# Patent

## Title
Methods of treating Alzheimer's disease and pharmaceutical compositions thereof

## Abstract

The present invention describes methods of treating dementia comprising administering an effective daily dose of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine to improve or augment the effect of an acetylcholinesterase inhibitor.

## Background
This application is a continuation of U.S. patent application Ser. No. 14/021,033, filed Sep. 9, 2013, which claims priority to U.S. Provisional Application Ser. Nos. 61/782,084, filed Mar. 14, 2013, and 61/698,664, filed Sep. 9, 2012.


FIELD OF THE INVENTION
The present invention describes methods of treating Alzheimer's disease comprising administering an effective dose of Compound I to improve or augment the effect of an acetylcholinesterase inhibitor such as donepezil or rivastigmine. The invention further provides pharmaceutical compositions comprising Compound I.
BACKGROUND ART
Dementia is a clinical syndrome characterized by deficits in multiple areas of cognition that cannot be explained by normal aging, a noticeable decline in function, and an absence of delirium. In addition, neuropsychiatric symptoms and focal neurological findings are usually present. Dementia is further classified based on etiology. Alzheimer's disease (AD) is the most common cause of dementia, followed by mixed AD and vascular dementia, vascular dementia, Lewy body dementia (DLB), and fronto-temporal dementia.
The incidence of Alzheimer's disease is expected to increase through the year 2050 with an estimated prevalence of 11 to 16 million cases. Currently, two classes of medications are FDA approved for managing symptoms of AD—acetylcholinesterase inhibitors (AChEIs) and an N-methyl-D-aspartase (NMDA) receptor antagonist. AChEIs are commonly used as initial treatment on diagnosis. The AChEIs—donepezil, rivastigmine, galantamine, and tacrine—are indicated for mild-to-moderate AD; only donepezil is approved for the severe stage.
Despite the available therapies, there are no treatments to cure AD or to prevent or stop disease progression. Acetylcholinesterase inhibitors do not help everyone who has Alzheimer's disease and in fact are not efficacious in many patients. Considering that AChEIs and memantine have only a modest symptomatic effect, and cannot prevent AD decline and slow disease progression, there is a high unmet need for more effective symptomatic treatments and for a disease modifying/slowing therapies.
The use of selective 5-HT6 receptor antagonists to treat cognitive dysfunction has been suggested and is based on several lines of reasoning. For example, selective 5-HT6 receptor antagonists have been shown to modulate cholinergic and glutamatergic neuronal function. The activity of selective 5-HT6 receptor antagonists has been demonstrated in animal models of cognitive function. Since the disclosure of the first selective 5-HT6 receptor antagonists, there have been several reports on the activity of these selective compounds in in-vivo models of cognitive function. N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine (herein referred to as “Compound I”) is a potent and selective 5-HT6 receptor antagonist which has been in clinical development for treating cognition impairment associated with schizophrenia and as a treatment for AD.




In November 2008, a multi-centre, randomised, double-blind, fixed-dose study (120 mg/day BID) was initiated to explore the efficacy and safety of Compound I as adjunctive treatment to risperidone in patients with schizophrenia. Overall improvement in schizophrenia symptoms was assessed by using the Positive and Negative Syndrome Scale (PANSS) total score. Compound I did not offer any treatment advantage over placebo as measured by the PANSS total score. In 2010, it was announced that there did not appear to be any treatment advantage over placebo in improving patients' overall neurocognitive performance as assessed using the BACS composite Z-score and the PANSS cognitive subscale scores.
In 2012, it was reported that a randomized, double blind, placebo controlled trial conducted in Europe, Canada and Australia met its primary endpoint in the treatment of AD. Data demonstrated that Compound I plus 10 mg/day of donepezil significantly improved cognitive function in 278 patients with Alzheimer's disease compared to placebo plus donepezil, when measured by Alzheimer's Disease Assessment Scale-cognitive sub-scale (ADAS-cog). Compound I showed positive results in secondary endpoints including measures of global impression and daily living activities compared to donepezil treated patients.
The daily dose of 90 mg of Compound I in the AD study was administered three times daily (3×30 mg) to overcome the relative short half-life observed in subjects in previous clinical studies. An issue for that dose selection was to ensure that the maximum exposure level fell under the maximum exposure limit which had been established from non-clinical toxicology studies. Accordingly, a fixed dose of three times in the study was introduced.
As the 5-HT6 receptor is a novel target predominately localized in the brain, a key problem in the development is to determine the amount of CNS penetration and the correlation with plasma exposure.
With CNS targets, further challenges exist that revolve around whether a drug will pass the blood brain barrier and whether it will reach the target at a suitable concentration and for a sufficient length of receptor occupancy.
Direct brain measures of 5-HT6 receptor occupancy may be valuable to many decision-making processes during the development of centrally acting drugs targeted at 5-HT6 to ensure adequate proof-of-concept testing and to optimize dosing regimens. In humans, tools such as positron emission tomography (PET) with specific radiolabeled ligands have been used to quantitatively assess in-vivo occupancy of a number of neurotransmitter receptors, including those for dopamine, serotonin, and benzodiazepines (Talbot, et al., European Neuropsychopharmacology, 2002, 12, 503-511).
The inventors discovered an effective PET ligand, [11C]-LuPET, which has since been successfully evaluated for human use. The ligand was subsequently used to determine the 5-HT6 receptor occupancy following multiple dose ranges of Compound I. In the assessment for receptor occupancy, human subjects were administered the compound for at least three days at several dosage regimens.
The inventors discovered that high levels of receptor occupancies were observed after multiple dosages of Compound I and that receptor occupancy was maintained 24 hrs post dose. Data generated from a separate Phase I PK study in the elderly and data generated from the above AD study have shown that the elimination half life of Compound I in the elderly population was longer (about 19 hours) compared to young healthy subjects (about 12 hours).
With these convergent discoveries, the inventors have identified improved methods of treating AD by introducing a new and improved dosage regime comprising once daily administration in a novel dosage range. Based on the findings described herein, the dose range contemplated is expected to be efficacious while providing exposure levels below the NOAEL, thus improving the safety ratio. The invention is described in described in greater detail below.
SUMMARY OF THE INVENTION
There remains a need for new treatments and therapies for Alzheimer's disease, and Alzheimer's disease-related disorders, such as dementia.
Thus, provided herein are methods of treating Alzheimer's disease as adjunctive therapy to acetylcholinesterase treatment comprising administering an effective daily dose of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine or a pharmaceutically acceptable salt to a patient in need of such treatment, wherein the effective daily dose administered to the patient is between about 30 and about 60 mg.
The invention further provides methods of treating Alzheimer's disease as adjunctive therapy to acetylcholinesterase treatment comprising administering an effective daily dose of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine or a pharmaceutically acceptable salt to a patient in need of such treatment, wherein the effective daily dose administered to the patient is between about 30 and about 60 mg.
One embodiment of the invention is directed to a method of treating mild to moderate Alzheimer's disease. In one embodiment, the pharmaceutically acceptable salt is the hydrochloride.
In another embodiment, the dose is administered as an immediate release formulation.
Yet another embodiment, the method is for treating mild to moderate Alzheimer's disease.
In another embodiment, the acetylcholinesterase inhibitor is donepezil.
In another embodiment, the acetylcholinesterase inhibitor is rivastigmine.
In another embodiment, the dose is administered once daily.
The aforementioned embodiment of the invention pertaining to once daily administration of Compound I has clear advantages for patients. Such advantages include but are not limited to ease of administration, convenience, and patient compliance with consistent dosing. However, certain embodiments of the invention also include, based on the Applicants' data herein, administration of Compound I more than once daily in amounts equivalent to the amounts disclosed herein within a twenty-four hour period.
Thus, embodiments of the invention also include the following:
In one embodiment, the effective daily dose is 30 mg.
In yet another embodiment, the dose is effective daily dose is 40 mg or less.
In one embodiment, the dose is effective daily dose is 50 mg or less.
In another embodiment, the dose is effective daily dose is 60 mg or less.
As used herein, Compound I is N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine and accordingly, the invention further provides a pharmaceutical composition comprising Compound I wherein the composition when administered to a human provides a blood plasma concentration of Compound I in a range of about 56 ng/mL to about 310 ng/mL at steady-state plasma level and when the composition is administered to provide an effective daily dose of Compound I of about 60 mg or less.
Further provided is a pharmaceutical composition comprising Compound I wherein the composition provides when administered to a human a receptor occupancy of Compound I greater than or equal to about 90% at the 5HT-6 receptor at a steady-state plasma level and when the composition is administered to provide an effective daily dose of Compound I of about 60 mg of less.
The invention further provides a pharmaceutical composition comprising 60 mg or less of Compound I, wherein the composition when administered to a human provides a blood plasma concentration of Compound I in a range of about 56 ng/mL to about 310 ng/mL at steady-state plasma level.
In one embodiment, the composition is an immediate release formulation.
In one embodiment, the effective daily dose is 30 mg.
In yet another embodiment, the effective daily dose is 40 mg or less.
In one embodiment, the effective daily dose is 50 mg or less.
The invention describes novel methods for treating and preventing dementia caused by vascular diseases; dementia associated with Parkinson's disease; Lewy Body dementia; AIDS dementia; mild cognitive impairments; age-associated memory impairments; cognitive impairments and/or dementia associated with neurologic and/or psychiatric conditions, including epilepsy, brain tumors, brain lesions, multiple sclerosis, Down's syndrome, Rett's syndrome, progressive supranuclear palsy, frontal lobe syndrome, and schizophrenia and related psychiatric disorders; cognitive impairments caused by traumatic brain injury, post coronary artery by-pass graft surgery, electroconvulsive shock therapy, and chemotherapy, comprising administering a therapeutically effective amount of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine to improve or augment the effect of a acetylcholinesterase inhibitor.
The invention also describes novel methods for treating and preventing delirium, Tourette's syndrome, myasthenia gravis, attention deficit hyperactivity disorder, autism, dyslexia, mania, depression, apathy, and myopathy associated with or caused by diabetes comprising administering a therapeutically effective amount of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine to improve or augment the effect of a acetylcholinesterase inhibitor. The invention further describes novel methods for delaying the onset of Alzheimer's disease, for enhancing cognitive functions, for treating and preventing sleep apnea, for alleviating tobacco withdrawal syndrome, and for treating the dysfunctions of Huntington's disease comprising administering a therapeutically effective amount of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine to improve or augment the effect of a cholinesterase inhibitor.
Provided herein are methods of treating Alzheimer's disease by improving or augmenting the effect of a acetylcholinesterase inhibitor comprising administering a dose of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine at least once daily. In one embodiment, the dose is administered every two days.
The invention is described in more detail below.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Time-activity curves (TACs) of selected brain regions.
FIG. 2: Time-profile of total radioactive metabolites of [11C]LuPET in plasma
FIG. 3: Histograms (with SEM bars) of distribution volume (VT) in cerebellum
FIG. 4: Histograms (with SEM bars) of test-retest variability (TRV)
FIG. 5: Histograms (with SEM bars) of distribution volume VT in selected brain regions
FIG. 6: Histogram of displacement (%) of [11C]LuPET by one single dose of 10 mg of olanzapine in selected brain regions
FIG. 7: Histograms (mean with SE bars) of occupancy of 5-HT6 receptors by Compound I by dosing scheme for 3 hour and second post-dose time points.
FIG. 8: Occupancy-PK plasma concentration of Compound I plots for putamen, caudate nucleus, and ventral striatum at 3 hour post-dose time point
FIG. 9: Occupancy-PK plasma concentration of Compound I plots for putamen, caudate nucleus, and ventral striatum at second post-dose time points.
FIG. 10: Occupancy-PK plasma concentration of Compound I plots for putamen, caudate nucleus, and ventral striatum pooling two post-dose time points
FIG. 11: Trans-axial images of binding potential at the level showing putamen and caudate nucleus for baseline, and 51 hours post-dose scans
FIG. 12: Plasma concentrations of Compound I vs. 5-HT6 receptor occupancy
FIG. 13: Simulation of 5-HT6 occupancy in caudate nucleus at steady-state for an AD population



DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention in detail, it may be helpful to provide definitions of certain terms to be used herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.
N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine is a potent and selective 5-HT6 receptor antagonist in clinical development for treating AD and is referred to as Compound I. The synthesis of Compound I, its use for the treatment of cognitive dysfunction disorders, and pharmaceutical compositions comprising this substance are disclosed in U.S. Pat. No. 7,157,488. Unless otherwise specified, or clearly indicated by the text, reference to Compound I useful in the therapy of the invention includes both the free base and all pharmaceutically acceptable salts of the compounds. A preferred salt of Compound I is the hydrochloride.
The AD study cited in the Background section is referred to in this application as study 12936A. The schizophrenia study cited in the Background section is referred to in this application as study 12450A.
In one embodiment of the invention, provided herein are methods of treating Alzheimer's disease by improving or augmenting the effect of a acetylcholinesterase inhibitor comprising administering a once daily effective dose of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine or a pharmaceutically acceptable salt to a patient in need of treatment thereof, wherein the dose range is between about 30 and about 60 mg. The invention further provides methods of treating AD Alzheimer's disease with Compound I as adjunctive therapy to acetylcholinesterase inhibitors.
One embodiment of the invention is directed to methods of treating mild Alzheimer's disease while a separate embodiment relates to methods of treating moderate Alzheimer's disease.
Yet another embodiment relates to methods of treating severe Alzheimer's disease.
One embodiment relates to methods of treating mild to moderate Alzheimer's disease.
In one embodiment, Compound I is administered as an immediate release formulation.
In another embodiment, Compound I is administered as a pharmaceutically acceptable salt.
In one embodiment, Compound I is administered as the hydrochloride salt.
In one embodiment, the acetylcholinesterase inhibitor is donepezil.
In another embodiment, the acetylcholinesterase inhibitor is rivastigmine.
In yet another embodiment, the acetylcholinesterase inhibitor is galantamine.
In one embodiment, the dose is an amount between 10 and 80 mg.
In a separate embodiment, the dose is an amount between 10 and 70 mg.
In one embodiment, the dose is an amount between 10 and 60 mg.
In one embodiment, the dose is an amount between 10 and 50 mg.
In one embodiment, the dose is an amount between 20 and 50 mg.
In one embodiment, the dose is an amount between 20 and 40 mg.
In a separate embodiment, the compound is administered in a 10 mg dose.
In one embodiment, Compound I administered in a 20 mg dose.
In one embodiment, Compound I is administered in a 30 mg dose.
In one embodiment, Compound I is administered in a 40 mg dose.
In another embodiment, Compound I is administered in a 50 mg dose.
In one embodiment, Compound I is administered in a 60 mg dose.
In yet another embodiment, Compound I is administered in a 70 mg dose.
In one embodiment, Compound I administered in a 80 mg dose.
In one embodiment, Compound I is administered in a 90 mg dose.
As used herein, the following terms shall have the meanings as set forth below:
A “therapeutically effective dose” of Compound I is an amount sufficient to provide an observable therapeutic benefit compared to baseline clinically observable signs and symptoms of Alzheimer's disease as measured by ADAS-cog, and Alzheimer's disease-related dementia treated in connection with the combination therapy.
“Immediate-release” is meant to include a conventional release, in which release of the drug starts immediately after administration. As used herein, the term “immediate release” includes dosage forms that allow the drug to dissolve in the gastrointestinal contents, with no intention of delaying or prolonging the dissolution or absorption of the drug. The objective is for the drug to be released rapidly after administration, for example for it to be possible to release at least 80% of the anti-dementia drug within approximately 30 minutes after commencement of dissolution in a dissolution test.
The term “acetylcholinesterase inhibitor” is known in those skilled in art and includes compounds selected from the group consisting of donepezil, rivastigmine, galantamine and tacrine. The FDA approved dosages of the acetylcholinesterase inhibitor are encompassed by the instant invention. For example, the methods cover the dosages of donepezil shown to be effective in controlled clinical trials of the treatment of mild to moderate Alzheimer's disease are 5 mg or 10 mg administered orally once per day. A 23 mg orally once daily dose of donepezil is also approved for treating moderate to severe AD.
The term “steady-state plasma level” means that a plasma level for Compound I has been achieved and which is maintained with subsequent doses of Compound I (preferably Css is maintained) at a level which is at or above the minimum effective therapeutic level and is below the minimum toxic plasma level for a given active agent(s).
The term “daily” means a given, continuous twenty-four (24) hour period.
The term “dose” is used herein to mean administration of Compound I in one dosage form to the patient being treated. In some embodiments, the dose is a single oral formulation. In some embodiments, the dose is formulated as a tablet, a capsule, a pill, or a patch administered to the patient
The term “effective daily dose” means the total amount of Compound I administered to a patient in need of therapy in a continuous, twenty-four (24) hour period. As a non-limiting example used herein solely to illustrate the meaning of the term, an effective daily dose of 90 mg shall mean and include administering a single dose of 90 mg in a twenty four hour period, administering two doses of 45 mg each within a twenty four hour period, and administering three doses of 30 mg each in a twenty four hour period, and so on. When administering Compound I in such a manner, i.e. more than once in a twenty four hour period, such administrations can be spread evenly through the twenty four hour period or even be administered simultaneously or nearly so.
The term “dose range” as used herein refers to an upper and a lower limit of an acceptable variation of the amount of agent specified. Typically, a dose of the agent in any amount within the specified range can be administered to patients undergoing treatment.
The term “treat” is used herein to mean to relieve, reduce or alleviate at least one symptom of a disease in a subject. For example, in relation to dementia, the term “treat” may mean to relieve or alleviate cognitive impairment (such as impairment of memory and/or orientation) or impairment of global functioning (overall functioning, including activities of daily living) and/or slow down or reverse the progressive deterioration in global or cognitive impairment.
The term “subject” is intended to include animals, which are capable of suffering from or afflicted with dementia associated with a CNS disorder, including without limitation neurodegenerative diseases such as Alzheimer's disease, Down's syndrome and cerebrovascular dementia, or any disorder involving, directly or indirectly, Alzheimer's disease. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from Alzheimer's disease or Alzheimer's disease-associated dementia, or Lewy body dementia.
The use of the terms “a” and “an” and “the” in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising, “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
The PET ligand used in the Positron Emission Topography study described in the Experimental Section is referred to as [11C]LuPET and has the following structure:




