostigil or a pharmaceutical salt thereof per dose (150-200 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 75 mg ladostigil or a pharmaceutical salt thereof per dose (150 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 80 mg ladostigil or a pharmaceutical salt thereof per dose (160 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 85 mg ladostigil or a pharmaceutical salt thereof per dose (170 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 90 mg ladostigil or a pharmaceutical salt thereof per dose (190 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 95 mg ladostigil or a pharmaceutical salt thereof per dose (190 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of ladostigil or a pharmaceutical salt thereof formulated for immediate release wherein each dose comprises 75-90 mg ladostigil or a pharmaceutical salt thereof per dose (150-180 mg/day), 75-85 mg ladostigil or a pharmaceutical salt thereof per dose (150-170 mg/day), 75- 80 mg ladostigil or a pharmaceutical salt thereof per dose (150-160 mg/day), 80-95 mg ladostigil or a pharmaceutical salt thereof per dose (160-190 mg/day), 80-90 mg ladostigil or a pharmaceutical salt thereof per dose (160-180 mg/day), 80-85 mg ladostigil or a pharmaceutical salt thereof per dose (160-170 mg/day), 85-95 mg ladostigil or a pharmaceutical salt thereof per dose (170-190 mg/day), 85-90 mg ladostigil or a pharmaceutical salt thereof per dose (170-180 mg/day) or 90-95 mg ladostigil or a pharmaceutical salt thereof per dose (180-190 mg/day).

In some embodiments, methods comprise chronic administration twice daily of 105-

125 mg ladostigil or a pharmaceutical salt thereof per dose (210-250 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 105 mg ladostigil or a pharmaceutical salt thereof per dose (210 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 1 10 mg ladostigil or a pharmaceutical salt thereof per dose (220 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 1 15 mg ladostigil or a pharmaceutical salt thereof per dose (230 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 120 mg ladostigil or a pharmaceutical salt thereof per dose (240 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of 125 mg ladostigil or a pharmaceutical salt thereof per dose (250 mg/day) formulated for immediate release. In some embodiments, methods comprise chronic administration twice daily of ladostigil or a pharmaceutical salt thereof formulated for immediate release wherein each dose comprises less than 105 mg ladostigil or a pharmaceutical salt thereof per dose.

In some embodiments, dosages and formulations such as any of the aforementioned dosages and formulations are prepared with release profile such that the maximum concentration of ladostigil's metabolites (R)-MCPAI and (R)-MCAI in blood plasma does not exceed 400 ng/ml and 80 ng/ml respectively. According to certain particular embodiments, the range of concentration of (R)-MCPAI in blood plasma lies between 1 0-450ng/ml. In other embodiments, the concentration of (R)-MCPAI in blood plasma is up to 300 or 350 ng/ml and is at least 100, 150 or 200 ng/ml. In a particular embodiment, the concentration of (R)-MCPAI in blood plasma is about 250 ng/ml. According to certain other particular embodiments, the maximum concentration of (R)-MCAI in blood plasma does not exceed 80, 50 or 25 ng/ml. In other embodiments, the concentration of (R)-MCAI in blood plasma is at least 15 ng/ml. In a particular embodiment, the concentration of (R)-MCAI in blood plasma is about 60 ng/ml. At such dosages, a clinically effective level of AChE inhibitory activity may be achieved while remaining significantly below doses that would ordinarily be considered acceptable from the standpoint of side effects. The AChE inhibitory activity observed when ladostigil or a pharmaceutical salt thereof is administered does not coincide with the level of ladostigil absorption at higher doses.

Thus, while ladostigil is taken up in a somewhat linear fashion, the observed inhibition of AChE activity does not increase at a corresponding rate. Lower level dosages result in fewer side effects without a proportional reduction in effectiveness. Unexpectedly, the drug's activity coincides with its level of absorption until it reaches about 40% AChE inhibition after which activity falls off relative to absorption level. Data obtained from a multiple dose pharmacokinetic (PK) and pharmacodynamic (PD) study indicated that at lower doses of ladostigil the percentage of AChE inhibition increases dose proportionally and with longer treatment duration. Yet, at high ladostigil doses, AChE inhibition levels off at 60-70%, in spite of increasing blood levels of ladostigil [(R)-CPAI] and its major active metabolites (R)- MCPAI and (R)-MCAI. Thus, the present invention surprisingly discloses that administering ladostigil at its maximal tolerable dose based on pharmacokinetics, previously considered clinically preferential, in fact does not provide an enhanced therapeutic effect. Rather, it is unexpectedly disclosed herein, that dosages and formulations such as any of the aforementioned dosages and formulations disclosed by the invention provide clinically effective therapeutic effects with minimal side effects. Pharmaceutical compositions may be administered by any route that provides the safe, clinically effective amounts of ladostigil or pharmaceutically acceptable salts thereof. In some embodiments, the drug is provided by oral administration.

In another embodiment, the pharmaceutical composition is an oral immediate release composition. In another embodiment, the term "immediate release" pharmaceutical formulation includes any formulation in which the rate of release of ladostigil or a pharmaceutically acceptable salt thereof from the formulation and/or the absorption of drug, is neither appreciably, nor intentionally, retarded by galenic manipulations. In the present case, immediate release may be provided for by way of an appropriate pharmaceutically acceptable diluent or carrier, which diluent or carrier does not prolong, to an appreciable extent, the rate of drug release and/or absorption. In another embodiment, the term excludes formulations which are adapted to provide for "modified", "controlled", "sustained", "prolonged", "extended" or "delayed" release of ladostigil or a pharmaceutically acceptable salt thereof.

Pharmaceutical compositions that comprise ladostigil or pharmaceutically active salts thereof and pharmaceutically acceptable carriers or diluents are provided. The pharmaceutical compositions may be formulated by one having ordinary skill in the art. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field, which is incorporated herein by reference.

Unit dosage forms comprise the active ingredient ladostigil or a pharmaceutically active salt thereof ladostigil and pharmaceutically excipients or carriers such as fillers, disintegrants, lubricants, glidants, and soluble and insoluble polymers. In another embodiment, a pharmaceutical composition of the invention comprises a metabolite of ladostigil. In another embodiment, a pharmaceutical composition of the invention comprises a metabolite of ladostigil and is free of the parent drug.

Examples of fillers include water-soluble compressible carbohydrates such as sugars

(e.g., dextrose, sucrose, maltose, and lactose); sugar-alcohols ( e.g., mannitol, sorbitol, malitol, xylitol); starch hydro lysates (e.g., dextrins, maltodextrins, and the like); water insoluble plastically deforming materials (e.g., microcrystalline cellulose or other cellulosic derivatives); and water-insoluble brittle fracture materials (e.g., dicalcium phosphate, tricalcium phosphate and the like and mixtures thereof).

Examples of binders include dry binders such as polyvinyl pyrrolidone, hydroxypropylmethylcellulose, and the like; wet binders such as water-soluble polymers, including hydrocolloids such as acacia, alginates, agar, guar gum, locust bean, carrageenan, carboxymethylcellulose, tara, gum arabic, tragacanth, pectin, xanthan, gellan, gelatin, maltodextrin, galactomannan, pusstulan, laminarin, scleroglucan, inulin, whelan, rhamsan, zooglan, methylan, chitin, cyclodextrin, chitosan, polyvinyl pyrrolidone, cellulosics, sucrose, starches, and the like; and derivatives and mixtures thereof.

Examples of disintegrants for making a core or core portion by compression include sodium starch glycolate, cross-linked polyvinylpyrrolidone, cross-linked carboxymethylcellulose, starches, microcrystalline cellulose, and the like.

Examples of lubricants for making a core or core portion by compression include long chain fatty acids and their salts, such as magnesium stearate and stearic acid, talc, glycerides and waxes.

Examples of glidants for making a core or core portion by compression include colloidal silicon dioxide, and the like.

