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  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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• • A—HUMAN NECESSITIES
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  • A61P25/00—Drugs for disorders of the nervous system
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  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P9/00—Drugs for disorders of the cardiovascular system
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
  • G01N33/743—Steroid hormones
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
  • G01N2800/2821—Alzheimer

Definitions

• the present invention concerns diagnostic methods for detecting
• neuropathologies and particularly Alzheimer's disease, by testing for increased levels
• DHEN central nervous system dehydroepiandrosterone
• DHEN Dehydroepiandrosterone
• DHEN DHEN sulfate
• Neurosteroids have been implicated in a number of different clinical conditions including memory (9, 26, 85), depression (84), and pre-menstrual syndrome (67). They have also been proposed to be potential drugs for the treatment of anxiety (36, 37, 40, 46) and epilepsy (35, 41, 47) via their ability to potentiate GABA inhibition (65). Although several studies have looked at the regional distribution of neurosteroids in the human brain (30, 31, 38), there is no evidence as to where these steroids were synthesized, hi addition, there have been no published studies examining the mechanisms of regulation of neurosteroid biosynthesis in either rodent or human brain, assuming it occurs. Despite the lack of information regarding the role of neurosteroids in the brain
• peripheral DHEA levels as a predictor of future neuropathology.
• central nervous system (CNS) DHEA is increased in
• DHEA is synthesized in the brain via an alternative
• neuropathologic diseases and in particular, Alzheimer's disease.
• the methods of the invention involve measuring the amounts of neurosteroids
• ⁇ -amyloid protein are also included, given the finding reported herein that ⁇ -amyloid
• protein and other agents or conditions inducive of oxidative stress may have a
• FIG. 1 Neurosteroid Levels in Alzheimer's Disease (AD) and Control Brain Endogenous neurosteroid levels in human brain were measured by specific
• P450cl7 is expressed in the cortex of both control and AD patients, although
• Brain homogenates were separated by SDS-PAGE and blotted with 4G8, a
• AD frontal cortex A ⁇ - immunoreactivity is about 30 kDa. There is some A ⁇ -immunoreactivity in control
• FIG. 4 DHEA and A ⁇ Levels in Cerebrospinal Fluid (CSF) from AD and Control Patients
• DHEA was purified from CSF extracts by HPLC and measured by specific
• AD patients have only a 98 kDa species, while AD patients have AP-immunoreactivity occurring at a wide range of molecular weights from 46-98 kDa.
• AD patients do not have a DHEA precursor that can be acted upon by oxidizing agents. AD patients do not have a DHEA precursor that can be acted upon by oxidizing agents. AD patients do not have a DHEA precursor that can be acted upon by oxidizing agents. AD patients do not have a DHEA precursor that can be acted upon by oxidizing agents. AD patients do not have a DHEA precursor that can be acted upon by oxidizing agents. AD patients do not have oxidizing agents.
• Figure 6 Human brain cells have an alternative pathway for D synthesis.
• axis indicate mM dose of FeSO4; SU, SU/3 and SU/10 indicate the addition of 5 mM SU
• FeSO4 treatment causes a significant increase in MGM-1 and ⁇ HN cellular ROS
• Figure 8 ⁇ -Amyloid increases cellular ROS and D synthesis.
• MGM-1 cells were treated with ⁇ -amyloid (A ⁇ ) alone or with A ⁇ and Vitamin E
• the present inventors have found that patients in the early stages of oxidative stress-related neuropathological diseases can be identified by virtue of increased levels of DHEA in the brain, and that the DHEA in these patients is synthesized in the brain by an alternative metabolic pathway (16).
• This pathway may be regulated by intracellular free radicals and reactive oxygen species through a novel precursor, as the absence or decrease of this precursor parallels the observed increase in DHEA in the CNS of these patients.
• the precise identity of the precursor is not yet known, its presence can be measured by the addition of FeSO 4 and other reducing agents which presumedly cause a classical Fenton reaction and formation of DHEA if the precursor is present (15).
• oxygen-free radicals by the addition of Fe 2+ to cells has been previously described (88, 89).
• Generation of oxygen free radicals has been implicated in neurodegeneration (44), and evidence of oxidative stress has been shown in Alzheimer's disease - brain (20, 21, 29), where oxidative stress contributes to the formation of amyloid plaques and neurofibrillaiy tangles (45, 83).
• ⁇ -amyloid is a component of Alzheimer's disease plaques (18) and can cause increases in reactive oxygen species (ROS) via several mechanisms (55, 58, 59).
• the present invention includes a method of detecting oxidative damage
• DHEA Dehydroepiandrosterone
• identity of the precursor is still unknown, its presence can be determined by treating cells
• one diagnostic method according to the present invention includes a
• Oxidative stress-mediated DHEA formation refers particularly to DHEA
• hydroperoxide intermediates and particularly a C- 17 or C-20 oxygenated steroid
• ROS reactive oxygen species
• astrocytes can make DHEA via this pathway, but neurons do not. Thus, a patient in the
• Oligodendricyte cells and astrocytes can be identified by the presence of markers
• MBP myelin baisc protein
• 2',3'-cyclic nucleotide-3'-phosphodiesterase are oligodendrocyte-specific markers, whereas glial
• GFAP fibrillary acidic protein
• FeSO 4 has a
• MGM-1 derived from a glioblastoma multiforme tumor (70), also responds to FeSO 4
• Alzheimer's patients has already been converted to DHEA.
