WO2009046338A1

WO2009046338A1 - Method for increasing cerebral blood flow with (+)-nilvadipine enantiomer

Info

Publication number: WO2009046338A1
Application number: PCT/US2008/078803
Authority: WO
Inventors: Michael J. Mullan; Daniel Paris; Robert A. Ivey, Iii
Original assignee: Roskamp Research LLC
Current assignee: Roskamp Research LLC
Priority date: 2007-10-05
Filing date: 2008-10-03
Publication date: 2009-04-09
Anticipated expiration: 2010-04-05

Abstract

The present invention provides methods for treating, preventing, or ameliorating the symptoms of low cerebral blood flow in a human or animal by administering therapeutically effective amounts of the (S)-enantiomer of the dihydropyridine compound nilvadipine, also known as (+)-nilvadipine, to a human or animal in need thereof.

Description

METHOD FOR INCREASING CEREBRAL BLOOD FLOW WITH (+)-NIL VADIPINE

ENANTIOMER

This application claims priority to US provisional patent application 60/978,023, filed October 5, 2007, and the entire contents of the priority application are incorporated by reference.

Field of the Invention

The present invention relates to a method for treating the pathophysiological effects of reduced cerebral blood flow. More specifically, the method involves administering a specific enantiomer of the dihydropyridine compound nilvadipine. The (+)-enantiomer of nilvadipine opposes pathophysiological effects in the brain of animals or humans afflicted with diseases associated with decreased cerebral blood flow by increasing cerebral blood flow and is more effective at increasing cerebral blood flow than the racemic mixture of nilvadipine.

BACKGROUND OF THE INVENTION

Description of Related Art

Alzheimer's disease (AD) is the most common neurodegenerative disorder of aging, afflicting approximately 1% of the population over the age of 65. Characteristic features of the disease include the progressive accumulation of intracellular neurofibrillary tangles, extracellular parenchymal senile plaques, and cerebrovascular deposits in the brain. The principal component of senile plaques and cerebrovascular deposits is the 39-43 amino acid β-amyloid peptide (Aβ), which is proteolytically derived from amyloid precursor protein (APP), a transmembrane glycoprotein. APP is a single-transmembrane protein with a 590-680 amino acid extracellular amino terminal domain and an approximately 55 amino acid cytoplasmic tail. Messenger RNA from the APP gene on chromosome 21 undergoes alternative splicing to yield eight possible iso forms, three of which (the 695, 751 and 770 amino acid iso forms) predominate in the brain. APP undergoes proteolytic processing via three enzymatic activities, termed α-, β- and γ-secretase. Alpha-secretase cleaves APP at amino acid 17 of the Aβ domain, thus releasing the large soluble amino-terminal fragment α-APP for secretion. Because α-secretase cleaves within the Aβ domain, this cleavage precludes Aβ formation. Alternatively, APP can be cleaved by β-secretase to define the amino terminus of Aβ and to generate the soluble amino- terminal fragment β-APP. Subsequent cleavage of the intracellular carboxy-terminal domain of APP by γ-secretase results in the generation of multiple peptides, the two most common being 40-amino acid Aβ (Aβ40) and 42-amino acid Aβ (Aβ42). Aβ40 comprises 90-95% of the secreted Aβ and is the predominant species recovered from cerebrospinal fluid (Seubert et al, Nature, 359:325-7, 1992). In contrast, less than 10% of secreted Aβ is Aβ42. Despite the relative paucity of Aβ42 production, Aβ42 is the predominant species found in plaques and is deposited initially, perhaps due to its ability to form insoluble amyloid aggregates more rapidly than Aβ40 (Jarrett et al., Biochemistry, 32:4693-7, 1993). The abnormal accumulation of Aβ in the brain is believed due to either over-expression or altered processing of APP.

Aβ peptides are thus believed to play a critical role in the pathobiology of AD, as all the mutations associated with the familial form of AD result in altered processing of these peptides from APP. Indeed, deposits of insoluble, or aggregated, fibrils of Aβ in the brain are a prominent neuropathological feature of all forms of AD, regardless of the genetic predisposition of the subject.

Concomitant with Aβ deposition, there exists robust activation of inflammatory pathways in AD brain, including production of pro-inflammatory cytokines and acute-phase reactants in and around Aβ deposits (McGeer et al, J Leukocyte Biol, 65:409-15, 1999). Activation of the brain's resident innate immune cells, the microglia, is thought to be intimately involved in this inflammatory cascade. It has been demonstrated that reactive microglia produce proinflammatory cytokines, such as inflammatory proteins and acute phase reactants, such as alpha- 1-antichymotrypsin, transforming growth factor β, apolipoprotein E and complement factors, all of which have been shown to be localized to Aβ plaques and to promote Aβ plaque "condensation" or maturation (Nilsson et al., J. Neurosci. 21 : 1444-5, 2001), and which at high levels promote neurodegeneration. Epidemiological studies have shown that patients using non-steroidal anti-inflammatory drugs (NSAIDS) have as much as a 50% reduced risk for AD (Rogers et al., Neurobiol. Aging 17:681-6, 1996), and post-mortem evaluation of AD patients who underwent NSAID treatment has demonstrated that risk reduction is associated with diminished numbers of activated microglia (Mackenzie et al., Neurology 50:986-90, 1998). Further, when Tg APP_SW mice, a mouse model for Alzheimer's disease, are given an NSAID (ibuprofen), these animals show reduction in Aβ deposits, astrocytosis, and dystrophic neuritis correlating with decreased microglial activation (Lim et al., J. Neurosci. 20:5709-14, 2000).

Products of the inflammatory process in the AD brain therefore may exacerbate AD pathology. Furthermore, there is evidence that activated microglia in AD brain, instead of clearing Aβ, are pathogenic by promoting Aβ fϊbrillogenesis and consequent deposition as senile plaques (Wegiel et al, Acta Neuropathol. (Berl.) 100:356-64, 2000).

It also has been suggested that AD pathogenesis is due to the neurotoxic properties of Aβ. The cytotoxicity of Aβ was first established in primary cell cultures from rodent brains and also in human cell cultures. The work of Mattson et al. (J. Neurosci., 12:376-389, 1992) indicates that Aβ, in the presence of the excitatory neurotransmitter glutamate, causes an immediate pathological increase in intracellular calcium, which is believed to be very toxic to the cell through its greatly increased second messenger activities.