Examples of polymers include hydrophilic polymers and materials, insoluble polymers and materials, pH-dependent polymers, and the like. Hydrophilic materials include: water swellable cellulose derivatives, polyalkalene glycols, thermoplastic polyalkalene oxides, acrylic polymers, hydrocolloids, clays, gelling starches, and swelling cross-linked polymers, and derivatives, copolymers, and combinations thereof. Examples of cellulose derivatives include sodium carboxymethylcellulose, cross-linked hydroxypropylcellulose, hydroxypropyl cellulose (HPC), hydroxypropylmethylcellulose (HPMC), hydroxyisopropylcellulose, hydroxybutylcellulose, hydroxyphenylcellulose, hydroxyethylcellulose (HEC), hydroxypentylcellulose, hydroxypropylethylcellulose, hydroxypropylbutylcellulose, hydroxypropylethylcellulose. Examples of polyalkalene glycols include polyethylene glycol. Examples of thermoplastic polyalkalene oxides include poly (ethylene oxide). Examples of acrylic polymers include potassium methacrylatedivinylbenzene copolymer, polymethylmethacrylate, CARBOPOL (high-molecular weight cross-linked acrylic acid homopolymers and copolymers), and the like. Examples of hydrocolloids include alginates, agar, guar gum, locust bean gum, kappa carrageenan, iota carrageenan, tara, gum arabic, tragacanth, pectin, xanthan gum, gellan gum, maltodextrin, galactomannan, pusstulan, laminarin, scleroglucan, gum arabic, inulin, pectin, gelatin, whelan, rhamsan, zooglan, methylan, chitin, cyclodextrin, chitosan. Examples of clays include smectites such as bentonite, kaolin, and laponite; magnesium trisilicate, magnesium aluminum silicate, and the like, and derivatives and mixtures thereof. Examples of gelling starches include acid hydrolyzed starches, swelling starches such as sodium starch glycolate, and derivatives thereof. Examples of cross-linked polymers include cross-linked polyvinyl pyrrolidone, cross- linked agar, and cross-linked carboxymethylcellose sodium.

Examples of insoluble materials include water-insoluble polymers, and low-melting hydrophobic materials. Examples of water-insoluble polymers include ethylcellulose, polyvinyl alcohols, polyvinyl acetate, polycaprolactones, cellulose acetate and its derivatives, acrylates, methacrylates, acrylic acid copolymers; and the like and derivatives, copolymers, and combinations thereof. Examples of low-melting hydrophobic materials include fats, fatty acid esters, phospholipids, and waxes. Examples of fats include hydrogenated vegetable oils such as for example cocoa butter, hydrogenated palm kernel oil, hydrogenated cottonseed oil, hydrogenated sunflower oil, and hydrogenated soybean oil; and free fatty acids and their salts. Examples of fatty acid esters include sucrose fatty acid esters, mono, di, and triglycerides, glyceryl behenate, glyceryl palmitostearate, glyceryl monostearate, glyceryl tristearate, glyceryl trilaurylate, glyceryl myristate, GlycoWax-932, lauroyl macrogol-32 glycerides, and stearoyl macrogol-32 glycerides. Examples of phospholipids include phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, and phosphatidic acid. Examples of suitable waxes include carnauba wax, spermaceti wax, beeswax, candelilla wax, shellac wax, microcrystalline wax, and paraffin wax; fat-containing mixtures such as chocolate; and the like.

pH-dependent polymers include enteric cellulose derivatives, for example hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, cellulose acetate phthalate; natural resins such as shellac and zein; enteric acetate derivatives such as for example polyvinylacetate phthalate, cellulose acetate phthalate, acetaldehyde dimethylcellulose acetate; and enteric acrylate derivatives such as for example polymethacrylate-based polymers such as poly(methacrylic acid, methyl methacrylate) 1 :2, and poly(methacrylic acid, methyl methacrylate) 1 : 1, and the like, and derivatives, salts, copolymers, and combinations thereof.

Other excipients may include preservatives; sweeteners such as aspartame, acesulfame potassium, sucralose, and saccharin; flavourants; colourants; antioxidants; surfactants; wetting agents; and the like and mixtures thereof. Dosage unit forms may be coated with polishes and the like.

In one embodiment, the oral dosage form comprises predefined release profile. In one embodiment, the oral dosage form of the present invention comprises an extended release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form of the present invention comprises a slow release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form of the present invention comprises an immediate release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form is formulated according to the desired release profile of the pharmaceutical active ingredient as known to one skilled in the art.

Peroral compositions, in some embodiments, comprise liquid solutions, emulsions, suspensions, and the like. In some embodiments, pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art. In some embodiments, liquid oral compositions comprise from about 0.001% to about 0.933% of ladostigil or a salt thereof, or in another embodiment, from about 0.01% to about 10 %.

In some embodiments, compositions for use in the methods of this invention comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of the compounds of the present invention and optionally, other compounds, intended for topical intranasal administration. In some embodiments, the compositions comprise from about 0.001% to about 10.0% w/v of ladostigil or a salt thereof, more preferably from about 00.1% to about 2.0, which is used for systemic delivery of ladostigil or a salt thereof by the intranasal route.

In another embodiment, the pharmaceutical compositions are administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation. In some embodiments, liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment, the pharmaceutical compositions are administered intravenously, and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially, and are thus formulated in a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical compositions are administered intramuscularly, and are thus formulated in a form suitable for intramuscular administration.

In one embodiment, pharmaceutical compositions of the present invention are manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. In one embodiment, pharmaceutical compositions for use in accordance with the present invention is formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. In one embodiment, formulation is dependent upon the route of administration chosen.

In one embodiment, injectables, of the invention are formulated in aqueous solutions. In one embodiment, injectables, of the invention are formulated in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. In some embodiments, for transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

In one embodiment, the preparations described herein are formulated for parenteral administration, e.g., by bolus injection or continuous infusion. In some embodiments, formulations for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some embodiments, compositions are suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

The compositions also comprise, in some embodiments, preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulphate and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases to adjust the pH of these aqueous compositions as needed. The compositions also comprise, in some embodiments, local anaesthetics or other actives. The compositions can be used as sprays, mists, drops, and the like.

In some embodiments, pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients, in some embodiments, are prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include, in some embodiments, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions contain, in some embodiments, substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In another embodiment, the suspension also contains suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

Example 1 - Preclinical and phase I studies: Ladostigil tablets comprising ladostigil tartrate as the active ingredient have been used in preclinical and clinical studies. These studies had shown that ladostigil [(R)-CPAI] and its major metabolites, in particular (R)- MCPAI and (R)-MCAI, inhibit both AChE and BuChE. Although ladostigil has only very weak MAO-A and MAO-B inhibitory activity in vitro, on chronic oral administration it was found to inhibit both MAO-A and B selectively in the brain, due to the formation of an active metabolite, (R)-HPAI. Ladostigil has neuroprotective activity in vitro and in different animal models and stimulates conversion of amyloid precursor protein (APP) via a-secretase to the non-amyloidogenic sAPP. It also protects against drug-induced memory deficits and demonstrates anti-depressant activity. These unique pharmacodynamic properties indicate that treatment with ladostigil may improve not only cognitive function but also the behavioural and psychological symptoms frequently seen in AD patients.

Phase I (^"tolerability and safety in healthy volunteers): In a double-blind, placebo controlled study the tolerability and safety, pharmacokinetics, and pharmacodynamics of a single-dose of ladostigil administered over a range of 2.5 to 200 mg, were evaluated in 90 healthy male volunteers aged 18-40. The Maximal Tolerated Dose (MTD) was defined as the dose immediately below the minimum un-tolerated dose based on multiple-dose administration of the drug to panels of 6 patients receiving progressively higher doses. From these assessments it was apparent that ladostigil could be considered to be safe and well tolerated over the dose range of 2.5 to 175 mg and the 175 mg dose was thus considered to be the MTD. Most of the adverse events after active treatment occurred in the subjects receiving 175 and 200 mg. The main symptoms were nausea and vomiting. Dizziness, pallor and increased sweating also occurred and are all consistent with treatment with AChE inhibitors.

All adverse events (AE) had resolved at the follow-up visit, which was conducted at least five days after drug administration. The incidence of AEs correlated with the degree of inhibition of AChE activity in plasma, which reached an average of about 50-55% in groups dosed with 175 and 200 mg base.

Example 2 - Phase lb study

The current study was a multi-centre, double blind, placebo-controlled, randomized, sequential cohort, escalating dose study design aimed at assessing the tolerability and safety of ladostigil in AD patients based on changes in vital signs, ECG, laboratory parameters and adverse events reports following a dose titration phase and a 6-week maintenance phase.

The dose range selected was based on the aforementioned single dose study in which doses of up to 175 mg were well tolerated and on a previous multiple-dose study in healthy subjects, in which doses up to 140 mg b.i.d. administered for 14 days were also well tolerated. A titration phase may lead to a higher tolerability, as found in studies in monkeys in which dose escalation was found to increase tolerability for higher doses of ladostigil. Moreover, it has been determined that higher doses (about two fold) of some ChE inhibitor based drugs are better tolerated in AD patients than in healthy young volunteers. Because of the dose escalation employed in this study it was not possible to define the MTD. However, as some subjects appeared to be more sensitive to cholinergic side effects, down titration was allowed during the study period at the discretion of the investigator.

Initially, there were to be four sequential cohorts, each cohort having two study phases: a dose escalation phase and a maintenance phase. Each cohort, except for the last one, consisted of at least 30 subjects with an approximate 3: 1 randomization to ladostigil and placebo respectively. Testing on the fourth cohort was discontinued for reasons independent of safety or efficacy.