Pharmaceutically Acceptable Salts

The present invention also comprises salts of Compound I, typically, pharmaceutically acceptable salts. Such salts include pharmaceutically acceptable acid addition salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, sulfamic, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, itaconic, lactic, methanesulfonic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methane sulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids, theophylline acetic acids, as well as the 8-halotheophyllines, for example 8-bromotheophylline and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in Berge, et al., J. Pharm. Sci. 1977, 66, 2, the contents of which are hereby incorporated by reference.
Furthermore, Compound I and salts thereof may exist in unsolvated as well as in solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like.
Pharmaceutical Compositions
The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of Compound I and optionally a pharmaceutically acceptable carrier or diluent. Compound I may be administered alone or in combination with pharmaceutically acceptable carriers, diluents or excipients, in either single or multiple doses. The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, German), Ed., Mack Publishing Co., Easton, Pa., 1995.
Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, agar, pectin, acacia, stearic acid and lower alkyl ethers of cellulose corn starch, potato starch, talcum, magnesium stearate, gelatine, lactose, gums, and the like. Other adjuvants or additives usually used for such purposes such as colourings, flavourings, preservatives etc. may be used provided that they are compatible with the active ingredients.
The pharmaceutical compositions formed by combining Compound I and the pharmaceutical acceptable carriers are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration. The formulations may be presented in dosage form by methods known in the art of pharmacy.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules or tablets, each containing a predetermined amount of the active ingredient, and which may include one or more suitable excipients. The orally available formulations may be in the form of a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion. If a solid carrier is used for oral administration, the preparation may be tabletted, placed in a hard gelatine capsule in powder or pellet form or it can be in the form of a troche or lozenge.
Without limiting the scope of the invention, an example of an immediate release formulation of a once daily 30 mg dose of a pharmaceutically acceptable salt of Compound I is the following:
















Compound I monohydrochloride
32.75
mg



Calcium Phospohate Dibasic
222.0
mg



Colloidal Slilcon Dioxide NF (Aerosil 200)
3.900
mg



Magnesium Sterate NF (Vegatable Grade)
1.300
mg









The formulation can be encapsulated in a Gelatin Capsule Size #3.
In a similar manner, pharmaceutical compositions may be prepared comprising the administration of Compound I wherein the dose ranges administered are between about 30 mg and about 60 mg.
Methods of Treatment
Provided herein is a combination therapy useful for the treatment of mild, moderate and severe Alzheimer's disease, as well as symptoms associated with mild to moderate Alzheimer's disease. As discussed below, the methods provided herein have a number of advantages.
The term “Alzheimer's disease” refers to a progressive disease of the human central nervous system. It is manifested by dementia typically in the elderly, by disorientation, loss of memory, difficulty with language, calculation, or visual-spatial skills, and by psychiatric manifestations. It is associated with degenerating neurons in several regions of the brain. The term “dementia” as used herein includes, but is not restricted to, Alzheimer's dementia with or without psychotic symptoms.
In a particular embodiment, the therapeutic methods provided herein re effective for the treatment of mild, moderate and severe Alzheimer's disease in a subject. Phases of Alzheimer's further include “moderately severe cognitive decline,” also referred to as “moderate or mid-stage Alzheimer's disease,” “severe cognitive decline,” also referred to as “moderately severe or mid-stage Alzheimer's disease,” and “very severe cognitive decline,” also referred to as “severe or late-stage Alzheimer's disease.” Moderately severe cognitive decline is characterized by major gaps in memory and deficits in cognitive function emerge. At this stage, some assistance with day-to-day activities becomes essential. In severe cognitive decline, memory difficulties continue to worsen, significant personality changes may emerge and affected individuals need extensive help with customary daily activities. Late stage Alzheimer's disease or very severe cognitive decline is the final stage of the disease when individuals lose the ability to respond to their environment, the ability to speak and, ultimately, the ability to control movement.
In another embodiment, the patient to be treated by the combination therapy of the invention has an MMSE score between 12 and 22. “MMSE” refers to the Mini-Mental State Examination used in the cognitive assessment community.
EXPERIMENTAL SECTION










Table of Abbreviations
















5-HT2A
5-hydroxytryptamine 2A receptor


5-HT6
5-hydroxytryptamine 6 receptor


%
Percentage


AD
Alzheimer's Disease


AIC
Akaike information criterion


ANOVA
analysis of variance


A(T)
radioactivity concentration of the radioligand in a



brain region at time t after the injection (units)


Bavail
density of receptors available (unoccupied) to



bind radioligand in vivo (nmol · L−1 or nmol



receptor · 1,000 cm−3 tissue)


BPND
in vivo binding potential (unitless)


° C.
degrees Celsius


CIAS
Cognitive Impairment Associated with Schizophrenia


COV
coefficient of variation


CRO
contract research organization


C(t)
radioactivity concentration of the radioligand in plasma



at time, t after the injection


D
BPND post-dose


ECG
electrocardiogram


EC50
effective concentration that causes 50%



of maximum occupancy


eg
exempli gratia (for example)


fND
free fraction in nondisplaceable compartment (unitless)


FWHM
Full width at half maximum


g
Grams


H
Hour


HPLC
high-performance liquid chromatography


HRRT
high resolution research tomography


ie
id est (that is)