U.S. Patent Application No. 2005/0009885 (January 13, 2005) (Mullan et al.) discloses a method for reducing Aβ deposition using nilvadipine, as well as methods of diagnosing cerebral amyloidogenic diseases using nilvadipine. In addition, U.S. Patent 4,338,322 describes nilvadipine for its antihypertensive effects. Nilvadipine (NIV ADIL™) has received regulatory approval in Ireland for treatment of hypertension at a dose of 8 mg per day, or 16 mg per day if an adequate anti-hypertensive effect is not achieved with 8 mg per day. See also U.S. Patent 5,508,413, which discloses antihypertensive effects of the (+)-enantiomer of nilvadipine. The effects of racemic nilvadipine on cerebral blood flow in human subjects are reported in Hanyu et al. (Nuclear Medicine Communications, vol. 28, no. 4, pages 281-287, April 2007). In addition, the role of racemic nilvadipine in antagonizing Aβ vasoactivity and reduced cerebral blood flow has been reported in Paris et al. (Brain Research, vol. 999, pages 53-61, 2004). Despite the reports indicated above, there exists a need for treatment, amelioration, prophylaxis, or prevention of the symptoms of diseases associated with decreased cerebral blood flow, for example in the inexorable progression of brain degeneration that is a hallmark of AD, wherein the prophylaxis addresses the Aβ deposition, Aβ neurotoxicity, microglial- activated inflammation, and altered or overexpression of APP that is seen in AD patients with therapeutically effective treatment with minimal side effects.

SUMMARY OF THE INVENTION

In order to meet this need, the present invention provides for methods for increasing cerebral blood flow in an animal or human suffering from, or at risk of suffering from, a disease associated with decreased cerebral blood flow, comprising administering to the animal or human a therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine. In particular, the present invention provides for administration of enantiomerically-enriched (+)- nilvadipine yielding increased cerebral blood flow compared to the increase in cerebral blood flow elicited by the same amount of racemic nilvadipine or (-)-nilvadipine. In preferred embodiments, the (+)-enantiomer is present in excess in the administered composition, and the enantiomeric excess is preferably 90% or more, 95% or more, and more preferably 98% or more, including 100%, up to the detectable limit of purity. In one embodiment, such diseases are cerebral amyloidogenic diseases, particularly AD, including early stage or mild forms of the disease, such as mild cognitive impairment (MCI). In other embodiments, diseases and disorders that may be treated in accordance with methods according to the invention include stroke, ischemia, cerebral vasospasm, giant cell arteritis, and depression. The present invention also provides methods for diagnosing a disease associated with decreased cerebral blood flow in an animal or human, wherein the disease is a cerebral amyloidogenic disease, or determining if the animal or human is at risk for developing cerebral amyloidogenic disease, comprising: taking a first measurement of the plasma concentration of Aβ in the peripheral circulation of the animal or human; administering a therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine to the animal or human; taking a second measurement of the plasma concentration of Aβ in the peripheral circulation of the animal or human; and calculating the difference between the first measurement and the second measurement. An increase in the plasma concentration of Aβ in the second measurement compared to the first measurement indicates a risk of developing and/or a possible diagnosis of a cerebral amyloidogenic disease in the animal or human. In addition, administration of enantiomerically-enriched (+)-nilvadipine may result in lowered dosages compared to administration of racemic nilvadipine due to improved specificity by the active agent. In preferred embodiments, the (+)-enantiomer is present in excess in the administered composition, and the enantiomeric excess is preferably 90% or more, 95% or more, and more preferably 98% or more, including 100%, up to the detectable limit of purity.

The present invention further provides methods for reducing the risk of cognitive impairment in animals or humans suffering from, or at risk of suffering from, a disease or disorder associated with cognitive impairment or a risk thereof. Such diseases include traumatic brain injury as well as cerebral amyloidogenic diseases or conditions associated with traumatic brain injury. In certain embodiments, the method comprises administering to the animal or human in need thereof a therapeutically effective amount of enantiomerically-enriched (+)- nilvadipine following a traumatic brain injury and continuing enantiomerically-enriched (+)- nilvadipine treatment for a prescribed period of time thereafter. In particular, administration of enantiomerically-enriched (+)-nilvadipine results in an increase in cerebral blood flow compared to the same amount of racemic nilvadipine. In addition, administration of enantiomerically-enriched (+)-nilvadipine may permit reduced dosages compared to administration of racemic nilvadipine due to an increase in specificity for achieving increased cerebral blood flow. In preferred embodiments, the (+)-enantiomer is present in excess in the administered composition, and the enantiomeric excess is preferably 90% or more, 95% or more, and more preferably 98% or more, including 100%, up to the detectable limit of purity.

In certain embodiments, methods of the invention are employed to treat or reduce the effects and sequelae of cerebral events such as an ischemic stroke, traumatic brain injury, or brain hemorrhage.

The present invention also provides for methods for reducing the risk of developing a disease or disorder associated with reduced cerebral blood flow, comprising administering to the animal or human a therapeutically effective amount of enantiomerically-enriched (+)- nilvadipine, wherein the enantiomerically-enriched (+)-nilvadipine administration begins after the diagnosis of risk for developing the disease or condition associated with reduced cerebral blood flow. In particular, the present invention provides for administration of enantiomerically-enriched (+)-nilvadipine with an increased effect on cerebral blood flow compared to the same amount of racemic nilvadipine. In addition, administration of enantiomerically-enriched (+)-nilvadipine may permit reduced dosages that achieve the same cerebral blood flow effect compared to administration of racemic nilvadipine due to an increase in the specificity for the effect. In preferred embodiments, the (+)-enantiomer is present in excess in the administered composition, and the enantiomeric excess is preferably 90% or more, 95% or more, and more preferably 98% or more, including 100%, up to the detectable limit of purity.

The present invention provides for treating the clinical profile of a disease or disorder associated with reduced cerebral blood flow encompassing one or more or all of the cognitive and behavioral traits associated with the reduced cerebral blood flow. For example, if the disease or disorder is AD, such traits may include a non-limiting list of symptoms such as profound memory loss, difficulty in performing familiar tasks, problems with language, disorientation, decreased judgment, impaired abstract thinking, changes in personality, mood, or behavior, and/or characteristic scores in a battery of cognitive tests. Such cognitive tests include the Wechsler Memory Scale Revised (WMS-R), the Clinical Dementia Rating (CDR), the Mini-Mental State Examination (MMSE) and/or the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS).

The present invention provides for treatment with enantiomerically-enriched (+)-nilvadipine which may yield a specific clinical outcome. One endpoint for treatment is a measurable improvement in one or more disease symptoms in those affected by reduced cerebral blood flow. The improvement may result in an asymptomatic patient, or may reflect an improvement in ability compared to one or more symptoms prior to treatment. Alternatively, one endpoint for treatment may be to maintain a baseline symptomatic level. In other words, this endpoint represents stabilization of the disease or one or more symptoms of the disease permanently or for a period of time. In addition, one endpoint for treatment may be a reduction in the rate of disease progression compared to an untreated control. Furthermore, treatment may include pretreatment of an individual considered to be at risk for development of reduced cerebral blood flow, but prior to clinical manifestation of one or more symptoms. Reference to treatment of disease also encompasses treatment of one or more symptoms, where the treatment is palliative rather than curative.

The present invention also provides for methods of antagonizing Aβ vasoactivity and reduced cerebral blood flow comprising administering an amount of enantiomerically-enriched (+)- nilvadipine. In particular, the present invention provides for administration of enantiomerically-enriched (+)-nilvadipine with an increased effect on cerebral blood flow compared to the cerebral blood flow associated with administration of the same amount of racemic nilvadipine or (-)-nilvadipine. In the data presented herein, for example as shown in FIGS. 1 IA and B, (+)-nilvadipine begins to exhibit vasoactive properties at a much lower dose in the FPL64176 rat aorta model of vasoconstriction compared to (-)-nilvadipine.