Recruitment to the second, third and fourth cohorts were initiated after safety and tolerability assessment of the preceding cohort, 10 days after the beginning of the maintenance phase. The review of each cohort was blinded, unless serious safety issues were involved. In this case procedures were incorporated to keep the blinding to those involved in the management of the study prior to unblinding the data.

This trial originally included 4 sequential subject cohorts. In all cohorts, subjects were randomly assigned to ladostigil or placebo to achieve a ratio of 3: 1, respectively. Only subjects who at the baseline visit met the eligibility criteria were allocated to a treatment group, based on a randomization scheme using blocks stratified by centres and cohorts. Individual subject safety and tolerability were closely followed to assure each dose escalation. In case of low tolerability, the dose could be lowered to a previously tolerated dose after which renewed attempts for escalation could be performed. The maintenance dose was determined when the subject reached the target dose and tolerated it, or when the investigator decided not to further escalate the dose.

The safety results for subjects in all 3 cohorts (total of 103 individuals) show that no deaths occurred and only 8 severe adverse events (SAE) were reported for 5 subjects (6.8%) in the ladostigil group, only one of which was considered by the investigator to be probably related to the drug itself. All SAEs were linked to the higher doses with 2 subjects experiencing a total of 5 events in Cohort 2 and 3 subjects each experiencing a single event in Cohort 3. Among the ladostigil treated patients 52 subjects (70.3%) experienced any AE, compared to the 16 (55.2%) among those who had received the placebo. Most of the AEs observed among ladostigil-treated subjects, including in particular nausea, vomiting, or diarrhoea, are typical of AChE inhibitors. A total of 13 (17.6%) subjects on ladostigil terminated the study early due to an AE, compared to 2 (6.9%) in the placebo group. The distribution of the incidences of AEs, AE-related discontinuations, laboratory findings and vital signs among the three cohorts did not show any dose-safety correlation. The lack of a dose-safety relationship prevented definition of an MTD in the current study. At all doses tested up to and including 210 mg b.i.d., ladostigil's safety profile equals that of, or is even better than another carbamate AChE inhibitor, rivastigmine, when tested at its therapeutic doses.

The pharmacodynamics (PD) of AChE inhibition data was shown to support previous findings with regards to the non-linearity observed between ladostigil administered dose and AChE inhibitory activity, which does not coincide with the level of ladostigil absorption at higher doses and levels off at 60-70% in spite of increasing the blood levels of ladostigil. A non dose-proportional PD effect was seen across the treatment cohorts and indicates that the Cohort 3 dose level is approaching this plateau value. For the lowest dose cohort, six to seven weeks of 100 mg b.i.d ladostigil maintenance dosing (i.e. after initial dose escalation) inhibits plasma ChE with peak values (2 hrs post dose) of close to 50%, and this inhibition persists at levels above 30% for at least 12 hrs. Furthermore, with this dose regimen and at higher ladostigil doses (i.e. Cohorts 2 & 3), at least 40% AChE inhibition is achieved in 100% of the study population, which, according to literature data, is sufficient to sustain long-term improvement of cognitive function in AD patients. Also in agreement with the earlier data was the determination that ladostigil dosing results in high levels of inhibition of MAO-A and -B activities. This is the first demonstration of significant MAO inhibition within AD patients who also had adequate AChE inhibition. This also indicates ladostigil's potential antidepressant activity and neuro-protection against oxidative stress.

The current study was not designed with the power to prove the psychometric efficacy of ladostigil.

The data presented herein show that a dosing regimen with proper titration escalating into a maintenance dose of 100 mg bid appears sufficient to evoke and sustain an AChE inhibition of at least 40%, and thereby elicit a therapeutic effect in AD patients.

Detailed drug administration: Table 1 outlines the dose titration and maintenance for each cohort during the different phases of the study. Escalation 1, escalation 2, escalation 3, escalation 4, escalation 5, etc. visits (dose titration) were designated Esc-1 , Esc-2, Esc-3, Esc- 4, Esc-5 etc. Maintenance 1 and maintenance 2 visits were designated Maint-1 and Maint-2.

Table 1: Dose titration and maintenance per cohort

* The maintenance dose was determined when the subject reached the target dose^ when the dose had been lowered due to intolerability to previously tolerated dose or when the investigator decided to not further escalate the dose.

Ladostigil 20 mg, 50 mg and 80 mg tablets and their matching placebo that were manufactured according to Good Manufacturing Practice (GMP) principles and guidelines applicable to Investigational Medicinal Products (IMP) were provided to the investigators as follows.

Ladostigil:

Active ingredient: Ladostigil tartrate; Chemical name: Ethyl-methyl-carbamic acid (R)-3-prop-2-ynylamino-indan-5-yl ester, (2R, 3R)-2, 3-dihydroxy-succinic acid (2: 1), in short [(R)-CPAI] ₂ tartrate; Abbreviated chemical name: [R)-CPAI]₂ Tartrate; Pharmaceutical form: Tablet; Administration route: Oral; Strength: Ladostigil tablets 20 mg; Each tablet contains 20 mg of ladostigil (as tartrate). Ladostigil tablets 50 mg: Each tablet contains 50 mg of ladostigil (as tartrate). Ladostigil tablets 80 mg: Each tablet contains 80 mg of ladostigil (as tartrate). Excipients: Mannitol, starch pregelatinised, silicon dioxide, povidone, mannitol granulate (only in 20 mg tablets), stearic acid and talc. Description: 20 and 50 mg tablets: White to off-white, round, flat, bevelled, round tablets, scored on one side and plain on the other side. 80 mg tablets: White to white-off, round, convex tablets, scored on one side.

Placebo: Active ingredient: None, Chemical name: not applicable, Pharmaceutical form: Tablet, Administration route: Oral, Dosage or strength: "Small" placebo tablets, identical in appearance to ladostigil 20 mg and 50 mg tablets.

"Big" placebo tablets, identical in appearance to ladostigil 80 mg tablets, Excipients: Colloidal silicon dioxide, starch pregelatinised, mannitol, stearic acid and talc. Description: Physical appearance identical to that of the matching active drug.

Subjects were instructed to administer ladostigil tablets with a meal and at least 100 ml fluids (Cohorts 3 and 4) once or twice daily according to study stage (cohort number, titration, maintenance etc.). At study visits, the investigator would ask subjects and caregivers from Cohort 1 and 2 for information regarding time of IMP administration in relation to food.

For each of the cohorts the initial dose started as daily administration for 7±4 days of:

Cohort 1 : 70 mg/placebo once daily,

Cohort 2: 100 mg/placebo once daily

Cohort 3: 50 mg/placebo twice daily

Cohort 4: 50 mg/placebo once daily, followed by 2-5 (or more) titration steps to reach the maximal target dose of:

Cohort 1 : 100 mg/placebo twice daily

Cohort 2: 150 mg/placebo twice daily

Cohort 3: 210 mg/placebo twice daily Cohort 4: 210 mg/placebo once daily

During the escalation phase of the study the drug dose could be adjusted, if needed, to reach the individual maximal tolerated dose.

During the maintenance phase, which lasted 6 weeks, each subject was treated with the maximal dose reached during the escalation phase. This phase had 2 visits two weeks apart (Maint-1 and Maint-2). If not tolerated, the dose could be adjusted at the discretion of the investigator during the maintenance period.

Cohort 1 : After randomization 21 subjects received an initial dose of 70 mg ladostigil qd and received matching placebo from day 1 until day 7 (±4 days). Depending upon the tolerability, there were two escalation steps, each within one week of each other to reach the dose of 100 mg/placebo b.i.d:

1. 70 mg/placebo b.i.d for 7±4 days.

2. 100 mg/placebo b.i.d for 7±4 days.

Thereafter, the subjects entered into a five-week (35±4 days) maintenance phase.

Cohort 2: After randomization, 29 subjects received an initial dose of 100 mg ladostigil qd and 11 received matching placebo from day 1 until day 7 (±4 days). Depending upon the tolerability, there were three escalation steps, each step within one week of each other, to reach the dose of 150 mg/placebo b.i.d.:

1. 100 mg/placebo b.i.d.

2. 130 mg/placebo b.i.d, and

3. 150 mg/placebo b.i.d.

Thereafter, the subjects entered into a five-week (3 ±4 days) maintenance phase.

Cohort 3: After randomization 22 subjects received an initial dose of 50 mg ladostigil qd and 9 received matching placebo from day 1 until day 7 (±4 days). Depending upon the tolerability, there were five escalation steps, each step within one week of each other, to reach the dose of 210 mg/placebo b.i.d.:

1. 80 mg/placebo b.i.d,

2. 120 mg/placebo b.i.d,

3. 150 mg/placebo b.i.d,

4. 180 mg/placebo b.i.d, and

5. 210 mg/placebo bid.