IRB
institutional review board


iv
intravenous


JHU
Johns Hopkins University


K1
rate constant for transfer from arterial plasma to tissue



(mL/min/g or mL/min/cm3)


k2
fractional brain-to-blood clearance rate constant of the



radioligand (min−1)


k2′
efflux constant back to plasma; see Koeppe et al., 1991


k3
association constant of the radioligand (min−1)


k4
dissociation rate constant of the radioligand (min−1)


KD
dissociation constant


μg
microgram


mCi
millicurie


mg
milligram


mL
milliliter


min
Minute


MRI
Magnetic Resonance Imaging


n
number


ng
Nanogram


OBD
optimum bed density, a trademark of Waters



Corporation, Milford, MA


Omax
maximum occupancy


OTCM
one tissue compartmental model


PET
Positron Emission Tomography


pH
reverse logarithmic representation of relative



hydrogen proton (H+) concentration


PI
Principal Investigator


PK
pharmacokinetic


po
per os, oral


PRGA
Plasma Reference Graphical Analysis


RO
receptor occupancy


R2
coefficient of determination for linear regression


SPGR
spoiled gradient, a type of data acquisition setting for MRI


T
tesla


TACs
time-(radio)activity curves


TTCM-UC
two-tissue compartment model approach with 5 parameters


TTCM-C
constrained two-tissue compartment model approach



with 5 parameters


v0
vascular volume in tissue (mL/mL)


VND
non-displaceable distribution volume, given as the K1-k2



ratio in TTCM-UC and TTCM-C (mL/mL)


VT
total volume of distribution (mL/mL)


VOI
volumes of interest







Brain structures








Am
amygdala


Cb
cerebellum


Cg
cingulate cortex


CN
caudate nucleus


Fr
frontal cortex


Fs
fusiform gyrus


GP
globus pallidus


Hp
hippocampus


In
insula cortex


Oc
occipital cortex


Pa
parietal cortex


PH
parahippocampus


Pu
putamen


vS
ventral striatum


Th
thalamus


Tp
temporal cortex









Example 1
Preparation of [11C]LuPET
The precursor is 3-phenylsulfonyl-8-(piperazin-1-yl)quinoline (C19H19N3O2S; MW: 353.4) and is known and publically disclosed compound. The precursor was dissolved in acetonitrile and transferred to a BioScan Autoloop System and reacted with [11C]-Iodomethane prepared as follows. 11CO2 prepared from bombardment of high purity nitrogen gas containing 0.5 to 1.0% oxygen with accelerated protons was reacted with hydrogen on a molecular sieve:nickel catalyst column at 380° C. to produce 11CH4, which was reacted with iodine vapor heated to 740° C. to form 11CH3I. [11C]-Iodomethane was swept through a furnace containing silver triflate to convert the radiolabeled iodomethane to [11C]-methyl triflate. [11C]-methyl triflate was swept into the loop methylation system using helium gas at a flow rate of about 20 mL/min at ambient temperature. The accumulation of [11C]-radioactivity in the loop was monitored with a local radiation monitor until the radioactivity reaches a plateau. The reaction mixture in the loop was allowed to remain at room temperature for 4.5 minutes. Crude [11C]LuPET was purified by preparative high-pressure liquid chromatography (HPLC) using Waters XBridge Prep OBD C18 10 μm 10×150 mm column with 30% acetonitrile:70% aqueous buffer (57 mM TEA adjusted to pH 7.2 with o-phosphoric acid) using a 10 mL/min flow rate. The [11C]LuPET fraction as determined with an in-line radiometric detector was collected in a reservoir of water. The reservoir was pressurized to load the [11C]LuPET onto the C18 Sep-Pak. The C18 Sep-Pak was then washed with 10 mL 0.9% Sodium Chloride for Injection. The [11C]LuPET is eluted from the C18 Sep-Pak with 1 mL of Ethanol followed by 10 mL of 0.9% Sodium Chloride Injection, through a sterilizing 0.24 filter in a sterile, pyrogen-free vial that has been preloaded with 4 mL Sodium Chloride Injection.
Example 2
Positron Emission Topography Experiments (Parts A and B)
As the radioligand has not previously been dosed to humans it was initially evaluated in the human brain to identify an optimal method for quantification and validate the radioligand as a PET tracer (Part A). The primary objective of this study was to assess occupancy of the 5-HT6 receptors after multiple oral doses of Compound I in healthy subjects using PET with [11C]LuPET as the radioligand (Part B).
Subjects
Eight healthy male subjects (Age: 30.6±7.7 years; range: 22-44 years) participated in Part A, and sixteen healthy male subjects (Age: 32.3±7.6 years; range: 21-44 years) participated in Part B of this study.
PET Experiments
PET studies were performed on the GE Advance Tomograph (GE Medical Systems, Waukesha, Wis., USA). Subjects had one venous catheter for the radioligand injection, and one arterial catheter to obtain arterial blood samples for the determination of radioactivity in plasma. Then, subjects were positioned in the scanner with the head restrained with a custom-made thermoplastic mask to reduce the head motion during PET data acquisition. Then, a 10 minute attenuation scan was performed using a rotating 68Ge source for attenuation correction. Dynamic PET acquisition was then performed in a three-dimensional mode for 90 min following an intravenous bolus injection of [11C]LuPET. A total of 30 PET frames were obtained (4×15, 4×30, 3×60, 2×120, 5×240, and 12×300 seconds). Arterial blood samples were collected at very short intervals (<5 s) initially and gradually prolonged intervals (every 15 min after 30 min) throughout the PET study for determination of plasma radioactivity. Selected samples taken at 0, 5, 10, 30, 45, 60, and 90 min were analyzed by HPLC for the presence of the radioligand and its radioactive metabolites as described elsewhere (Hilton et al., 2000).
In Part A, baseline scans were repeated on the same day (n=1) or separate days (n=7) ranging from 1 to 18 days apart to test reproducibility of PET outcome variables. A third scan was administered 5 hours after one single dose (10 mg) of olanzapine (n=5), utilized as a standard compound with high 5-HT6 receptor affinity.
In Part B, one baseline scan was followed by one post-dose scan at 3 hours (3H scan) and at 10, 11, 27, or 51 hours (2nd post dose scan, P2) following a minimum of three days of oral dosing of Compound I.
Reconstruction of PET data: Emission PET scans were reconstructed using the back projection algorithm with a ramp filter using the software provided by the manufacturer correcting for attenuation, scatter, and dead-time (Kinahan and Rogers, 1989). The radioactivity was corrected for physical decay to the injection time. Each PET frame consisted of 128 (left-to-right) by 128 (nasion-to-inion) by 35 (neck-to-cranium) voxels. Expected spatial resolution in this reconstruction setting was 5.5 and 6.1 mm full width at half maximum (FWHM) in the radical and tangential directions, respectively, at 10 cm radius from the center of the field-of-view (Lewellen et al., 1996).
MRI Acquisition
On a separate occasion, a spoiled gradient (SPGR) sequence MRI was obtained on each subject for anatomical identification of the structures of interest using the following parameters: Repetition time, 35 ms; echo time, 6 ms; flip angle, 458°; slice thickness, 1.5 mm with no gap; field of view, 24×18 cm2; image acquisition matrix, 256×192, reformatted to 256×256.
PET Data Analysis
Volumes of interest (VOIs): Cortical VOIs were automatically defined using Freesurfer software and combined into standard regions including: frontal (Fr), temporal (Tp), parietal (Pa), and occipital (Oc) cortices, fusiform gyms (Fs), cingulate (Cg), and insula (In). Subcortical regions were defined with FIRST software (Patenaude et al., 2011) and manually adjusted on individual MRIs. Subcortical regions included putamen (Pu), caudate nucleus (CN), ventral striatum (vS), globus pallidus (GP), thalamus (Th), hippocampus (Hp), and amygdala (Am). VOIs were transferred from MRI to PET spaces following MRI-to-PET coregistration parameters given by SPM5 (Ashbumer J, Friston 2003; Maes et al., 1997) to obtain time-activity curves (TACs) of regions. A total of 25 regions per scan were employed for methodological evaluations (Part A) while occupancy calculation was limited to Pu, CN, and vS (Part B).
Derivation of PET outcome variables: Primary PET outcome variables are total distribution volume, VT and binding potential, BPND (=fND·Bavail/KD, where fND refers to the fraction of non-displaceable compartment, Bavail stands for the density of available (unoccupied) 5-HT6 receptors, and KD stands for the dissociation constant; Innis et al., 2007).
A set of standard plasma input methods were employed to identify the optimal method for derivation of regional distribution volume (VT) for [11C]LuPET including, a one tissue compartmental model (OTCM) with three parameters (K1, and k2′; See Koeppe et al., 1991 for the definitions, and v0, tissue vascular volume), two tissue compartmental models with five parameters (K1, k2, k3, k4, and v0; See Innis et al., 2007 for the definitions), without and with constraining the K1-k2 ratio (non-displaceable distribution volume, VND (Abi-Dargham et al., 1994) to the cerebellum estimate (TTCM-UC and TTCM-C, respectively), and the plasma reference graphical analysis (PRGA; Logan et al., 1990). In TTCM-UC and TTCM-C, BPND was given as the k3-k4 ratio. In PRGA, BPND can be obtained as the region-to-Cb VT ratio less one, if it was confirmed that VT of Cb was not affected by the administration of Compound I. Metabolite-corrected plasma TACs were obtained by applying percent parent ligand time-profiles given by HPLC analysis to total plasma TACs, after interpolating at plasma sample times using the piecewise cubic Hermite interpolation implemented in Matlab (Mathworks, Cambridge, Mass., USA) and used in plasma input methods.
In addition, tissue reference methods, namely the multilinear reference tissue method with 2 parameters (MRTM2; Ichise et al., 2002) and reference tissue graphical analysis (RTGA; Logan et al., 1996) were applied. In RTGA, k2R (the brain-to-blood clearance rate constant of Cb) was set at 0.076 min−1, a mean k2 value (across baseline scans) given by TTCM-UC.
Test and retest scans of Part A and baseline scans of Part B were used for this section.
Independent estimation of distribution volume of nondisplaceable compartment, VND: It has been shown that a scatter plot of ΔVT (baseline minus post-dose) versus baseline VT, often referred to as Lassen plot can yield ‘theoretically correct’ VND as x-intercept of the regression line if the plot aligns linearly (Lassen et al., 1995; Cunningham et al., 2010). Note that this method yields one VND value that is common to all regions of the baseline and post-dose scans. The use of Lassen plot may be granted for occupancy calculation only if the plot aligns linearly. VND values given by the plot is used to evaluate whether VT of Cb may be used as an estimate of VND (i.e., negligible 5-HT6 receptors in Cb) in PRGA to obtain BPND and receptor occupancy.
Test-Retest Variability: The reproducibility of VT and BPND of [11C]LuPET was evaluated using the test-retest variability (TRV) which is given by the following formula (e.g., Sudo et al., 2001):