The present invention also provides methods for treating transplantable neuronal stem cells, comprising administering an amount of enantiomerically-enriched (+)-nilvadipine to the neuronal stem cells prior to transplantation of the stem cells in the central nervous system of an animal or human afflicted with a cerebral amyloidogenic disease, such as AD. The administered amount is the amount that achieves the desired cytoprotective effect. In preferred embodiments, the (+)-enantiomer is present in excess in the administered composition, and the enantiomeric excess is preferably 90% or more, 95% or more, and more preferably 98% or more, including 100%, up to the detectable limit of purity.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a bar graph that illustrates the effect of chronic administration of racemic nilvadipine on Aβ deposition (Aβ burden) in different regions of the brain of TgAPP_sw mice using a 4G8 immunostaining technique.

FIG. 2 is a bar graph that illustrates the effect of chronic administration of racemic nilvadipine on microglial activation in TgAPP_sw mice in three regions of the brain using a CD45 immunostaining technique that determines the number of CD45+microglia.

FIG. 3 is a bar graph that illustrates the effect of racemic nilvadipine on microglial activation in N9 murine microglial cells in vitro activated with lipopolysaccharide (LPS) for 24 hours. Microglial activation is determined by TNF-α production (pg/ml) measured by ELISA.

FIG. 4 is a bar graph that illustrates the effect of racemic nilvadipine administration on Aβ neurotoxicity using HPNC cells treated for three days with 30 μM of pre-aggregated Aβl-40 (AgAβ). Neurotoxicity is assessed by measuring the amount of lactic dehydrogenase (LDH) released from cells.

FIGS. 5A and 5B are bar graphs that illustrate the effect of racemic nilvadipine on APP processing using human glioblastoma cells transfected with APP_SW. Cells were treated with 50 nM and 250 nM nilvadipine for 24 hours (FIG. 5A) and for 48 hours (FIG. 5B). Production of Aβ 1-40 in the culture medium was measured by ELISA. FIG. 6: A-D) Two-dimensional color-coded (presented in gray-scale) microvascular flow maps of the brain of Tg APPsw recorded with a laser Doppler imager. Representative Laser Doppler imaging flow data depicting the variation of regional CBF in the cortex of TgAPPsw (line 2576) implanted with a placebo pellet and with a pellet delivering 56 mg/Kg/day of (-)-nilvadipine.

E) Histogram representing the values of cerebral blood flow expressed as a % of the cerebral blood flow values measured in the cortex of Tg APPsw mice implanted with a placebo pellet. No significant effect of (-)-nilvadipine was observed on the cerebral blood flow of the animals receiving a dosage of 30 mg/Kg/Day or 56 mg/Kg/Day of (-)-nilvadipine.

FIG. 7 is a dose response curve showing the effect of pure enantiomeric forms of nilvadipine (nilvadipine 1 and nilvadipine 2) as well as a mixture of the 2 enantiomers in equal proportion (N1+N2) on Aβl-40 production by 7 W WT APP751 Chinese hamster ovary cells following 24 hours treatment. Both enantiomers appear to dose dependently inhibit Aβl-40 production in a similar fashion.

FIG. 8 is a dose response curve showing the effect of pure enantiomeric forms of nilvadipine (nilvadipine 1 and nilvadipine 2) as well as a mixture of the 2 enantiomers in equal proportion (N1+N2) on Aβl-42 production by 7 W WT APP751 Chinese hamster ovary cells following 24 hours treatment. Note that the pure enantiomer nilvadipine 2 as well as the racemic mixture of nilvadipine (N1+N2) slightly stimulate Aβl-42 at low dose whereas the enantiomer nilvadipine 1 is deprived of such effect. FIG. 9 is a chiral chromatograph showing the separation of the enantiomers of nilvadipine. Nilva Peak l corresponds to (-)-nilvadipine (nilvadipine 1); Nilva_Peak_2 corresponds to (+)-nilvadipine (nilvadipine 2). Nilva lO, which refers to the original racemic mixture of nilvadipine, is included for illustrative purposes.

FIG. 10 shows the effect of (-)-nilvadipine (nilvadipine 1) and (+)-nilvadipine (nilvadipine 2) on FPL64176 induced vasoconstriction in rat aortae. Data show that (+)-nilvadipine completely antagonizes FPL64176-induced vasoconstriction whereas (-)-nilvadipine does not affect the vasoactive effect of the L-type calcium channel agonist, showing that (+)- nilvadipine is an L-type calcium channel blocker whereas (-)-nilvadipine is deprived of such effect.

FIGS. 1 IA-B show the vasoactive properties of (+)-nilvadipine versus (-)-nilvadipine over a range of drug dosages.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides prophylactic methods and therapeutic methods for increasing cerebral blood flow in an animal or human suffering from, or at risk of suffering from, a disease associated with decreased cerebral blood flow or that can be treated or managed by increased cerebral blood flow, comprising administering to the animal or human a therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine. Without wishing to be bound by theory, the administration of enantiomerically-enriched (+)- nilvadipine yields increased cerebral blood flow compared to administration of the same amount of racemic nilvadipine. For example, the invention provides for the halting or slowing of the progression of brain degeneration that is a hallmark of certain cerebral amyloidogenic diseases, such as, Alzheimer's disease (AD), in animals and humans, by administering enantiomerically-enriched (+)-nilvadipine (isopropyl-3-methyl-2-cyano-l,4- dihydro-6-methyl-4-(m-nitrophenyl)-3,5-pyridine-dicarboxylate; MW 385.4). Treatment, prevention, and amelioration of symptoms are encompassed by methods according to the invention.

Diseases associated with decreased cerebral blood flow can include without limitation stroke, such as ischemic stroke, ischemia, depression, including subcortical ischemic depression, giant cell arteritis, temporal arteritis, cerebral vasospasm, infarction, obstruction of a cerebral blood vessel, hemorrhage, such as subarachnoid hemorrhage, or any other indication related to restricted cerebral blood flow.

Unless otherwise specified, the term nilvadipine as used herein refers to the racemic mixture. The term "enantiomerically enriched" as used herein refers to a compound that is a mixture of enantiomers in which the (+)-enantiomer is present in excess, and preferably present to the extent of 90% or more, 95% or more, and more preferably 98% or more, including 100%, up to the detectable limit of purity. For example, purity can be determined by detection with chiral HPLC methods. In one embodiment, enantiomeric excess is calculated by subtracting the minor component from the major component. For example, a mixture of enantiomers with 98% (+)-enantiomer and 2% (-)-enantiomer would be calculated as a 96% enantiomeric excess of the (+)-enantiomer. In particular, one embodiment of the present invention provides a method for treating, preventing, or ameliorating the symptoms of a cerebral amyloidogenic disease or condition in an animal or human suffering or at risk of suffering from a cerebral amyloidogenic disease or condition by administering therapeutically effective amounts of enantiomerically-enriched (+)-nilvadipine. Because most cerebral amyloidogenic diseases, such as AD, are chronic, progressive, intractable brain dementias, it is contemplated that the duration of enantiomerically-enriched (+)-nilvadipine treatment will last for up to the lifetime of the animal or human. The cerebral amyloidogenic diseases or conditions include without limitation Alzheimer's disease, transmissible spongiform encephalopathy, scrapie, traumatic brain injury, cerebral amyloid angiopathy, and Gerstmann-Straussler-Scheinker syndrome. Methods of the invention also include treatment, prevention, or management of mild cognitive impairment (MCI), which may progress to AD. As such, methods for preventing progression of MCI to AD, or in general, the progression of AD and other cerebral amyloidogenic diseases and conditions, are included.