Thereafter, the subjects entered into a five-week (35±4 days) maintenance phase. Cohort 4: After randomization only 2 subjects were entered in this cohort. Both subjects received an initial dose of 50 mg ladostigil qd once daily. Depending upon the tolerability, there were five escalation steps, each step within one week of each other, to reach the dose of 210 mg/placebo qd:

1. 80 mg/placebo qd,

2. 120 mg/placebo qd,

3. 150 mg/placebo qd,

4. 180 mg/placebo qd, and

5. 210 mg/placebo qd.

Thereafter, the subjects entered into a five week (35±4 days) maintenance phase.

All doses of ladostigil or placebo were administered while subjects were in the study unit under the supervision of study personnel. Records were maintained on the timing and manner of drug administered to each subject. Pharmacokinetic assessments:

Blood samples (approximately 9 ml) for measurement of plasma levels of (R) CPAI (ladostigil) and its metabolites were collected in a subpopulation of subjects according to the following timelines:

Maint-1 visit. (Cohorts 1 & 2) 10±4 days after the last escalation visit (days 24±4 and 31 ±4 in cohorts 1 and 2 respectively):

Pre-dose (immediately prior to drug administration, 0 hr (12 hr post last dose) and 15, 30 min, and 1 , 2, and 3 hr post dose.

Termination visit. Blood samples were collected for all 4 cohorts at the following time intervals over a 24 hr period (Day 59±4 for Cohort 1, Day 66±4 for Cohort 2 and Day 80±4 for Cohorts 3 and 4).

Pre-dose (immediately prior to drug administration, 0 hr) and 15 and 30 min, and 1 , 2, 3, 4, 6, 8 hr (Cohort 3 & 4) and 24 hr post dose.

Independent pharmacokinetic (PK) parameters were determined wherever possible for ladostigil and its metabolites based on their plasma concentration data:

Cmax_> T_max, AUC(O-T) (units of ng*hr*mL-l and nN*hr*mL-l), AUC_∞, λ₂, and T .

Tmax was determined as the time point with the highest concentration value in any series of at least 5 consecutive time points starting from pre-dosing (0 hr), provided this time point was followed by at least one with a lower value. Two data time points following T_max on a close to linear segment of the semi-log mean concentration-time curve was considered the minimum requirement for calculating Ty_2, which was considered valid only if the resulting value >T_max.

Pharmacodynamic assessment:

Blood samples for MAO and DHPG determination were withdrawn at Baseline

(before drug), Termination and Follow-up (4 weeks after termination) visits. Samples for ChE determination were collected at the following visits and time points presented in Table 2.

Table 2: Timing of assay of ChE inhibition

Preparation of Platelets for MAO Determination

Nine ml of blood were withdrawn into a 9 ml vaccutainer tube containing 3.2% sodium citrate and placed on ice. The sample was centrifuged within one hour at 4°C for 10 minutes at 200 x g. The supernatant was transferred to a new rube and centrifuged for an additional 10 minutes at 2000 x g at 4°C. After discarding the supernatant 1 mL 0.3 M sucrose was added to the pellet to preserve its activity. Samples were stored at -20°C until shipment on dry ice to the site for analysis.

Plasma Preparation for DHPG

Five ml of blood were withdrawn into an EDTA containing vaccutainer tube, mixed gently and put on ice, and 100 μΤ of sodium metabisulphite solution (15%w/v in water) was added immediately. The blood was centrifuged within 30 minutes for 10 minutes at 2000 x g at 4°C. The plasma supernatant was separated and stored in 2 polypropylene tubes at -70°C and shipped on dry ice to the site for analysis.

Blood Haemolysate Preparation for ChE

One-and-a-half (1.5) ml blood was withdrawn into an EDTA containing tube, put on ice, and duplicate samples of 0.5 ml blood were immediately haemolysed into 10 mM phosphate buffer pH 6.0. The haemolysate was frozen immediately at -70°C and shipped in dry ice to the site for analysis.

Methods of analysis:

Determination of Acetylcholinesterase and Butyrylcholinesterase Activity:

Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) activities were determined by modification of the Ellman method, using acetylthiocholine (ATC) and butyryltiocholine (BTC) as substrates and 5,5' dithiobis (2-nitrobenzoic acid) (DTNB;

Ellman's reagent) to develop the chromophore. The increase of OD at 412 nm over 3 minutes at 28°C was monitored. The slope of the straight line obtained by linear regression provided the activity in terms of average changes in OD over time, AOD/min. The AOD/min of the blank (without ATC or BTC) was subtracted to give the net reading attributed to enzyme activity.

Calculations of results:

The BuChE activity was determined using BTC.

From this the AChE activity was calculated as follows:

Activity on BTC

AchE Activity = Activity on ATC

1 9 *

* 1.9 = the ratio of BTC/ATC activity of BuChE from human plasma, determined in preliminary experiments.

Conversion of activity from AOD/min to activity in units/L:

A unit of enzyme activity is the amount of enzyme, which produces 1 μπιοΐ product from a substrate in 1 minute. Under the current assay conditions a factor of 41.75 was calculated for transformation of the results of AOD/min into units/L:

Activity Units/L = Activity in AOD/min*41.75

The AChE and BuChE activity was normalised per initial OD of blank reaction assay ) that can serve as an index of the blood concentration in the haemolysate.

Normalized activity = Activity/OD (OD = initial OD in the blank reaction). The percentage of inhibition for every time point was calculated in comparison to activity of the baseline sample of the individual.

MAO-B Assay

MAO-B activity in platelets was measured according to the modified methods of

Otsuka and Kubayashi using C14 PEA as substrate. Protein in the platelets samples was determined by the Lowry method.

Calculation of results: Decays Per Minute (Dpm) data were converted into nN/mg protein/hour. All samples from the same subject were analysed on the same day. For each subject the percentage of inhibition was calculated relative to that from the baseline visit sample (=100%).

DHPG Determination

Determination of DHPG in the samples of human plasma treated with EDTA and metabisulfite was performed after purification of DHPG by fixation on alumina under basic conditions, followed by washing with water, then defixation under acid conditions. The acid eluate was analysed on a reversed phase HPLC column Prevail C18 using electrochemical detection. The method was validated for the determination of NA and DHPG in human plasma. Results were calculated and expressed in pg/ml. The lower limit of quantification of the method is 50 pg/ml. For each subject, the percentage of inhibition was calculated in comparison to his/her values at his baseline visit.

Pharmacokinetic Results

Blood samples collected at maintenance phase:

Blood samples were collected from 13 ladostigil treated subjects and 6 untreated ones in Cohort 1 and from 20 ladostigil treated subjects and 7 untreated ones in Cohort 2.

Data of the following subjects whose blood had been collected (all Cohort 2) were excluded from all analyses at Maintenance:

Subjects 10733, 20436, 30536and 40237 - No reliable data PK were available.

Subject 40239 - No readings at 2 and 3 hrs.

Cmax and T_max values of the parent drug and the primary metabolites could be calculated in Cohort 1 for 12 out of the 13 ladostigil-treated subjects with valid PK data, and of the secondary metabolites, (R)-MCAI and (R) -ECAI, for 8 and 9 ladostigil-treated subjects, respectively (Table 3). In Cohort 2, C_max and T_max values of the parent drug could be calculated for all 15 ladostigil-treated subjects, of the primary metabolites for 10-12 subjects, and for the two secondary metabolites for 3 and 7 subjects. In all other subjects the highest concentration value was recorded at the last time point, which made it unclear whether this represented T_maxJC_max. No Ti_/2, AUC_(0-T) and AUC₂₄ results are presented because collection of blood samples up to 3 hr post dose only prevented reliable calculation of most values.

No PK parameter values could be calculated for any of the untreated subjects, because in all plasma samples the concentrations for (R) -CPAI and all its metabolites were BQL.

The data in Table 3 show, for those subjects for whom Tmax could unequivocally be established, similar relative patterns of T_max and C_max values for ladostigil and its metabolites in both cohorts. In both cohorts T_max was the shortest for (R)-CPAI and (R)-HCPAI, whereas in Cohortl, T_max was the latest for both secondary metabolites (R)-MCAI and (R)-ECAI, and in Cohort 2 for (R)-ECAI [the single value for (R)-MCAI did not allow to evaluate its position in- the order of T_max values]. In both cohorts, (R)-CAI and (R)-HPAI tended to show later T_max values than (R)-CPAI and the other primary metabolites. The highest C_max values were obtained for (R)-HCPAI, which had not been determined in previous clinical studies, followed by (R)-CPAI and (R)-MCPAI, whereas the lowest values were noted for (R)-ECAL In general, T_max tended to be later and C_max higher in Cohort 2 compared to Cohort 1. The differences in T_max were statistically significant (P<0.05) for (R)-CPAI, (R)-MCPAI, (R)- ECPAI, and (R) -HPAI, whereas a statistically difference (P<0.01) for C_max was observed for (R)-HPAI only. The fact that the T_max could be averaged only for those subjects whose T_max occurred before the 3 hr data time point, effectively resulted in a certain bias towards lower max values in other subjects for those metabolites whose T_max had not been unequivocally attained by the last blood collection.