  
    
      
        
          TRV
          =
          
            ⁢
            
              
                ∑
                
                  i
                  =
                  1
                
                n
              
              ⁢
              
                
                  
                    
                      
                      
                        
                          ⁢
                          
                            V
                            test
                          
                        
                        -
                        
                          V
                          retest
                        
                      
                      
                    
                    
                      
                        (
                        
                          
                            V
                            test
                          
                          +
                          
                            V
                            retest
                          
                        
                        )
                      
                      /
                      2
                    
                  
                  /
                  n
                
                ⁢
              
            
          
        
        ⁢
        
          
        
      
    
    
      
        (
        1
        )
      
    
  




where vtest and vretest refer to estimates of VT or BPND of test and retest scans in the region, respectively. A number of papers employed a TRV of 10% as a criterion of radioligand acceptable reproducibility (Hirvonen et al., 2009). Therefore this level was used as reference in this report. Test and retest scans of Part A alone were used for this section.

Occupancy and occupancy-PK relationships: Occupancy of 5HT6 receptors (RO in %) by Compound I (Part B) and olanzapine (Part A) was calculated with the following equation:




  
    
      
        
          RO
          =
          
            
              (
              
                1
                -
                
                  
                    
                      BP
                      ND
                      D
                    
                    ⁢
                  
                  
                    ⁢
                    
                      BP
                      ND
                      B
                    
                  
                
              
              )
            
            ×
            100
          
        
        ⁢
        
          
        
      
    
    
      
        (
        2
        )
      
    
  




where superscripts indicate BPND of baseline (B) and post-dose (D), respectively.

A single oral dose of 10 mg olanzapine has shown to result in a dopamine D2 receptor occupancy of approximately 60% in healthy male subjects (Nyberg et al., 1997). The olanzapine human in vitro receptor affinity for the 5-HT6 and dopamine D2 receptors are reported to be comparable with Ki values of approximately 10 nM and 30 nM, respectively (Kroeze et al, 2003), so an at least comparable 5-HT6 occupancy (=60%) could be expected at the 10 mg dose. As olanzapine has even higher affinity for the 5-HT2A receptor, some contribution from 5-HT2A to the measured total displacement following the olanzapine dose must be anticipated. In the striatum the density of 5-HT6 is high (Woolley et al., 2004) and density of 5-HT2A is low (Pompeiano et al., 1994), and consequently the main contribution will be from displacement from 5-HT6 receptors in this region.
The occupancy-PK (the concentration of Compound I in plasma) relationship was fitted by the following modified first-order Hill equation.




  
    
      
        
          RO
          =
          
            
              
                ·
                
                  O
                  max
                
              
              ⁢
            
            
              PK
              +
              
                
                  EC
                  50
                
                ⁢
              
            
          
        
        ⁢
        
          
        
      
    
    
      
        (
        3
        )
      
    
  




where Omax stands for the maximal attainable occupancy, and EC50 refers to the PK that achieves 50% of Omax. Akaike information criteria (AIC; Akaike 1974; Burnham and Anderson 2004) was used to examine goodness of fit for compartmental models and for the examination of occupancy-PK relationships using Equation 3.