In another embodiment of the present invention, a method is provided for reducing the risk of cognitive impairment or for treating cognitive impairment in animals or humans suffering from traumatic brain injury (TBI) by administering to the animal or human a therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine immediately after the TBI and continuing the enantiomerically-enriched (+)-nilvadipine treatment for a prescribed period of time thereafter. The duration of enantiomerically-enriched (+)-nilvadipine treatment that is contemplated for those animals or humans suffering from a TBI can last for between about one hour to five years, preferably between about two weeks to three years, and most preferably between about six months and twelve months. In another embodiment of the present invention, a method is provided for increasing cerebral blood flow in an animal or human to improve cognition or slow the progress of an impairment of cognition. Such methods are irrespective of the status of the animal or human with respect to cerebral amyloidogenic diseases, such as AD. It is contemplated that the duration of enantiomerically-enriched (+)-nilvadipine treatment in such methods will last for up to the lifetime of the animal or human. Changes in cognition may be quantified by methods known in the art. For example, cognition may be measured by the Logical Memory subscore of the Wechsler Memory Scale Revised (WMS-R), by Clinical Dementia Rating (CDR), by Mini-Mental State Examination (MMSE), by the Alzheimer's disease Assessment Scale-Cognitive Subscale (ADAS-Jcog), and the like. See, for example, methods of assessment described in Hanyu et al., Nuclear Medicine Communications, vol. 28, no. 4, pages 281-287, April 2007, the contents of which are herein incorporated by reference. Administration of enantiomerically-enriched (+)-nilvadipine according to the invention increases cognitive ability or reduces or slows a decline in cognitive ability by an amount selected from 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, between 20-30%, between 30-40%, between 40-50%, and above, and any value (integer or non-integer) in between, over a time period of days, weeks, months, or years, up to the lifetime of the animal or human. Measurement of cognitive ability may be measured at the same time as treatment with enantiomerically-enriched (+)- nilvadipine, or may be measured after treatment in the event that treatment has been discontinued.

In a further embodiment of the present invention, a method is provided for diagnosing or determining the risk for developing a cerebral amyloidogenic diseases, such as AD, in an animal or human, by taking a first measurement of the plasma concentration of Aβ in the peripheral circulation of the animal or human; administering a therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine form to the animal or human; taking a second measurement of the plasma concentration of Aβ in the peripheral circulation of the animal or human at a later time; and then calculating the difference between the first measurement and the second measurement. An increase in the plasma concentration of Aβ in the second measurement compared to the first measurement indicates a risk of developing or a possible diagnosis of a cerebral amyloidogenic disease in the animal or human. The duration of time that enantiomerically-enriched (+)-nilvadipine is administered between the first and the second plasma Aβ concentration measurements can last for between about one day to twelve months, preferably between about one week to six months, and most preferably between about two weeks to four weeks. It is contemplated that a small increase in plasma Aβ concentration after enantiomerically-enriched (+)-nilvadipine administration would be indicative of a risk of developing AD and/or diagnostic of the beginning stages of AD. Larger increases in plasma Aβ concentration after enantiomerically-enriched (+)-nilvadipine administration would reflect higher concentrations of Aβ liberated from the brain into the peripheral circulation and thus would be more indicative of a positive diagnosis of AD.

In one or more embodiments, a method is provided for treating the clinical profile of a disease or disorder associated with reduced cerebral blood flow encompassing one or more or all of the cognitive and behavioral traits associated with the reduced cerebral blood flow. For example, if the disease or disorder is AD, such traits may include a non-limiting list of symptoms such as profound memory loss, difficulty in performing familiar tasks, problems with language, disorientation, decreased judgment, impaired abstract thinking, changes in personality, mood, or behavior, and/or characteristic scores in a battery of cognitive tests. Such cognitive tests include the Wechsler Memory Scale Revised (WMS-R), the Clinical Dementia Rating (CDR), the Mini-Mental State Examination (MMSE) and/or the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS).

In one or more embodiments, treatment with enantiomerically-enriched (+)-nilvadipine may yield a specific clinical outcome. One endpoint for treatment is a measurable improvement in one or more disease symptoms in those affected by reduced cerebral blood flow. The improvement may result in an asymptomatic patient, or may reflect an improvement in ability compared to one or more symptoms prior to treatment. Alternatively, one endpoint for treatment may be to maintain a baseline symptomatic level. In other words, this endpoint represents stabilization of the disease or one or more symptoms of the disease permanently or for a period of time. In addition, one endpoint for treatment may be a reduction in the rate of disease progression compared to an untreated control. Furthermore, treatment may include pretreatment of an individual considered to be at risk for development of reduced cerebral blood flow, but prior to clinical manifestation of one or more symptoms. Reference to treatment of disease also encompasses treatment of one or more symptoms, where the treatment is palliative rather than curative.

In one embodiment, the present invention provides for methods of antagonizing Aβ vasoactivity and reduced cerebral blood flow comprising administering an amount of enantiomerically-enriched (+)-nilvadipine. In particular, the present invention provides for administration of enantiomerically-enriched (+)-nilvadipine with an increased effect on cerebral blood flow compared to the same amount of racemic nilvadipine or (-)-nilvadipine. The therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine that is administered, optionally in unit dosage form, to animals or humans afflicted with, or at risk of developing, a disease associated with decreased cerebral blood flow, as well as administered for the purpose of determining the risk of developing and/or a diagnosis of a cerebral amyloidogenic disease in an animal or human, according to the methods of the present invention, can range from between about 0.05 mg to 30 mg per day, preferably from between about 2 mg to 20 mg per day, more preferably from between about 4 mg to 12 mg per day, and most preferably about 8 mg per day. The daily dosage can be administered in a single unit dose or divided into two, three or four unit doses per day. In another embodiment, the therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine is 1 mg per day, 2 mg per day, 3 mg per day, 4 mg per day, 5 mg per day, 6 mg per day, 7 mg per day, 8 mg per day, 9 mg per day, 10 mg per day, 11 mg per day, 12 mg per day, 13 mg per day, 14 mg per day, 15 mg per day, 16 mg per day, 17 mg per day, 18 mg per day, 19 mg per day, 20 mg per day, 21 mg per day, 22 mg per day, 23 mg per day, 24 mg per day, 25 mg per day, 26 mg per day, 27 mg per day, 28 mg per day, 29 mg per day, and 30 mg per day, all amounts including the term about, and any amount integer or otherwise, between the foregoing amounts. Ranges can vary. For example, non-limiting ranges include lower endpoints of 1, 2, 3, 4, and 5 mg per day, and upper endpoints of 15, 20, 25, and 30 mg per day, and any amount integer or otherwise, within the above mentioned amounts can serve as an endpoint.