Table 3: T_max and C_max values of (R)-CPAI and its metabolites in ladostigil-treated subjects at maintenance in Cohorts 1 and 2

2.00] 2.00]

64.3 ± 0.98 [0.25 - 102 ± 1.45 [1 .00 -

(R)-HPAI 12 20.2 2.00] 1 1 37** 2.00]*

1034 ± 0.58 [0.25 - 1 190 ± 1.04 [0.50 -

(R)-HCPAI 12 425 1.00] 12 307 2.00]*

56.4 ± 1.31 [0.50 - 71.9 ± 1.33 [1.00 -

(R)-MCAI 8 21.2 2.00] 3 43.6 2.00]

1.67 [0.50 - 1.86 [1.00 -

(R)-ECAI 9 20.7 ± 5.9 2.00] 7 25.6 ± 7.5 2.00]

a Number of subjects for whom it was possible to calculate C_max and T_max.

b Arithmetic means ± SD

c Arithmetic means [range]

^* Significantly different from corresponding value in Cohort 1 , PO.05

Significantly different from corresponding value in Cohort 1,P<0.01

Blood samples collected at termination phase:

At termination, blood samples were collected from 12 ladostigil treated subjects and from 6 untreated subjects in Cohort 1, from 8 ladostigil treated subjects and from 3 untreated subjects in Cohort 2, from 13 ladostigil-treated subjects and from 7 untreated subjects in Cohort 3, and from 1 ladostigil treated subject in Cohort 4. All subjects completed treatment. The above-noted subjects listed under Maintenance and Termination in Cohorts 1 and 2 were the same ones. Depending upon the metabolite in question, measurements were made on 1 1 or 10 ladostigil treated subjects in Cohort 1, on 7 or 6 ladostigil treated subjects in Cohorts 2, and on 9 or 7 ladostigil treated subjects in Cohort 3. For those subjects from whom blood samples were available at the appropriate time points, the mean concentration-time curves were plotted and depicted as a mean concentration-time curve for (R)-CPAI and each metabolite, whereas the main pharmacokinetic parameters, T_max, C_max, AUC(o_-T), AUC24, and T]/2, of (R)-CPAI and all its metabolites were computed and presented in Table 4.

No PK parameter values could be calculated for any of the untreated subjects, because in all plasma samples the concentrations for (R)-CPAI and all its metabolites were BQL. Table 4: Pharmacokinetic parameters of (R)-CPAI and its metabolites at termination in Cohorts 1, 2 and 3

Secondary Metabolites

(R)-MCAI Tmax [hi] 2.50 [0.50 - 3.29 [2.00 - 2.86 [1.00 2.80

6.00] 6.00] - 4.00] [1.00 -

4.00]

C_max [ng/ml] 52.2 ± 14.0 56.9 ± 21.3 78.6 ± 81.8 ±

34.5* 29.0*

T [hr] 8.19 ± 2.75^b 7.71 ± 3.37 8.54 ± 8.62 ±

1.74° 1.61^e

AUC(O-x) 275 ± 74^a 277 ± 1 11^§§ 569 ± 574 ± [ng.hr/ml] 24₇c** 226^e**

AUC₂₄ 753 ± 209^b 766 ± 264^§ 1223 ± 1245 ± [ng.hr/ml] 572°* 525^e*

(R)-ECAI Tmax [hr] 2.58 [1.00 - 3.14 [2.00 - 2.57 [1.00 2.4

6.00] 6.00] - 4.00] [1.00 - 4.00]

Cm_ax [ng/ml] 19.6 ± 7.3 19.4 ± 6.0 ^§ 37.2 ± 34.1 ±

6.4** 8.3**

Ti/2 [hr] 6.63 ± 1.57^a 9.82 ± 4.34 7.78 ± 7.95 ±

3.62^c 3.33^e

AUC(O-T) 104 ± 39^a Q1 4- 30 236 239 ± [ng.hr/ml] 51^c** 47^e**

AUC₂₄ 286 ± 87^b 244 ± 69 ^§§ 486 ± 498 ± [ng.hr/ml] 7^c** 94^e** A11 data arithmetic means ± SD; arithmetic means [range] for T_max

AUC(O-T): τ = 6 hr for Cohorts 1 and 2, x = 8 hr for Cohort 3

^=1 1 ; ^=10; ^CN=6; ^DN=9; ^=7

Significantly different from values in Cohort 1 : *(P<0.05); **(P<0.01)

Significantly different from values in Cohort 3 : ^§(P<0.05); ^§i}(P<0.01 )

(RVCPAI

The structure of (R)-CPAI and the mean concentration-time curves for (R)-CPAI in the three cohorts are depicted in Figure 1. The parent drug was detected in almost all subjects 15 min after administration and peak levels were reached between 0.5 and 1 hr. In most subjects (R)-CPAI levels were below 50 ng/mL by 8 hr (Cohort 3), and in all but one subject in Cohort 3 no longer detectable after 24 hr. Because of the large inter subject variability there were no significant differences in the exposure to the drug in the three cohorts. The main pharmacokinetic parameters for (R)-MCPAI (Table 4) show that its mean max values ranged from 0.25-2 hr without significant differences across the three cohorts.

Plasma exposure of (R)-CPAI as assessed by C_max, AUC₂₄ and T_1/2 also did not change with doses increasing between 100 to 210 mg b.i.d, but the AUC(O-x) for Cohort 3 was

significantly greater than for Cohort 1 (P<0.05). These findings are in agreement with those at maintenance (Table 3) in which there also was no difference between T_max and C_max in the two cohorts. (R -MCPAI

The structure of (R)-MCPAI and the mean concentration time curves for of the primary metabolite (R)-MCPAI in the three cohorts are depicted in Figure 2. In most subjects (R)-MCPAI was detected 15 min after administration and peak levels were reached between 1 and 2 hr. The metabolite was still measurable after 8 hr (Cohort 3) but, except for one subject in Cohort 3, not after 24 hr. Inter subject variation was less than that for the parent drug, which enabled the detection of a more reliable difference in exposure between Cohort 1 and Cohorts 2 and 3.

The main pharmacokinetic parameters for (R)-MCPAI (Table 4) show that its mean Tmax values ranged from 0.5-3 hr without significant differences across the three cohorts. Mean C_max, AUC(O-x) and AUC₂4 values were significantly greater only in Cohort 3 [P<0.01 for C_max and AUC(O-T); PO.05 for AUC₂4] when compared to those in Cohort 1. The C_max and T_max values for (R)-MCPAI in Cohorts 1 and 2 at termination were similar to those observed during the maintenance phase (Table 3). (R)-ECPAI

The structure of (R)-ECPAI and the average concentration time curves for the primary metabolite (R)-ECPAI in the three cohorts are shown in Figure 3. (R)-ECPAI was detected 15 min after administration and peak levels were reached in most subjects between 1 and 2 hr. In Cohort 3 (R)-ECPAI was still detectable after 8 hr but, for all but one subject, not after 24 hr. There were significant differences in exposure to this metabolite among the three cohorts.

The main pharmacokinetic parameters of (R)-ECPAI (Table 4) show that its mean Tmax values ranged from 0.5-4 hr without significant differences across the three cohorts. Mean values of C_max and AUC(O-x) increased significantly with increasing dose, being greater in Cohort 2 than in Cohort 1 (PO.05), in Cohort 3 than in Cohort 2 (PO.05) and in Cohort 3 than in Cohort 1 (PO.01 ). Mean values of AUC₂₄ were significantly greater in Cohort 3 than in Cohort 1 (PO.05) but did not differ between Cohorts 1 and 2 or between 2 and 3. Ti/₂ values did not differ across the three cohorts. The mean values of C_max and T_max

42 for (R)-ECPAI in Cohorts 1 and 2 at termination were similar to those observed during the maintenance phase (Table 3).

(R)-CAI

The structure of (R)-CAI and the mean concentration time curves for the primary metabolite (R)-CAI in the three cohorts are shown in Figure 4. (R)-CAI was detected in almost all subjects at the 0 time reading i.e., 12 hr after the last twice daily dose of ladostigil. In most subjects peak levels were reached between 1 and 2 hr. (R)-CAI was still detectable after 8 hr (Cohort 3) and in about half of all subjects at 24 hr.