Results

The following results from Parts A and B refer to the figures in the Drawings of the Invention. Tissue TACs: On baseline scans, TACs of Pu and CN formed peaks before 10 min, and showed continued increases thereafter for the 90 min period (FIG. 1) while TACs of other brain regions reached respective peaks before 20 min and decreased monotonously thereafter. Cb showed the lowest accumulations of the radioactivity among brain regions. In post-dose scans, shapes of Pu and CN TACs became closer to TACs of other brain regions in a dose dependent manner (Panel B). Cb TACs remained relatively similar between baseline and post-dose scans.
FIG. 1: Time-activity curves (TACs) of selected brain regions: Plasma TACs: Time-activity curves (TACs) of selected brain regions (FIG. 1), as means of baseline scans of Part B (Panel A) and 3 H post-dose (B) scans. Putamen and caudate nucleus showed distinctively high accumulation of radioactivity while cerebellum showed the lowest accumulation among brain regions. Total radioactive metabolites in plasma increased as a function of time after the tracer injection (FIG. 2, Panel A), reaching 69±9% at 90 min. 3H and 2nd-post dose scans showed indistinguishable HPLC time-profiles to baseline scans. Total and metabolite-corrected plasma TACs are shown in Panel B. Both formed peaks within 1 min (inlet), and declined mono-exponentially thereafter. Note that mean HPLC time-profile of 8 scans with successful HPLC were used in data analysis of Part A because HPLC was not successful in remaining 12 scans due to technical problems. HPLC time profiles of individual scans were used in Part B.
FIG. 2: Time-profile of total radioactive metabolites of [11C]LuPET in plasma (Panel A), averaged across baseline scans of Part B. Time activity curves (TACs), total and metabolite corrected in plasma (B) expressed in SUV as averages of baseline scans, limiting y-axis 1000 nCi/mL to display later parts TACs clearly (FIG. 2). The inlet was intended to show peaks clearly. Evaluation of methods for PET outcome variables: AIC supported TTCM over OTCM (i.e., OTCM showed higher AIC values than TTCM-UC or TTCM-C in 99.8% or 99.1% of total of 434 regions, respectively) suggesting that the blood-brain-transfer can be kinetically separated from the association-dissociation processes for [11C]LuPET. Thus, OTCM was rejected for this radioligand. However, TTCM-UC and TTCM-C yielded outliers (defined arbitrary as: VT>20 mL/mL, the highest value by PRGA and BPND>15, 3 times greater than the highest value by PRGA) in 8.1% and 6.2% of total regions for VT and 7.2% and 4.4% for BPND. Therefore, it was concluded that TTCM-UC and TTCM-C were not sufficiently robust for estimation of VT and BPND with [11C]LuPET. PRGA plots approached asymptotes at least at 40 min and showed excellent linearity (R2, coefficients of determination >0.93 in all regions). Further evaluations of PRGA are provided in the next two sections.
Between the two reference tissue methods, RTGA (=x) showed stronger correlation to PRGA (y=0.72·x+0.073; R2=0.926) than MRTM2 (y=0.73·x+0.22; R2=0.878), although both methods suffered underestimation of BPND at high BPND regions.
Evaluation of Lassen plot and cerebellum VT: Compared to the baseline scan, VT of Cb decreased at 3H scan (t=−3.09; p<0.01; paired t-test) and at second post-dose scan (t=−2.56; p<0.05) as shown in FIG. 3, Panel A. No statistical differences were observed between 3H and second post-dose scans (t=1.06; p>0.3). Lassen plot was linear (R2>0.9) in all cases except for one case (baseline vs. 51 hour post-dose scans of Subject 502 who showed the lowest occupancy; R2=0.501) whose VND estimate was −0.39 mL/mL. VND estimates remained unchanged between 3H and P2 scans (Panel B). However, the presence of the outlier eliminated the use of Lassen plot for occupancy calculation from this study. When the outlier was excluded, VT of Cb (=y) correlated with VND given by Lassen plot (y=0.90·x+0.08; R2=0.945) but was lower than VND (t=−4.36; p<0.001). This finding (VT in Cb at 3H<VND given by Lassen plot) suggested slight overestimation of VND by the plot. Together with statistically significant but negligible differences (a mean difference of 0.39 mL/mL between baseline and 3H scans) relative to high VT values observed in striatum regions (VT  ̃10 mL/mL in target regions), evaluations in this section justified the use of PRGA for occupancy calculation with [11C]LuPET using Cb as reference region.
FIG. 3: Histograms (with SEM bars) of distribution volume (VT) in cerebellum, Cb (Panel A) given by PRGA for baseline (B) and 3 hours (3H) and second post-dose (P2) scans, and distribution volume of non-displaceable compartment, VND obtained by Lassen plots of regional VT of baseline and 3H scans (3H), and baseline and P2 scans (P2) (Panel B). Test-retest variability: PRGA showed low TRV values of VT (range: 12.7%-15.6%), achieving the desired 10% level in various regions, while TTCM-C (range: 13.3%-45.8%) showed higher TRV values. On BPND, TRV values of target regions (i.e., Pu and CN) were close to 20% by PRGA and slightly higher by TTCM-C (FIG. 4, Panel A). Tissue reference methods showed unacceptable TRV values in various cortical and subcortical regions (Panel B). However, RTGA showed TRV values in target regions that were very close to the 10% level.
FIG. 4: Histograms (with SEM bars) of test-retest variability (TRV) estimates of binding potential, BPND for plasma input methods (Panel A) and reference tissue methods (B) in selected brain regions. Dotted horizontal lines indicate the 10% level which is often considered to be desirable level of TRV. Results of the method evaluation section including test-retest variability estimation indicated that PRGA is the most appropriate method for the derivation of VT, BPND, and occupancy with [11C]LuPET among widely recognized PET data analysis methods. Therefore, results obtained by PRGA are mainly presented hereafter. Results of RTGA are also presented as needed because the method section also indicated that RTGA may be usable when insertion of the arterial catheter and arterial blood sampling could be confounding factors, although the method may suffer from underestimation of BPND at high BPND regions. Regional values of VT given by PRGA and BPND given by PRGA and RTGA are presented in FIG. 5 to indicate regional distributions of VT and BPND in healthy male subjects in the studied age ranges.
FIG. 5: Histograms (with SEM bars) of distribution volume VT in selected brain regions (Panel A) in selected brain regions given by PRGA, and binding potential, BPND given by PRGA and RTGA (B). Displacement of [11C]LuPET binding by olanzapine: One dose of 10 mg olanzapine displaced binding of [11C]LuPET about 80% across regions (FIG. 6), although some regional differences were observed. The findings were consistent with the expected displacement that was discussed in the occupancy and occupancy-PK relationship section of the method section. Note that relatively similar results were obtained whether test or retest scan was used as the baseline scan for the sake of displacement calculation.
FIG. 6: Histogram of displacement (%) of [11C]LuPET by one single dose of 10 mg of olanzapine in selected brain regions (Panel A). Trans-axial images of binding potential BPND at the level showing putamen (Pu) and caudate nucleus (CN) for baseline (B), and post-olanzapine (C) scans. Individual BPND images were spatially normalized and averaged across subjects (n=5). Occupancy of 5-HT6 receptors by Compound I and occupancy-PK relationships: Observed occupancy values for dosing schemes are shown in Table 2.
FIG. 7. Occupancy-PK plots of 3H scans are shown in FIG. 8, together with the best fits of the plots by Equation 3 (i.e., model prediction). AIC supported a two parameter fit (i.e., estimation of both Omax and EC50) in favor of a one parameter fit (fixing Omax at 100%) in three regions. Estimates of Omax, EC50, and PK that is predicted to exert an 80% occupancy (80% RO) are listed in Table 2.
FIG. 7: Histograms (mean with SE bars) of occupancy of 5-HT6 receptors by Compound I by dosing scheme for 3 hour (Panel A) and second (B) post-dose time points. Dosing schemes include 5 mg (5Q; Part B4) once daily or QD, 30 mg QD (30Q; Part B3), 30 mg b.i.d. (30B; Part B2), and 60 mg b.i.d (60B; Part B1). Scatter plots of occupancy (=y) versus concentrations of Compound I in plasma (mean from 30 to 90 min post-tracer injection) of 3H scans (FIG. 8) were fitted by Equation 3 assuming a 100% Omax (Model 1; one parameter, EC50 to estimate), and estimating both Omax and EC50 (Model 2). Model 2 was supported over Model 1 in Pu, CN, and vS by Akaike information criterion (AIC; The smaller the AIC value the better the fit) (Akaike 1974) and F-test comparing residual sums of squares (RSS) of the two models (Table 1).
FIG. 8: Occupancy-PK plasma concentration of Compound I (Lu AE58054) plots for putamen (Pu), caudate nucleus (CN), and ventral striatum (vS) at 3 hour post-dose time point. Model prediction curves (i.e., the best fits by Equation 3) are shown by dotted curves.
FIG. 9: Occupancy-PK plasma concentration of Compound I (Lu AE58054) plots for putamen (Pu), caudate nucleus (CN), and ventral striatum (vS) at second post-dose time points: Model prediction curves (i.e., the best fits by Equation 3) shown by dotted curves. The plots with all data points pooled were fitted better by Model 2 than by Model 1 in Pu and CN, suggesting that Omax might be identified uniquely in these structures. Between 3H data points alone (red dotted curves) and the pooled data points (black dotted curves), model predicted curves were essentially identical in Pu and CN. Extrapolations of model prediction curves of P2 data alone (green dotted curves) also agreed with prediction curves of the pooled data.
The plots with all data points pooled were fitted better by Model 2 than by Model 1 in Pu and CN, suggesting that Omax might be identified uniquely in these structures. Between 3H data points alone (red dotted curves) and the pooled data points (black dotted curves), model predicted curves were essentially identical in Pu and CN. Extrapolations of model prediction curves of P2 data alone (green dotted curves) also agreed with prediction curves of the pooled data. Accordingly, estimates of Omax and EC50 were consistent across 3H, P2 and pooled data sets. These findings were not conclusive for vS presumably reflecting unstable estimates of occupancy in this region.
RTGA which does not require plasma TACs yielded comparable estimates of EC50 in Pu, Cn, and vS, but slightly lower estimates of Omax than PRGA also lists PK values that were predicted to generate 80% occupancy, assuming that this occupancy level might generate optimal clinical effect. The PK values were about 120 ng/mL for CN and vS, and about 50 ng/mL for Pu by PRGA. The PK value was about 100 ng/mL for CN by RTGA but not available for Pu and vS due to the fact that estimates of Omax were lower than 80% in these structures.
FIG. 10: Occupancy-PK (plasma concentration of Compound I (Lu AE58054)) plots for putamen (Pu), caudate nucleus (CN), and ventral striatum (vS) pooling two post-dose time points. Model prediction curves (i.e., the best fits by Equation 3) are shown as indicated.
FIG. 11: Trans-axial images of binding potential BPND at the level showing putamen (Pu) and caudate nucleus (CN) for baseline, and 51 hours (n=4; 30 mg one single dose only; C) post-dose scans: Trans-axial images of binding potential, BPND at the level showing putamen (Pu) and caudate nucleus (CN) for baseline and 51 hours (n=4; 30 mg one single dose only; C) post-dose scans. A BPND image of [11C]MDL100,809 of healthy young subjects (n=8) are shown in Panel D for reference. Individual BPND images were spatially normalized and averaged across members.