While not wishing to be bound by theory, it is believed that increased cerebral blood flow may result in a reduction in deposition of Aβ material, or may serve as a flushing mechanism to help remove deposited Aβ material. Further, it is believed that the increased cerebral blood flow is a separate and distinct effect from (+)-nilvadipine's known anti-hypertensive effect, which is derived from L-type calcium channel blocking. In other words, where (+)- nilvadipine may have a common anti-hypertensive effect with other L-type calcium channel blocking, the increased cerebral blood flow effect of (+)-nilvadipine may result from activity other than L-type calcium channel blocking, and may not be common to other L-type calcium channel blockers.

In one embodiment according to the invention, the therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine increases cerebral blood flow by an amount selected from the group consisting of greater than about 1%, greater than about 5%, greater than about 10%, and above, up to the maximum cerebral blood flow that can be safely tolerated by a human or animal, following administration of the enantiomerically-enriched (+)-nilvadipine compared to the pretreatment cerebral blood flow. The increase in cerebral blood flow may be in comparison to the normal population, or may be in comparison to the treated individual's pre-treatment cerebral blood flow. In one embodiment, the post-treatment cerebral blood flow is measured after a period of treatment such that a steady-state change in cerebral blood flow has been established. For example, post-treatment cerebral blood flow may be measured 1 hour after treatment, or for example, after about 1 week of treatment, about 4 weeks of treatment, about 12 weeks of treatment, about 6 months of treatment, and the like. In one embodiment, administration of (+)-nilvadipine restores normal or near normal cerebral blood flow in an individual suffering from a disease state with decreased cerebral blood flow. In another embodiment, administration of (+)-nilvadipine results in a decrease in symptoms associated with reduced cerebral blood flow. Cerebral blood flow can be measured in a variety of ways. For example, cerebral blood flow may be measured with MRI techniques. In one embodiment, cerebral blood flow may be measured with single photon emission computed tomography (SPECT) (see, for example, methods reported in Hanyu et al., Nuclear Medicine Communications, vol. 28, no. 4, pages 281-287, April 2007, the contents of which are herein incorporated by reference). Increased cerebral blood flow may be global (i.e. the entire brain), or it may be regional, for example, in one or more parts of the brain such as the left frontal lobe, the right temporal lobe, the left and/or right middle frontal gyri, and the left superior parietal lobule.

In still another embodiment of the present invention is a method for pre -treating transplantable human or xenogenic neuronal stem cells by administering a therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine to the neuronal stem cells prior to transplantation of the cells in the central nervous system of an animal or human that may be afflicted with a cerebral amyloidogenic disease, such as AD. Presumably, neuronal stem cells themselves would not have a significant deposition of Aβ. However, if the neuronal transplant is intended for an Aβ-burdened environment, pre-treatment of the neuronal stem cells should enhance the ability of the transplanted neurons to survive in their new environment by increasing the local cerebral blood flow. The effective amount of enantiomerically-enriched (+)-nilvadipine that is administered for pre-treating the neuronal stem cells can range from between about 1 nM to 3 μM, preferably between about 10 nM to 2 μM, and most preferably between about 100 nM to 1 μM. It is known that stem cells, when directed to differentiate into specific cell types, such as neuronal cells, offer the possibility of a renewable source of replacement cells and tissues to treat diseases and conditions, such Alzheimer's disease, Parkinson's disease or spinal cord injury. When such cells are transplanted/implanted into a patient, it is advisable not only to pre-treat the cells with enantiomerically-enriched (+)-nilvadipine but to begin therapeutic treatment of the patient with enantiomerically-enriched (+)-nilvadipine post-implantation as well. It is contemplated that the methods of the present invention may be used on transgenic animal models for AD, such as the PDAPP and TgAPP_sw mouse models, which may be eventually useful for treating, preventing and/or inhibiting conditions associated with amyloid deposition, Aβ neurotoxicity and microgliosis in the central nervous system of such animals or in humans. Thus, the present invention provides for transgenic animal models for AD which are constructed using standard methods known in the art and as set forth in U.S. Pat. Nos. 5,487,992; 5,464,764; 5,387,742; 5,360,735; 5,347,075; 5,298,422; 5,288,846; 5,221,778; 5,175,385; 5,175,384; 5,175,383; and 4,736,866.

Enantiomerically-enriched (+)-nilvadipine can be administered to a patient via various routes including parenterally, orally or intraperitoneally. Parenteral administration includes the following routes: intravenous; intramuscular; interstitial; intra-arterial; subcutaneous; intraocular; intracranial; intraventricular; intrasynovial; transepithelial, including transdermal, pulmonary via inhalation, ophthalmic, sublingual and buccal; topical, including ophthalmic, dermal, ocular, rectal, or nasal inhalation via insufflation or nebulization.

Enantiomerically-enriched (+)-nilvadipine that is orally administered can be enclosed in hard or soft shell gelatin capsules, or compressed into tablets. Enantiomerically-enriched (+)- nilvadipine also can be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, sachets, lozenges, elixirs, suspensions, syrups, wafers, and the like. Further, enantiomerically-enriched (+)-nilvadipine can be in the form of a powder or granule, a solution or suspension in an aqueous liquid or non-aqueous liquid, or in an oil-in-water or water-in-oil emulsion. The tablets, troches, pills, capsules and the like also can contain, for example, a binder, such as gum tragacanth, acacia, corn starch; gelating excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; a sweetening agent, such as sucrose, lactose or saccharin; or a flavoring agent. The active ingredient may be formulated or administered in a unit dosage form or in a non-unit dosage form. When a dosage unit form is a capsule, it can contain, in addition to the materials described above, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain nilvadipine, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring. Additionally, enantiomerically-enriched (+)-nilvadipine can be incorporated into sustained-release preparations and formulations.

Enantiomerically-enriched (+)-nilvadipine can be administered to the CNS, parenterally or intraperitoneally. Solutions of nilvadipine as a free base or a pharmaceutically acceptable salt can be prepared in water mixed with a suitable surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative and/or antioxidants to prevent the growth of microorganisms or chemical degeneration.

The desired (+)-nilvadipine enantiomer has the (S) configuration at its chiral center. The compound provided herein may be enantiomerically pure, or be a stereoisomeric mixture with some amount of (-)(R)-nilvadipine. It is understood that the disclosure of enantiomerically-enriched (+)-nilvadipine herein encompasses any optically active, polymorphic, or stereoisomeric form, or mixtures thereof, which preferably possesses the useful properties described herein, it being well known in the art how to prepare optically active forms and how to determine activity using the standard tests described herein, or using other similar tests which are will known in the art. The term "enantiomerically enriched" as used herein, refers to a compound that is a mixture of enantiomers in which the (+)- enantiomer is present in excess, and preferably present to the extent of 90% or more, 95% or more, and more preferably 98% or more, including 100%, up to the detectable limit of purity. For example, purity can be determined by detection with chiral HPLC methods.