The main pharmacokinetic parameters of (R)-CAI (Table 4) show that its mean T_max values ranged from 0.5-4 hr without significant differences across the three cohorts. Mean values of C_max and AUC(O-T) were significantly higher in Cohort 3 than in Cohorts 1 (P<0.01) and 2 (P<0.05). Mean v AUC₂₄ values were significantly greater (PO.05) in Cohorts 2 and 3 than in Cohort 1, but did not differ between Cohorts 2 and 3. Values of Ti_/2 did not differ among the three cohorts. The C_max and T_max values for (R)-CAI in Cohorts 1 and 2 at termination were similar to those observed during the maintenance phase (Table 3).

(R)-HPAI

The structure of (R)-HPAI and the mean concentration time curves for the primary metabolite (R)-HPAI in the three cohorts are shown in Figure 5. (R)-HPAI was barely detectable 15 min after administration. Peak levels were reached in most subjects between 1 and 2 hr. (R)-HPAI was still measurable after 8 hr (Cohort 3) but not at 24 hr. Low inter subject variability in the exposure to (R)-FIPAI enabled the detection of significant differences in exposure to this metabolite among the three cohorts.

The mean pharmacokinetic parameters for the primary metabolite (R)-HPAI (Table 4) show that its mean T_max values ranged from 0.5-3 hr without significant differences across the three cohorts. Mean values of C_max and AUC₂₄ were similar in Cohorts- 2 and 3 and significantly greater than in Cohort 1 (P<0.01). Mean values of AUC(O-x) showed a dose related increase among the three cohorts with the values for Cohorts 2 and 3 being higher than those in Cohort 1 (P<0.01), and those in Cohort 3 being higher than those in Cohort 2 (PO.05). Mean values of Ti_/2 did not differ among the three cohorts. The C_max and T_max values for (R)-HPAI in Cohorts 1 and 2 at termination were similar to those observed during the maintenance phase (Table 3).

43 (R)-HCPAI

The structure of (R)-HCPAI and the mean concentration time curves for the primary metabolite(R)-HCPAI in the three cohorts are shown in Figure 6. (R)-HCPAI was measurable 15 min after administration. (R)-HCPAI levels were higher than reached by the parent drug and all the other metabolites in each cohort. Peak levels were reached in most subjects between 1 and 2 hr. (R)-HCPAI was still measurable after 8 hr (Cohort 3) but, except for one subject, not at 24 hr.

The mean pharmacokinetic parameters of (R)-HCPAI (Table 4) show that its mean ma values ranged from 0.5-2 hr without significant differences across the three cohorts. Mean values of C_max and AUC(O-x) were significantly lower (PO.01) in Cohort 1 than those in Cohorts 2 and 3, but similar in Cohorts 2 and 3. Mean values of AUC₂₄ in Cohort 1 too were significantly lower than those in both Cohorts 2 and 3 (PO.01), but did not differ between Cohorts 2 and 3. Mean values of T1_/2 did not differ among the three cohorts. The values of C_max and T_max for (R)-HCPAI in Cohorts 1 and 2 at termination were similar to those observed during the maintenance phase (Table 3).

(R)-MCAI

The structure of (R)-MCAI and the mean concentration time curves for the secondary metabolite (R)-MCAI in the three cohorts are shown in Figure 7. At time 0 (12 hr after the previous dose) plasma (R)-MCAI levels were about 50% of C_max in all three cohorts. (R)-MCAI reached peak levels between 1 and 6 hr and was still measurable in all subjects after 24 hr.

The main pharmacokinetic parameters of (R)-MCAI (Table 4) show that its mean

Tmax values ranged from 0.5-6 hr without significant differences across the three cohorts. Mean values of C_max were significantly higher in Cohort 3 compared to Cohorts 1 (P .01) and 2 (P<0.05), but did not differ between Cohorts 1 and 2. AUC(O-T) (PO.01) and AUC₂ (PO.05)_, were significantly higher in Cohort 3 than in Cohorts 1 and 2. Mean values of T]_/2 did not differ in the three cohorts. The values of C_max and T_max for (R)-MCAI in Cohort 1 at termination were similar to those observed during the maintenance phase. For Cohort 2 too few data were available during maintenance to determine a difference in these parameters.

44 (R)-ECAI

The structure of (R)-ECAI and the mean concentration time curves for the secondary metabolite (R)-ECAI in the three cohorts are shown in Figure 8. At time 0 (12 hr after the previous dose) plasma (R)-ECAI levels were about 50% of C_max in all three cohorts. (R)-ECAI reached peak levels between 1 and 6 hr and was still measurable in most subjects after 24 hr.

The main pharmacokinetic parameters for (R)-ECAI (Table 4) show that its mean Tmax values ranged from 1.0-6 hr without significant differences across the three cohorts. Mean values of C_max were significantly higher in Cohort 3 than in Cohorts 1 (PO.01) and 2 (PO.05), and those of AUC(O-T) and AUC₂₄ were significantly higher (PO.01) in Cohort 3 than in Cohorts 1 and 2. The mean values of C_max and T_max for (R)-ECAI in Cohort 1 at termination were similar to those observed during the maintenance phase. Mean values of Ti/₂ did not differ in the three cohorts. There were too few maintenance data to determine whether there was a difference in these parameters in Cohort 2.

In general, the data in Table 5 show similar relative patterns of C_max and T_max values for ladostigil and its metabolites in all three cohorts. Similarly to the observations made during maintenance (Table 4) and in agreement with their proposed status as secondary metabolites, (R)-MCAI and (R)-ECAI displayed the slowest kinetics with the latest T_max and highest Ti ₂ values. The kinetics of (R)-CAI were still slower and the Ti/₂ values higher than of (R)-CPAI and the other primary metabolites. Remarkably, T_max values tended to be later in Cohort 2 than in both Cohorts 1 and 3. Again, at termination the highest C_max values were obtained for (R)-HCPAI followed by (R)-CPA1 and (R)-MCPAl, whereas the lowest values were noted for (R)-ECAI. C_max increased with dose for all metabolites, except for (R)-ECAI between Cohorts 1 and 2.

The metabolic ratios (MR) of (R)-CPAI and its metabolites for the three cohorts at termination are presented in Table 5. The highest MRs were observed for (R)-HCPAI followed by (R)-CPAI and (R)-MCPAI, whereas the lowest values were noted for (R)-ECAI and (R)-ECPAI. This order resembles those observed for C_max values at maintenance (Table 4) and at termination (Table 5). With the exception of (R)-ECPAI, no significant differences in metabolic ratios were observed among the three cohorts.

45 Table 5: Metabolic ratios of ladostigil and its metabolites in the three study cohorts

*Metabolic ratios (MR) are presented as the percentage of the ratio of AUC(O-T) (nmol.hr/mL) for each compound to the total AUC(O-T) (nmol.hr/mL) of the parent drug and metabolites.

** Significantly different (PO.01 ) from values in Cohorts 1 and 2.

Pharmacodynamic Results

An earlier report on the effect of ladostigil on PD activities presents data for all subjects among the ITT population with at least one result in addition to Baseline. These included 16 ladostigil treated subjects and 7 untreated subjects in Cohort 1 , from 25 ladostigil treated subjects and 8 untreated subjects in Cohort 2, and from 14 ladostigil-treated subjects [all 16 for AChE, 9 for DHPG and 9 (7 identical) for MAO-B] and 7 untreated subjects in Cohort 3, at Baseline, Maintenance, Termination, and/or Follow-up, i.e., four weeks after termination. In the current analysis were included only data from subjects, who completed the full intended dose for the cohort within the required time period. Moreover, for AChE and BuChE only subjects whose results were available at all time points during both Maintenance and Termination were included. This enabled accurate comparison on the same group of subjects, for each enzyme tested, the data collected per cohort, either between various time points at either Maintenance or Termination, or between the same time points at Maintenance and Termination. Consequently, the number of subjects is lower, and the data reported here may differ slightly from those observed earlier.