TABLE 1







Statistical evaluation of two models of the occupancy-PK equation (Equation 3)











Putamen (Pu)
Caudate nucleus (CN)
Ventral striatum (vS)














AIC

AIC

AIC




Model 1 vs. 2
F test
Model 1 vs. 2
F test
Model 1 vs. 2
F test

















3H
86.56 >> 64.69
48.25**
75.26 >> 66.26
13.84**
86.78~75.45
18.19**


P2
82.20 > 79.66
4.06
83.87~85.44
0.38
100.36~102.13
0.36


3H + P2
166.82 >> 146.20
31.17**
160.09 > 156.55
5.67*
193.33~191.56
3.74





AIC: >> strongly supports Model 2; > supports Model 2; ~ not clearly different Degree of freedom for F-test were (1,14), (1,13), and (1,29) for 3H, P2, and 3H + P2, respectively. Asterisks indicate levels of significance:


*for p < 0.05;


**for p < 0.001.






The same plots for P2 scans are shown in FIG. 9. Both AIC and F-test did not support Model 2 over Model 1 potentially because sufficient numbers of ‘close-to saturation’ data points were not observed to estimate Omax accurately at these later time points.







TABLE 2







Estimates of Omax, EC50, and PK that is predicted to exert 80% occupancy











Putamen (Pu)
Caudate nucleus (CN)
Ventral striatum (vS)

















Omax
EC50
80% RO
Omax
EC50
80% RO
Omax
EC50
80% RO



(%)
(ng/mL)
(ng/mL)
(%)
(ng/mL)
(ng/mL)
(%)
(ng/mL)
(ng/mL)













PRGA
















3H
82.8
4.9
137.8
90.3
6.4
50.1
85.5
4.6
66.5


P2
83.8
5.5
116.7
93.9
8.4
48.0
89.7
16.2
133.5


3H + P2
83.2
5.2
131.7
91.3
7.2
51.1
86.0
9.0
121.0









RTGA
















3H
77.5
5.4
—
85.2
6.7
102.8
78.2
4.6
—


P2
76.6
6.0
—
87.1
9.0
101.5
72.1
4.9
—


3H + P2
77.3
5.8
—
85.7
7.8
108.4
76.5
5.2
—









Images of BPND obtained by voxel-by-voxel application of PRGA are presented in FIG. 11 to visually display changes in BPND after administration of Compound I.
Discussion
The methodological evaluation section of this study identified PRGA as the optimal method for deriving VT, BPND, and occupancy from the radioligand PET data among widely recognized standard PET data analysis methods. The use of Cb as the reference region was justified for [11C]LuPET because VT estimates of Cb were slightly lower (but with excellent correlations to) than VND estimates given by Lassen plots for cases where the plot was successful.
It should be noted that TRV estimates of plasma input methods (TTCM and PRGA) could not be as accurate as they should be because of the use of mean HPLC time profiles for Part A scans due to unsuccessful HPLC in 60% of scans. Nevertheless, when individual scans' HPLC data were used in Part B, the goodness of fit (by means of AIC values) of occupancy-PK plots to the theoretical expectations (i.e., Equation 3) observed in this study were as good as AIC values observed in other receptor occupancy studies in our experience, which in turn ensures observed Omax and EC50 values obtained in Part B were accurate.
Distributions of BPND in the human brain of [11C]LuPET were strikingly different from BPND distributions of [11C]MDL100,907, a high-affinity antagonist ligand for 5-HT2A receptors (See FIG. 11): Pu and CN showed the highest BPND with [11C]LuPET but were relatively spared with [11C]MDL100,907 while cortical regions showed high BPND of [11C]MDL100,907 but relatively low BPND with [11C]LuPET.
Occupancy-PK plots agreed with the first order Hill's equation (Equation 3) for 3H and P2 scans separately or with 3H and P2 scans pooled together. Furthermore, prediction curves of these three data sets were essentially identical. These findings indicated that occupancy-PK relationships agreed between P2 scans (post-dose duration range: 10-51 hours; PK range: 0.54-204 ng/mL) and 3H scans in which PK values fell within the PK range of P2 scans in 12 out of 15 subjects. Therefore it appeared that Compound I was associated with relatively fast dissociation from receptors and clearance from the brain (i.e., no evidence of prolonged binding).
Based on results of Pu and CN which showed convincing statistics, this study predicted that oral administration of Compound I would be associated with Omax of about 90% and EC50 of slightly greater than 6 ng/mL. It should be noted that CN showed about 10% higher estimates of Omax than Pu. In fact, occupancy values were statistically higher for CN across all post-dose scans (t=2.09; p<0.05; df=30; paired t-test). Lastly, RTGA which does not require arterial blood sampling yielded comparable EC50 values to more invasive PRGA. It was speculated that slight but influential blocking in the reference region (Cb) may explain the underestimation of Omax by RTGA compared to PRGA. Thus, RTGA maybe applicable to drug occupancy studies in studying patient population on whom arterial blood sampling is not preferable Finally, it should be mentioned that vS suffered uncertainty (i.e., AIC scores indicated worse fits by Equation 3) probably due to smaller volumes ( ̃0.8 mL per side) than Pu and CN (>3 mL).
5-HT6 receptor occupancy following administration of Compound I at 60 mg BID, 30 mg BID and 30 mg QD was high; i.e. >90%, >85% and  ̃80%, respectively, at Cmax and only decreased slightly 24 hours post dose.
In conclusion, this study estimated Omax to be about 90% and EC50 to be about 6.5 ng/mL with an oral administration of an amount Compound I. These estimates contribute to estimating occupancy of 5-HT6 receptors that exerts optimal clinical efficacy when the optimal therapeutic doses and associated plasma concentrations of Compound I become available in its clinical applications.
Example 3
Population Pharmacokinetics Modeling & PK/PD Model 5-HT6 Occupancy Simulation
Using the data generated from the PET study of example 2, an objective of the modeling and simulation exercise was to estimate the 5-HT6 receptor occupancies after multiple administrations of Compound I at once daily dosages in the range of 5.0-30 mg in an Alzheimer's population accordingly to age.
Population Pharmacokinetics (popPK) Model
Data from a Phase I study in elderly and data from the AD study (study 12936A) have shown that the clearance of Compound I is reduced in the elderly and hence elderly subjects having higher plasma concentrations compared to younger subjects for the same dose. This age effect has been incorporated in the PopPK model. In total, the dataset used in the popPK model consisted of 265 patients. The doses administered, single or multiple, were in the range of 9.0-300 mg.
PK/PD Model 5-HT6 Occupancy
12 healthy subjects were included in the assessment of 5-HT6 occupancy after multiple administrations of Compound I. The subjects were administered Compound I for at least three days at the doses 120 (60 BID), 60 (30 BID), 30 mg/day (QD) or 5 mg (QD) with four subjects in each dose cohort. In total three PET scans were performed per subject, the first at baseline, the second around tmax on last day of dosing and the last in the interval 10-51 hours post dose on the last day of dosing.
Compound I plasma concentration versus 5-HT6 receptor occupancy in the caudate nuclei region is shown in FIG. 11. The PK/PD relation was modelled with an Emax model on the form Occ=Emax*Cp/(EC50+Cp) where Emax is the maximal occupancy, EC50 the Compound I plasma concentration giving rise to half Emax and Cp is the Compound I plasma concentration. In the clinical PET study, the occupancies for the second scan after baseline (performed 10-51 hours after last Compound I administration) did not obviously deviate more from the fitted plasma concentrations versus 5-HT6 receptor occupancy curve than the occupancies for the first scan after baseline (performed around Cmax). This indicates that no pronounced hysteresis or lag-time exists between PK and PD, which can be the case if for example the off-rate for the receptor is longer than the plasma elimination rate.
In order to allow for estimation of the inter-subject variability in the occupancy, non-linear mixed effect modelling was performed where the inter-subject variability was modelled as an exponential term on EC50. Emax and EC50 for the caudate nucleus region were estimated to 91% and 6.5 ng/mL, respectively. The uncertainties of the estimated values were low, in terms of relative standard errors they were 1.1% (Emax) and 15% (EC50). The reason for Emax not reached 100% is may be due to the fact that the radioligand used ([11C]LuPET) also binds to the 5-HT2A receptor. In the PK/PD simulations, Emax was set to 100%.
PK/PD Model
An Alzheimer's population in terms of age to the one in the study 12936A was simulated (range 54-90 years and median 75 years). Steady-state plasma profiles of Compound I and corresponding 5-HT6 receptor occupancies were simulated for the doses 5, 10, 15, 20, 25 and 30 mg/day and with 1000 patients per dose. The average 5-HT6 receptor occupancy in the caudate nucleus region during one day at steady-state was estimated for each patient. The median, 5% and 95% percentiles were estimated from the individual values for each dose group. Hence, 90% of the patients will be in the interval between the 5% and 95% percentiles.