Examples of methods that can be used to obtain optical isomers of the compounds include the following:

i) physical separation of crystals - a technique whereby macroscopic crystals of the individual enantiomers are manually separated. This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct;

ii) simultaneous crystallization - a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state;

iii) enzymatic resolutions - a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme

iv) enzymatic asymmetric synthesis, a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer; v) chemical asymmetric synthesis - a synthetic technique whereby the desired enantiomer is synthesized from an achiral precursor under conditions that produce asymmetry (i.e., chirality) in the product, which may be achieved using chiral catalysts or chiral auxiliaries;

vi) diastereomer separations - a technique whereby a racemic compound, precursor, or semisynthetic intermediate is reacted with an enantiomerically pure reagent (the chiral auxiliary) that converts the individual enantiomers to diastereomers. The resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer (see, for example, U.S. patent 5,508,413 assigned to Fujisawa Pharmaceutical Co., Ltd);

vii) first- and second-order asymmetric transformations a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer. The desired enantiomer is then released from the diastereomer;

viii) kinetic resolutions - this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions;

ix) enantiospecific synthesis from non-racemic precursors - a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis; x) chiral liquid chromatography - a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase. The stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions;

xi) chiral gas chromatography - a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase;

xii) extraction with chiral solvents - a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; and

xiii) transport across chiral membranes - a technique whereby a racemate is placed in contact with a thin membrane barrier. The barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane which allows only one enantiomer of the racemate to pass through.

The enantiomers of nilvadipine have been separated as follows:

Nilvadipine 1 (-)-Nilvadipine

αJ _D -219.6° (c = 1.0, MeOH)

Observed: [α]² _D ⁵ -99.0° (c = 0.057, MeO H) Nilvadipine 2 (+)-Nilvadipine

Literatu re: [α]² _D° +222.4° (c = 1.0, MeOH) Observed: [α]² _D ⁵ +170.1° (c = 0.05, MeOH)

The literature values for optical rotations of the enantiomers of nilvadipine are reported in Satoh, Y.; Okumura, K.; Shiokawa, Y. Chem. Pharm. Bull. 42(4) 950-952 (1994). See Figure 9 for data regarding the enantiomeric purity of the separated compounds.

By the term "about" is meant within +10% of the stated amount, or within experimental error of the measuring technique. The phrase "a composition consisting essentially of is meant to encompass a composition in which the active pharmaceutical ingredient is as indicated. In one embodiment, the phrase "consisting essentially of excludes non-listed active pharmaceutical ingredients, but does not exclude pharmaceutically acceptable vehicles, carriers, or diluents, or the manner in which the active pharmaceutical ingredient is formulated. In one embodiment, for example in a method of treatment, the phrase "consisting essentially of may encompass administration of one or more active pharmaceutical ingredients as the sole therapeutic agent(s) for that particular indication, while not excluding therapeutic agents administered for other reasons or indications.

EXAMPLES The methods of the present invention for reducing the pathological effects of Aβ in animals or humans suffering from diseases associated with amyloidosis, such as AD, will be described in more detail in the following non- limiting examples. Examples 1-6 provide data for racemic nilvadipine for comparative purposes.

Chromatographic Purification of Nilvadipine Enantiomers

Chromatographic purification of both enantiomers of nilvadipine was achieved via High Performance Liquid Chromatography (HPLC) using a stationary phase of modified cellulose (Chiral Technologies, West Chester, PA). The chromatographic conditions were as follows: column internal diameter, 10 mm; column length, 250 mm; mobile phase, 95:5 volumetric ratio of hexanes to ethanol; flow rate, 2.5 ml/min; column temperature, 7°C. Repeated injections of racemic nilvadipine using signal-based fraction collection yielded purified enantiomers for the examples described herein. See Figure 8.

EXAMPLE 1

Chronic Administration of Racemic Nilvadipine on Aβ deposition (Amyloid Burden)

The effect of chronic administration of racemic nilvadipine on Aβ deposition (amyloid burden) in different regions of the brain of TgAPP_sw mice was examined using a 4G8 anti-Aβ monoclonal antibody immunostaining technique. The 4G8 immunostaining technique was chosen for determining the Aβ burden because of its robust signal and optimal results for quantitative analysis of Aβ deposition. Briefly, paraffin sections were subjected to immunohistochemistry as described previously (Nakagawa, Y et al., Exp. Neurol., 163:244- 252, 2000). Sections were deparaffmized in xylene, hydrated in a series of ethanol and deionized water, and subjected to an antigen retrieval step by immersing sections in 88% formic acid for 60 min before immunohistochemistry for Aβ. Sections were washed in water, and endogenous peroxidases were quenched using a freshly prepared mixture of methanol (150 ml) plus hydrogen peroxide (33%, 30 ml). The avidin-biotin complex method was used according to the instructions of the vendor (Vector Laboratories, Burlingame, Calif). Amyloid burden was assessed by determining the percentage of the brain region that stained positive for Aβ. Negative controls included the application of the same immunohistochemistry protocol to sections, except preimmune serum was applied instead of primary antibody. TgAPP_sw mice were divided into an experimental group that received an effective amount of nilvadipine (n=7) and a control group that received a vehicle (n=5).

As shown in FIG. 1, treatment with racemic nilvadipine reduced the Aβ burden about 62% in the visual cortex compared to controls, about 65% in the parietal cortex compared to controls, about 58% in the motor cortex compared to controls, about 58% in the pyriform cortex compared to controls, about 52% in the CAl region of the hippocampus compared to controls, and about 50% in the CA2-CA3 region of the hippocampus compared to controls.

EXAMPLE 2

Chronic Administration of Racemic Nilvadipine on Microglial Activation

The effect of chronic administration of racemic nilvadipine on microglial activation in TgAPP₈W mice was examined in three regions of the mouse brain using a CD45 immunostaining technique in which the number of CD45+microglia was determined. Briefly, immuno histochemistry for CD45, a specific marker for leukocytes, was conducted on the cryostat brain sections. CD45-positive microglial cells were immunolocalized by incubation with a mouse monoclonal antibody against CD45 (Chemicon International) overnight at 4⁰C, followed by application of a biotinylated rabbit anti-mouse secondary antibody for 30 minutes. Detection of CD45 was completed with diaminobenzidine chromogen substrate, which produces a brown cell surface stain on CD45 -positive microglial cells.

As shown in FIG. 2, racemic nilvadipine treatment administered in an effective dosage amount reduced microglial activation about 33% in the hippocampus, about 43% in the parietal cortex, and about 27% in the motor cortex, when compared to controls.