Cholinesterase Inhibition

Table 6 presents the mean percentages of inhibition of AChE and BuChE obtained for ladostigil treated subjects after administration of ladostigil during Maintenance and at Termination. The cohort means were averaged from the percentages of inhibition for each subject in the cohort relative to its own value from the baseline visit. No significant differences in AChE and BuChE inhibition levels, at equal time points, were observed between 10 days after initiating the maintenance dose and approximately 32 days later at Termination in each of Cohorts 1 and 2, showing the consistency of maintenance therapy. However, a clear trend to increased mean AChE levels at higher ladostigil doses could be discerned when comparing the 3 cohorts at the same time points. These differences were statistically significant when comparing Cohort 3, at 2 hr (P<0.05) and at 6 hr (P<0.01) after drug administration, to Cohort 1. This difference in AChE inhibition reflects the difference in the levels of the most potent metabolites, (R)-MCPAI, (R)-CAI and (R)-MCAI, in the cohorts (see Pharmacokinetics section)

Table 6: Acetylcholinesterase inhibition at Maintenance and at

Termination in subjects of Cohorts 1 and 2

* 0 hr pre-dose values actually represent the 12 hr values following the previous b.i.d. dose **NA: Not applicable

^Significantly different from Cohort 1, PO.05; ^Significantly different from Cohort 1, PO.01

All results are averages of the percent of inhibition obtained for each subject of the cohort relative to its own baseline values.

Maintenance: 10 days following the start of maintenance therapy.

Termination: After the last maintenance dose

47 In all cohorts the pre -dose (0 hr; 12 hr after the preceding dose) percentages of AChE inhibition at Maintenance and Termination ranged 33.5-44.8% (70-75% of their subsequent peak levels). Though the PK profiles of successive doses may vary, this indicates persistence of very high levels of inhibition 12 hr after each dose.

The PK profile of AChE inhibition in Cohort 3 at termination, depicted in Figure 9, shows significant inhibition at 30.3 ± 5.4% (50.8% of the peak level at 2 hr) even at 24 hr following administration of the last dose. Comparison of the pre-dose/12 hr data in Table 6 and 24 hr data in Figure 9 to the kinetic profiles and data in the Pharmacokinetic Results, shows that AChE inhibition in plasma continued far beyond the presence of significant levels of the parent drug and most of its metabolites. Although not wishing to be bound by any theory, this difference in kinetics may be explained by several potential factors: a) The relatively slow kinetics of some of the carbamate metabolites, e.g., (R)-CAI and (R)-MCAI (Figure 4; Figure 7; Table 5), considered to be more potent AChE inhibitors and b) the pseudo reversible nature of AChE inhibition by carbamates. For example, the rate of decarbamylation of AChE in vitro after inhibition by (R)-CAI, (present in Cohort 3 in concentrations that can cause significant AChE inhibition), is very slow with a half life of more than 30 hr.

The data in Table 6 and the kinetics for Cohort 3 in Figure 9 show a much more rapid decline of BuChE inhibition compared to that of AChE, at predose (0 hr; 12 hr after the preceding dose) ranging 25.4-32.8% (46-57% of the peak values), and 12.1 ± 7.9% (18.8% of the 2 hr peak level) at 24 hr. This difference in kinetic profile is in accordance with the findings in the kinetics studies in which the decarbamylation rate after BuChE inhibition by ladostigil and its metabolites was significantly higher than that of AChE. The mean levels of BuChE inhibition at Termination at 2 and 6hr after drug administration, were significantly higher in Cohort 2 (P<0.05), and in Cohort 3 (P<0.01 ), than in Cohort 1.

The above data show that administration of a maintenance dose of 100 mg ladostigil b.i.d. caused 2 hr peak inhibition levels of blood AChE and BuChE of close to 50% at both Maintenance and Termination, whereas levels of >40% inhibition of both ChEs were maintained for at least 6 hrs following administration. The pre-dose data (actually determined on blood samples collected 12 hrs following an earlier dose) indicate that even 12 hrs after a 100 mg dose, levels of inhibition of approximately 33% and 25% were still sustained for AChE and BuChE, respectively.

48 Twenty-four hours after the last 210 mg ladostigil dose the levels of residual AChE and BuChE inhibition were 29.4% and 12.6%, respectively. Extrapolation of the kinetics for the 100 mg dose based on the kinetic profile for the 210 mg dose indicates that even 24 hr following the last of a daily 100 mg b.i.d. dose of ladostigil a residual AChE inhibition of approximately 23% may still be anticipated. The rate of decrease in AChE inhibition reported here at Termination appears to be slightly slower than earlier observations after 14 days b.i.d. administration of 90 or 1 10 mg ladostigil.

The following percentages inhibition ± SEM (N subjects) were recorded at Termination at pre-dose (0 hr) for control subjects completed at maximum placebo dose in Cohorts 1 , 2 and 3: AChE - -7.2±5.5 (4), 1.0±1.1 (4) and -1.3±1.9 (7), respectively; and BuChE—4.6±3.8 (4), -5.6±6.9 (4) and -1.6±5.0, respectively. Close to zero values of similar order of magnitude were observed in Cohorts 1 and 2 at Maintenance (data not shown).

Monoamine Oxidase Inhibition

The inhibition of MAO-B activity in platelets (% of baseline level), and the reduction (%) in plasma levels of DHPG, a metabolite of NA that reflects MAO-A activity, were measured in the three cohorts only at Termination at 2 hr after the last dose of ladostigil, and at follow-up, four weeks after the last dose.

Figure 10 shows a dose dependent reduction in the levels DHPG and MAO-B at termination. Platelet MAO-B inhibition was significantly greater in Cohorts 2 (P<0.05) and 3 (P<0.01) than in Cohort 1 but did not differ between Cohorts 2 and 3. The mean reduction in plasma DHPG was significantly greater in Cohort 3 than in Cohort 1 (P<0.05) but did not differ between Cohorts 1 and 2 or 2 and 3. The smaller differences between cohorts in the reduction in DHPG levels than in MAO-B inhibition may be attributed to the lower number of subjects with valid DHPG data. The reductions in MAO activity reflect the increase in the levels of (R)-HPAI, the only ladostigil metabolite with MAO-A and B inhibitory at the concentrations found in plasma and which were also significantly higher in Cohorts 2 and 3 than in Cohort 1 (Table 5). Four weeks after treatment cessation, DHPG levels [% inhibition ± SEM (N)] in Cohorts 1, 2 and 3 were still significantly reduced by 23.7±1 1.9 (6), 38.3±6.2 (6) and 28.2± 1 1.3 (4), respectively, whereas MAO-B activities approached baseline values at below percentages inhibition of -24.1±24.8 (9) and -8.2±12.7 (6), for Cohorts 2 and 3, respectively. Some statistically insignificant residual inhibition [22.2±26.5 (6)] was observed only in Cohort 1. The lower rate of decrease by DHPG may be explained by the fact that, whereas MAO-B is located in platelets, DHPG reduced is the result of inhibition of neuronal

49 MAO-A which could take longer to recover as it is dependent on the synthesis of new enzyme. With the exception of MAO-B in Cohort 6 [17.7±13.5 (6)], no significant changes in MAO-B activity or DHPG levels in any of the placebo-treated subjects were observed.

Conclusions

The results for MAO inhibition presented above show that even the lowest dose regime (Cohort 1) induced sufficient MAO inhibition to induce a potential antidepressant effect resembling that seen with other MAO-inhibitors.

The pharmacodynamic data presented here show that even the lowest maintenance dose (100 mg bid) of ladostigil causes plasma AChE inhibition with 2 hr peak values of close to 50% and values of over 30% persisting for at least 12 hrs following administration. The combined PD and PK results also indicate that the upper limit of AChE inhibition is approximately 70%. Higher ladostigil doses or longer treatment are unlikely to further increase AChE inhibitory activity. A second daily dose could be given in order to ensure AChE inhibition of more than 30% for a period of 24 hours. The brain selective MAO-A inhibitory activity of ladostigil indicates potential antidepressant activity, whereas MAO-B inhibition may help to maintain DA levels in AD patients with extrapyramidal symptoms and also provide neuroprotection against oxidative stress.

Example 3 - Phase I single dose study (TV-3326/101): The tolerability and safety of ladostigil or a placebo were evaluated in a randomized, double-blind, placebo-controlled Phase 1 ascending-single-dose study (2.5, 5, 10, 25, 50, 100, 125, 150, 175 and 200 mg of ladostigil base), in 90 healthy young (age: 18-40 years) male volunteers in 10 groups (nine subjects per group; six on active drug and three on placebo). Adverse events (AEs), safety laboratory parameters, physical examination, vital signs, 12-lead ECG, cardiac telemetry, oximetry and respiratory rate were monitored in the study.

All the AEs reported were mild or moderate; no serious adverse events (SAEs) occurred. Overall, 15 (16.7%) of 90 subjects experienced a total of 46 AEs in this study; 10 (16.7%)) of the ladostigil-treated volunteers (60 subjects) and five ( 16.7%) of the placebo- treated subjects (30 subjects).