TABLE 3







Summary of steady state exposures in an elderly population


(age 50-90 yearsa) simulated from the popPK model












Dose
Cmax (ng/mL)
Cmin (ng/mL)
AUC (h · ng/mL)

















Gender
(mg)
Mean
Median
SD
Mean
Median
SD
Mean
Median
SD




















Men
30 QD
173
136
120
100
61
116
3157
2251
2883



60 QD
370
286
273
225
134
265
6903
4821
6575



30 TID
529
376
510
433
273
503
11658
7928
12192


Women
30 QD
185
148
123
97
56
117
3176
2247
2947



60 QD
393
310
280
217
124
268
6938
4812
6701



30 TID
543
385
528
427
262
519
11753
7920
12609





amean 75 years, SD 7 years






Brief Discussion

Using the data from PK/PD model, FIG. 12 displays the projected correlation between 5-HT6 receptor occupancy and plasma concentration of Compound I. The exposure (see highlighted range on X axis) given by the fixed dose of Compound I in the AD study shows that the subjects in the study experienced high receptor occupancy levels of the 5-HT6 receptor.
FIG. 13 shows the simulated 5-HT6 receptor occupancy in caudate nucleus at steady-state for an Alzheimer's population for the doses 5.0-30 mg/day. The median occupancies range from 56% for 5.0 mg to 92% for 30 mg. Based on the results from the PET study, the plasma concentration of Compound I versus 5-HT6 occupancy curve seems to be well described.
Thus, the data supports that finding of a daily dosing of an effective amount between about 30 and about 60 mg thereby improving patient compliance and avoiding complications seen in previous clinical trials.

## Claims

1. A method of treating Alzheimer's disease, comprising administering to a patient in need of such treatment and undergoing treatment with an acetylcholinesterase inhibitor a daily dose of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine or a pharmaceutically acceptable salt thereof effective for treating Alzheimer's disease, wherein the daily dose administered to the patient is between 10 and 50 mg and the dose is administered once daily.

2. The method of claim 1, wherein the pharmaceutically acceptable salt is the hydrochloride.

3. The method of claim 1, wherein the dose is administered as an immediate release formulation.

4. The method of claim 1, wherein the Alzheimer's disease is mild to moderate Alzheimer's disease.

5. The method of claim 1, wherein the acetylcholinesterase inhibitor is donepezil.

6. The method of claim 1, wherein the acetylcholinesterase inhibitor is rivastigmine.

7. The method of claim 1, wherein the administration of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine improves or augments the effect of the acetylcholinesterase inhibitor.

8. The method of claim 1, wherein the dose is administered orally.

9. A method of treating Alzheimer's disease as adjunctive therapy to acetylcholinesterase inhibitor treatment, comprising:
administering an effective daily dose of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine or a pharmaceutically acceptable salt thereof to a patient in need of such treatment, wherein the daily dose administered to the patient is between 10 and 50 mg and the dose is administered once daily; and
administering an acetylcholinesterase inhibitor to the patient.

10. The method of claim 9, wherein the pharmaceutically acceptable salt is the hydrochloride.

11. The method of claim 9, wherein the dose is administered as an immediate release formulation.

12. The method of claim 9, wherein the Alzheimer's disease is mild to moderate Alzheimer's disease.

13. The method of claim 9, wherein the acetylcholinesterase inhibitor is donepezil.

14. The method of claim 9, wherein the acetylcholinesterase inhibitor is rivastigmine.

15. The method of claim 9, wherein the administration of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine improves or augments the effect of the acetylcholinesterase inhibitor.

16. The method of claim 9, wherein the dose is administered orally.

17. A method of treating Alzheimer's disease, comprising administering to a patient in need of such treatment and undergoing treatment with an acetylcholinesterase inhibitor a daily dose of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine or a pharmaceutically acceptable salt thereof effective for treating Alzheimer's disease, wherein the daily dose administered to the patient is 10 mg once daily.

18. The method of claim 17, wherein the pharmaceutically acceptable salt is the hydrochloride.

19. The method of claim 17, wherein the dose is administered as an immediate release formulation.

20. The method of claim 17, wherein the Alzheimer's disease is mild to moderate Alzheimer's disease.

21. The method of claim 17, wherein the acetylcholinesterase inhibitor is donepezil.

22. The method of claim 17, wherein the acetylcholinesterase inhibitor is rivastigmine.

23. The method of claim 17, wherein the administration of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine improves or augments the effect of the acetylcholinesterase inhibitor.

24. The method of claim 17, wherein the dose is administered orally.

25. A method of treating Alzheimer's disease as adjunctive therapy to acetylcholinesterase inhibitor treatment, comprising administering an effective daily dose of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine or a pharmaceutically acceptable salt thereof to a patient in need of such treatment, wherein the daily dose administered to the patient is 10 mg and the dose is administered once daily;
and administering an acetylcholinesterase inhibitor to the patient.

26. The method of claim 25, wherein the pharmaceutically acceptable salt is the hydrochloride.

27. The method of claim 25, wherein the dose is administered as an immediate release formulation.

28. The method of claim 25, wherein the Alzheimer's disease is mild to moderate Alzheimer's disease.

29. The method of claim 25, wherein the acetylcholinesterase inhibitor is donepezil.

30. The method of claim 25, wherein the acetylcholinesterase inhibitor is rivastigmine.

31. The method of claim 25, wherein the administration of N-[2-(6-fluoro-1H-indol-3-yl)ethyl]-3-(2,2,3,3-tetrafluoropropoxy)benzylamine improves or augments the effect of the acetylcholinesterase inhibitor.

32. The method of claim 25, wherein the dose is administered orally.