EXAMPLE 3

The Effect of Racemic Nilvadipine Administration on Microglial Activation

The effect of racemic nilvadipine on microglial activation was examined in N9 murine microglial cells in vitro activated with lipopolysaccharide (LPS) for 24 hours. N9 murine microglial cells are well characterized scavenger murine microglial clones derived from embryonic mouse brain. The extent of microglial activation was determined by TNF-α production (pg/ml) measured by ELISA. As shown in FIG. 3, microglial cells not activated with LPS (control cells) produced about 40 pg/ml TNF-α. Microglial cells in the presence of 50 nM nilvadipine produced about 40 pg/ml TNF-α. Increasing nilvadipine administration 10-fold (500 nM) did not change TNF-α production. Microglial cells in the presence of 1 μg/ml LPS produced about 820 pg/ml TNF-α, an increase of about 95% from the control cells and nilvadipine-administered cells. Microglial cells in the presence of both 1 μg/ml LPS plus 50 nM nilvadipine produced about 670 pg/ml TNF-α. LPS plus 500 nM nilvadipine administration decreased TNF-α production to about 610 pg/ml. Thus, nilvadipine opposed the LPS-induced microglial activation by about 20 to 25%.

EXAMPLE 4

The Effect of Nilvadipine Administration on Aβ Neurotoxicity

The effect of nilvadipine administration (10 nM and 100 nM) on Aβ neurotoxicity was examined using human neuronal progenitor cells (FINPC) treated for three days with 30 μM of pre-aggregated Aβl-40 (AgA). FINPC cells differentiate into neurons readily upon treatment with cyclic AMP. Cyclic AMP (1 mM) (Sigma) was added to the culture medium and the HNPC cells were incubated at 37⁰C for 48 hours or more under serum free conditions. This medium allowed differentiation of the progenitors into cells of neuronal lineage, as was confirmed by the staining of most of the cells with antibodies against the microtubule-associated protein, MAP-2. Neurotoxicity was assessed by measuring the amount of lactic dehydrogenase (LDH; an intracellular enzyme found in all cells) released from the cells.

As shown in FIG. 4, treatment of the cells with AgAβ produced about a 44% increase in LDH release compared to treatment of the cells with nilvadipine. There was no change in LDH release when 10 nM nilvadipine was added along with AgAβ. However, when the dosage amount of nilvadipine was increased 10-fold to 100 nM, the amount of LDH release was decreased by about 44%. EXAMPLE 5

The Effect of Nilvadipine Administration on APP Processing

The effect of nilvadipine on APP processing was examined using human glioblastoma cells transfected with APP_SW. The cells were treated with 50 nM and 250 nM nilvadipine for 24 and 48 hours, and production of Aβ 1-40 in the culture medium was measured by using a commercially available human Aβl-40 ELISA (Biosource, CA).

As shown in FIG. 5A, after 24 hours of treatment, 50 nM of nilvadipine reduced the production of Aβ 1-40 by about 9%, and 250 nM of nilvadipine reduced Aβl-40 production by about 15%. After 48 hours of treatment (FIG. 5B), 50 nM of nilvadipine reduced the production of Aβl-40 by about 18%, and 250 nM of nilvadipine reduced Aβl-40 production by about 5%.

EXAMPLE 6

The Effect of Nilvadipine Administration on Cerebral Blood Flow

64 week-old Tg APPsw (line 2576) were implanted subcutaneously with a biodegradable pellet of (-)-nilvadipine ensuring a slow release of 30 mg/Kg of body weight/Day of (-)- nilvadipine, with a pellet ensuring a release of 56 mg/Kg/Day of (-)-nilvadipine or with a placebo pellet. 26 days after the pellet implantation, the cerebral blood flow (CBF) was evaluated in the animals using a high resolution laser Doppler imager. Mice were anesthetized with a gas mixture of 3% isoflurane in oxygen. Animals were then immobilized on a mouse stereotaxic table and maintained under anesthesia with a mouse anesthetic mask (Kopf Instruments, Tunjunga, CA) delivering 3% isoflurane in oxygen. Rectal temperature was maintained at 37⁰C using a mouse homeothermic blanket system (Harvard Apparatus, Holliston, MA). An incision was made through the scalp and the skin retracted to expose the skull. The periosteal connective tissue, which adheres to the skull, was removed with a sterile cotton swab. Animals were maintained on a mixture of 1.5% isoflurane in oxygen. Cortical perfusion was measured with a Laser-Doppler Perfusion Imager from Moor Instruments (Wilmington, DE). A computer-controlled optical scanner directed a low-power He-Ne laser beam over the exposed cortex. The scanner head was positioned parallel to the cerebral cortex at a distance of 26 cm. The scanning procedure took 1 min and 21 sec for measurements of 5538 pixels covering an area of 0.8 x 0.8 cm. At each measuring site, the beam illuminated the tissue to a depth of 0.6 mm. An image color-coded to denote specific relative perfusion levels was displayed on a video monitor. All images were stored in computer memory for subsequent analysis. For each animal, CBF was measured in the entire cortex by manually delineating for each mouse the cortex area (0.51 to 0.54 cm² corresponding to 3504 to 3714 pixels). Relative perfusion values for each area studied were normalized against the CBF values obtained in control mice and expressed as a % of control CBF.

As shown in Figs. 6A-E, a 26 day-treatment with 30 mg/Kg/Day or 56 mg/Kg/Day of (-)- nilvadipine does not affect the cerebral blood flow of Tg APPsw mice.

EXAMPLE 7

The Effect of Varying Doses of Racemic and Isolated Enantiomers of Nilvadipine on Aβ Levels In Vitro The effect of pure enantiomeric forms of nilvadipine, (-)-nilvadipine (nilvadipine 1) and (+)- nilvadipine (nilvadipine 2) (FIG. 7) as well as a mixture of the two enantiomers (in equal proportion) on Aβl-40 and Aβl-42 production was examined using 7 W WT APP751 Chinese hamster ovary cells following 24 hours treatment. Production of Aβ 1-40 and Aβl- 42 in the culture medium was measured by using a commercially available human Aβl-40 ELISA (Biosource, CA) and commercially available human Aβl-42 ELISA (Biosource, CA), respectively.

As shown in FIG. 7, both enantiomers appear to dose dependently inhibit Aβl-40 production in a similar fashion. However, the (+)-nilvadipine (nilvadipine 2) as well as the racemic mixture of nilvadipine (N1+N2) slightly stimulate Aβl-42 at low dose whereas (-)- nilvadipine (nilvadipine 1) does not show this effect (FIG. 8).

EXAMPLE 8

Effect of Isolated Enantiomers of Nilvadipine on Vasoconstriction in Rat Aortae

The effect of pure enantiomeric forms of nilvadipine, (-)-nilvadipine (nilvadipine 1) and (+)- nilvadipine (nilvadipine 2) (FIG. 9) on FPL64176 induced vasoconstriction in rat aortae was examined (FIG. 10 and FIG. 11). Normal male Sprague-Dawley rats (7-8 months old) were humanely euthanatized, and freshly dissected rat aortae were segmented into 3 -mm rings and suspended in Kreb's buffer on hooks in a vessel bath apparatus. These hooks were connected to an isometric transducer linked to a MacLab system. Aortic rings were equilibrated in the tissue bath system for 2 h with the Kreb's buffer changed every 30 min. A baseline tension of 2g was applied to each aortic ring. Aortic rings were pretreated with 100 nM of (-)- nilvadipine (nilvadipine 1), 100 nM of (+)-nilvadipine (nilvadipine 2) or untreated 2 min prior to the addition of 1 μM of FPL64176 (2,5-dimethyl-4[2-(phenylmethyl)benzoyl]-lH- pyrrole-3-carboxylic acid methyl ester), a potent and selective agonist of L-type calcium channels. Aortic rings were constricted with FPL64176 for 30 minutes. The amount of contraction (in g) as compared to baseline was determined and the means and standard deviation of all such values were calculated.