Example 4 - Phase I multiple dose study (Study TV-3326/102): The tolerability and safety of ladostigil or a placebo were evaluated in a randomized, double-blind, placebo- controlled Phase I ascending-multiple-dose study (40, 60, 90, 110 and 140 mg ladostigil base), in 61 healthy (age: 40-65 years) male and female volunteers in five ascending groups (12 subjects per group; nine on active and three on placebo). Ladostigil was administered

50 once on Days 1 and 14 of the study, and twice daily doses were administered in the inclusive interval from Day 2 to Day 13.

Adverse events, safety laboratory parameters, physical examination, vital signs, 12-lead ECG cardiac telemetry, oximetry and respiratory rate were monitored during the study. Special cardiovascular safety measures, additional vital sign and ECG recordings, were taken on tyramine challenge days.

No SAE's were reported during the study. All the AEs reported were mild to moderate and did not require corrective treatment. Altogether 23 of the 46 active-treatment subjects (50%) reported a total of 100 AEs compared to seven of the 15 placebo subjects (47%) who reported 23 AEs. The most commonly reported AE with the largest difference in incidence between active treatment and placebo was nausea. Seven active-treatment subjects (15%) reported 29 incidences of nausea, but only in the treatment groups who received doses of 90 mg base and higher; no placebo subject reported nausea. The second highest difference in incidence of an AE between active-treatment and placebo was loose stools. Six active- treatment subjects (13%) reported eight episodes of loose stools; no placebo subjects reported it. It occurred in all of the active-treatment groups except for the lowest dose group, the 40 mg group, and occurred with the highest incidence (three of nine subjects) in the 140 mg group, the highest dose-treatment group. Except for one report, which was considered moderate in intensity, all other reports were mild, and none required corrective treatment. Four subjects (9%) in the active-treatment groups reported dizziness (total of 18 episodes) compared to one (7%) report in the placebo group. All reports of dizziness were mild to moderate in nature and none required corrective treatment. The number of AE episodes considered treatment-related after active treatment appeared to decrease over time, which may indicate the development of tolerance to the drug. There were no other AEs characterized by noteworthy differences between active treatment and placebo in either the number of subjects reporting a specific AE or in the number of reports for that AE. Overall, single and multiple doses of 40-140 mg b.i.d. of ladostigil were considered to be safe and well tolerated. There were no clinically significant changes from Baseline in any of the clinical laboratory, vital sign, ECG, oximetry and physical examination assessments for any of the subjects in either the active treatment or placebo groups.

Example 5 - Phase II study TV-3326/201 (Extent of Exposure): The tolerability and safety of ladostigil (final maintenance doses of 100, 150 and 210 mg base b.i.d.) was also evaluated in a multi-centre, randomized, double-blind, placebo-controlled Phase IIA

51 ascending-multiple-dose study in 103 (ladostigil =74; placebo =29) male and female AD patients (age: 60-87 years).

The primary purpose of this phase II study was to assess the tolerability, safety and maximum tolerated dose (MTD) of ladostigil in AD patients based upon changes in vital signs, ECG, laboratory parameters and reports of adverse events. In addition to safety, pharmacokinetic, and pharmacodynamic assessments, psychometric assessments were performed in subpopulations of the dose cohorts. The study included three completed cohorts with a total of 101 study subjects, distributed as follows: Cohort 1 at 100 mg b.i.d. (21 ladostigil versus nine placebo), Cohort 2 at 150 mg b.i.d. (29 ladostigil versus 1 1 placebo), and Cohort 3 at 210 mg b.i.d. (21 ladostigil versus nine placebo). Each cohort followed a different regimen of escalating dose titration phases followed by an approximately 6-week maintenance phase.

Example 6 - Open-label treatment period: Patients are titrated to the 80 mg b.i.d. dose level using the titration schedule utilized in the double-blind phase for the 80 mg treatment arm (i.e. 40 mg b.i.d. for seven days, followed by 60 mg morning dose and 40 mg evening dose for seven days, followed by 60 mg b.i.d. for seven days and then 80 mg b.i.d for the remaining 23 weeks).

Selection and timing of dose for each subject: Doses are taken twice daily every day by oral route. For each dosing, swallow one capsule with water, 30-45 minutes prior to eating. The morning dose should be taken immediately upon awakening, and evening dosing at least three hours prior to going to sleep, and preferably before 18:00 hours. Doses should always be taken at approximately the same time each day.

Table 7. Dose titration schedule, three weeks to reach final dosa e stren th.

Double-blind phase: The ladostigil dose level is titrated to the 80 mg b.i.d. dose level using the titration schedule presented in Table 7. Forty milligrams of ladostigil aree administered for seven days (one capsule in the morning and one capsule in the late

52 afternoon/early evening,) followed by a 60 mg morning dose and 40 mg evening dose for seven days, followed by 60 mg b.i.d. for seven days, followed by 80 mg b.i.d. for the remaining 23 weeks.

Open-label phase: At the end of the 26-week placebo-controlled component of the trial, patients are entering the open-label phase component of the trial and receive ladostigil treatment. When placebo patients are switched to the treatment arm, they are titrated to the 80 mg b.i.d. dose level using the same titration schedule utilized in the double-blind phase for the 80mg treatment arm (i.e. 40 mg b.i.d. for seven days, followed by 60mg morning dose and 40 mg evening dose for seven days, followed by 60 mg b.i.d. for seven days, followed by 80 mg b.i.d for the remaining 23 weeks). During the open-label period, patients visit the clinic at Week 39 and Week 52 for Visits 6 and 7, respectively. Figure 12 summarizes the treatment schedule for both phases of the study.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

53

Claims

CLAIMS What is claimed is:

1. A method for improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof, comprising administering to said subject for at least 28 continuous days, a daily dose of 60 to 200 mg ladostigil or a pharmaceutically active salt thereof, thereby improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof.

2. The method of claim 1 , wherein said daily dose is in one dose.

3. The method of claim 2, wherein said dose is 60 to 120 mg.

4. The method of claim 1, wherein said daily dose is divided to two daily doses.

5. The method of claim 4, wherein said two daily doses are equally divided doses.

6. The method of claim 4, wherein said daily dose is 120 to 130 mg.

7. The method of claim 4, wherein said daily dose is 150 to 190 mg.

8. The method of claim 4, wherein said daily dose is 200 mg.

9. The method of claim 5, wherein said daily dose is 160 to 200 mg.

10. The method of claim 5, wherein said daily dose is 160 mg.

1 1. The method of claim 1 , wherein said ladostigil or a pharmaceutically active salt thereof is formulated in an immediate release pharmaceutical composition.

12. The method of claim 1, wherein said pharmaceutically active salt thereof is ladostigil tartrate.

13. The method of claim 1 , wherein said subject in need thereof is a subject afflicted with a neurodegenerative disorder.

54

14. The method of claim 13, wherein said neurodegenerative disorder is Alzheimer's disease.

15. A method for improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof, comprising daily administering to said subject a first dose and a second dose of 30 to 40 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administering to said subject a first dose and a second dose of 40 to 60 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administering to said subject a first dose and a second dose of 50 to 70 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, followed by daily administering to said subject a first dose and a second dose of 70 to 95 mg ladostigil or a pharmaceutically active salt thereof for at least 4 days, wherein said second dose is administered at least 4 hours after said first dose, thereby improving cognitive function while minimizing side effects associated with the inhibition of cholinesterase activity in a subject in need thereof.

16. The method of claim 15, wherein said at least 4 days is at least 7 days.

17. The method of claim 15, wherein said twice a day is twice a day in equally divided doses.

18. The method of claim 15, wherein said 30 to 40 mg ladostigil or a pharmaceutically active salt thereof is 40 mg ladostigil or a pharmaceutically active salt thereof.

19. The method of claim 15, wherein said 40 to 60 mg ladostigil or a pharmaceutically active salt thereof is said first dose of 40 mg ladostigil or a pharmaceutically active salt thereof and said second dose of 60 mg ladostigil or a pharmaceutically active salt thereof.

20. The method of claim 15, wherein said 50 to 70 mg ladostigil or a pharmaceutically active salt thereof is 60 mg ladostigil or a pharmaceutically active salt thereof.

21. The method of claim 15, wherein said 70 to 95 mg ladostigil or a pharmaceutically active salt thereof is 80 mg ladostigil or a pharmaceutically active salt thereof.

55

22. The method of claim 15, wherein said ladostigil or a pharmaceutically active salt thereof is formulated in an immediate release pharmaceutical composition.

23. The method of claim 15, wherein said pharmaceutically active salt thereof is ladostigil tartrate.

24. The method of claim 15, wherein said subject in need thereof is a subject afflicted with a neurodegenerative disorder.

25. The method of claim 24, wherein said neurodegenerative disorder is Alzheimer's disease.

26. The method of claim 1 , wherein said administering is administering prior to eating.

27. The method of claim 15, wherein said twice a day is upon awakening and 3-6 hours before bed time.

56