As shown in FIG. 10, (+)-nilvadipine completely antagonizes FPL64176-induced vasoconstriction whereas (-)-nilvadipine does not produce the vasoactive effect of the L-type calcium channel agonist showing that (+)-nilvadipine is an L-type calcium channel blocker whereas (-)-nilvadipine is deprived of such effect. In a dose-dependent study, as shown in FIGS. 1 IA and B, (+)-nilvadipine begins to exhibit vasoactive properties at a much lower dose in the FPL64176 rat aorta model of vasoconstriction compared to (-)-nilvadipine.

General Conclusions

In view of the above data, it can be extrapolated that enantiomerically-enriched (+)- nilvadipine administration to animals or humans afflicted with a cerebral amyloidogenic disease, such as AD, can significantly decrease the amount of Aβ deposition in critical regions of the brain that characteristically demonstrate an abundance of such pathological deposits as well as reduce the amount of Aβ already deposited in the brain by increasing the cerebral blood flow. Additionally, enantiomerically-enriched (+)-nilvadipine administration may oppose the neurotoxic effects of Aβ, effects which are believed to be responsible for the widespread and devastating neuronal destruction seen with AD, as well as reduce microglial activation that causes the characteristic inflammatory response seen in the brains of AD patients. Moreover, enantiomerically-enriched (+)-nilvadipine treatment may reduce the concentration of already deposited Aβ in brains of animals or humans afflicted with cerebral amyloidogenic diseases such as AD at decreased dosages compared to racemic nilvadipine. In addition, data presented herein show that (+)-nilvadipine completely antagonizes FPL64176-induced vasoconstriction whereas (-)-nilvadipine does not affect the vasoactive effect of the L-type calcium channel agonist, showing that (+)-nilvadipine is an L-type calcium channel blocker whereas (-)-nilvadipine is deprived of such effect. Finally, data presented herein show the (+)-enantiomer of nilvadipine is more effective at increasing cerebral blood flow than the racemic mixture of nilvadipine or the (-)-nilvadipine enantiomer alone.

It will be apparent to those skilled in the art that various modifications and variations can be made in the methods of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention include modifications and variations that are within the scope of the appended claims and their equivalents. All references cited herein are incorporated by reference.

Claims

1. A method for increasing cerebral blood flow in an animal or human suffering from, or at risk of suffering from, a disease associated with decreased cerebral blood flow, comprising administering to the animal or human a therapeutically effective amount of enantiomerically- enriched (+)-nilvadipine.

2. A method for diagnosing a disease associated with decreased cerebral blood flow in an animal or human, wherein the disease is a cerebral amyloidogenic disease, comprising: taking a first measurement of the plasma concentration of Aβ in the peripheral circulation of the animal or human; administering a therapeutically effective amount of enantiomerically- enriched (+)-nilvadipine to the animal or human; taking a second measurement of the plasma concentration of Aβ in the peripheral circulation of the animal or human; and calculating the difference between the first measurement and the second measurement, wherein an increase in the plasma concentration of Aβ in the second measurement compared to the first measurement indicates a possible diagnosis of a cerebral amyloidogenic disease in the animal or human.

3. A method for treating or ameliorating the sequelae of traumatic brain injury in an animal or human, said method comprising increasing cerebral blood flow by administering to the animal or human an amount of enantiomerically-enriched (+)-nilvadipine effective to increase cerebral blood flow, wherein the enantiomerically-enriched (+)-nilvadipine administration begins following the traumatic brain injury.

4. A method for reducing the risk of developing symptoms associated with decreased cerebral blood flow in an animal or human diagnosed with a risk for developing symptoms associated with decreased cerebral blood flow by increasing cerebral blood flow in the animal or human, comprising administering to the animal or human in need thereof a therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine.

5. The method of any one of claims 1 to 4, wherein the disease associated with decreased cerebral blood flow is mild cognitive impairment, or a cerebral amyloidogenic disease or condition selected from the group consisting of Alzheimer's disease, transmissible spongiform encephalopathy, scrapie, traumatic brain injury, cerebral amyloid angiopathy and Gerstmann- Straussler- S cheinker syndrome .

6. The method of any one of claims 1 to 4, wherein the therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine is selected from the group consisting of between about 0.05-30 mg per day, about 2-15 mg per day, about 4-12 mg per day, about 2 mg per day, about 4 mg per day, about 6 mg per day, about 8 mg per day, about 10 mg per day, about 12 mg per day, about 14 mg per day, about 16 mg per day, about 18 mg per day, about 20 mg per day, about 22 mg per day, about 24 mg per day, about 26 mg per day, about 28 mg per day, and about 30 mg per day.

7. The method of any one of claims 1 to 4, wherein the therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine increases cerebral blood flow as measured by SPECT by an amount selected from the group consisting of greater than about 1%, greater than about 5%, and up to about 10%, following administration of enantiomerically-enriched (+)-nilvadipine.

8. The method of any one of claims 1 to 4, wherein the enantiomeric enrichment is selected from the group consisting of greater than about 95%, greater than about 98%, about 100%, and up to the limit of detection.

9. The method of any one of claims 1 to 4, wherein the duration of treatment with the therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine is selected from the group consisting of up to the lifetime of the animal or human, between one hour and 5 years, between one day and twelve months, between one week and six months, between two weeks and four weeks, between two weeks to three years, and between six months and twelve months.

10. The method of any one of claims 1 to 4, wherein the enantiomerically-enriched (+)- nilvadipine is administered in a unit dosage form which is selected from the group consisting of hard or soft shell gelatin capsules, tablets, troches, sachets, lozenges, elixirs, suspensions, syrups, wafers, powders, granules, solutions and emulsions.

11. The method of any one of claims 1 to 4, wherein the administration is via parenteral, oral, or intraperitoneal administration, and optionally wherein the parenteral route of administration selected from the group consisting of intravenous; intramuscular; interstitial; intra-arterial; subcutaneous; intraocular; intracranial; intraventricular; intrasynovial; transepithelial, including transdermal, pulmonary via inhalation, ophthalmic, sublingual and buccal; topical, including ophthalmic, dermal, ocular, rectal, and nasal inhalation via insufflation or nebulization, optionally selected from the group consisting of aerosols, atomizers, and nebulizers.

12. A method for treating a disease or condition associated with decreased cerebral blood flow in an animal or human afflicted with decreased cerebral blood flow, comprising administering to the animal or human a composition consisting essentially of a therapeutically effective amount of enantiomerically-enriched (+)-nilvadipine, and a pharmaceutically acceptable carrier.