• a patient in the beginning stages of a brain disorder might be diagnosed by showing decreased levels of
• CSF cerebrospinal fluid
• serum cerebrospinal fluid
• CSF fluid may be used as a
• samples wherein said samples are selected from the group consisting of cerebrospinal
• peptide and other reagents that induce the formation of ROS may be used to test for the
• DHEA quantities are conceivable methods for accurately measuring quantities of DHEA that are formed by the method of the invention.
• a preferred organic solvent is ether: ethylacetate (v/v), but any combination thereof.
• Radioimmunoassay kits for example, Radioimmunoassay kits for
• DHEA-S sulfated derivative
• the methods of the invention may be supplemented with further assays to verify the
• the samples may also be tested for an increase in
• radical oxygen species after treatment with the regulating agent e.g., by using 2,7 DCF
• the samples may also be
• the diagnostic method described above may also include steps whereby the cells
• SU 10603 is a known inhibitor of P450cl7 (64).
• P450cl7 is the
• present methods has the ability to synthesis DHEA via both pathways, treating with SU
• Trilostane (Stegram Pharmaceuticals, London, U.K.), a specific inhibitor of 3 ⁇ -
• HSD also inhibits the metabolism of pregnenolone by inhibiting its conversion into
• progesterone By treating a cell sample with both trilostane and SU 10603, it is possible to treat a cell sample with both trilostane and SU 10603.
• methylglutaryl CoA reductase inhibitor i.e., an inhibitor of cholesterol synthesis
• DHEA levels are significantly higher in the brains of Alzheimer's patients than in the
• Alzheimer's disease dementias in general, Alzheimer's disease, Parkinson's disease, ALS (Amylotropic Lateral)
• Hallervoden-Spatz (which, in some cases, may lead to increased reactive oxygen species), Hallervoden-Spatz
• Multi- fract dementia Pelizaeus-Merzbacher disease, Pick's disease, stroke, and
• traumatic brain injury may particularly benefit from the disclosed diagnostic assays due to the dramatic acute increase of oxidative stress due to the injury.
• Spinal cord injury and other peripheral nervous system injuries may
• the present invention generally includes a method of diagnosing the
• DHEA DHEA
• neurosteroid relative to a control will indicate the development of a neuropathologic disorder.
• the present invention also includes kits for practicing the disclosed diagnostic
• DHEA formation in a test sample of blood, body fluid or tissue
• a reagent may comprise a reagent
• kits of the invention can be designed with a vial containing an agent
• kits might also include at least one minicolumn or set of minicolumns to
• kits of the present invention may further, or alternatively, comprise a
• DHEA-specific antibody depending on whether the kit is designed for measuring levels
• DHEA via the oxidative stress-mediated metabolic pathway.
• kits of the invention may
• DHEA may be important in the aging process
• neuropathology or is acting to potentiate the disease process.
• amyloid peptide has the potential to stimulate the oxidative stress-induced synthesis of
• DHEA Alzheimer's disease
• DHEA plays an important role in neuropathology, or whether it is simply an epiphenomenon of the disease process.
• Alzheimer's disease and other neuropathologies it can serve as a useful marker of disease
• the present invention also includes a method of increasing or causing
• DHEA synthesis by cells comprising exposing said cells to ⁇ -amyloid peptide, wherein
• brain cells and particularly astrocytes and oligodendrocytes.
• astrocytes and oligodendrocytes.
• the present invention also includes methods of preventing or treating a
• module encompasses both inhibition of synthesis if the role of DHEA in neuropathology turns out to be harmful, and increased synthesis if the role of DHEA turns
• DHEA and its precursors may play different roles in
• the present invention also includes screening assays for identifying drug
• candidates potentially useful for treating a neuropathology comprising (a) exposing a composition comprising oligodendrocytes or astrocytes to a test compound to be screened
• modulating may vary depending on whether DHEA plays a destructive
• the human glioma cell line KG-l-C was obtained from the Riken Cell Bank
• NHA astrocytes
• hNT-PF neuro neurons
• Trilostane a specific inhibitor of 3 ⁇ -HSD, was a gift from Stegram Pharmaceuticals,
• KG-l-C is derived from a mixed glioma and has been described as having the
• MGM-1 and MGM-3 are derived from
• glioblastoma multiforme tumors 70. All three cell lines were grown in Dulbecco 's
• DMEM Eagle's Media
• Amphotericin B. h ⁇ T-PF cells are a post-mitotic, differentiated cell derived
• NT2 precursor cells can be induced
• hNT-PF cells were plated and maintained at 37C and 6% CO2, in hNT-PF neuron
• RNA was isolated from each cell type by the acid-guanidium thiocyanate-
• RNA from each cell type was reverse transcribed using oligod(T)s and MuLV reverse
• transcriptase according to the protocol provided by Perkin Elmer (Foster City, CA).
• Probes were synthesized by RT-
• Steroids were identified by respective retention times compared with
• oligodendrocytes and astrocytes were incubated with increasing concentrations of FeSO4
• Extracts were run on C18 TLC plates (Whatman, Clifton, NJ) with a mobile phase of
• TLC fractions were extracted with 2 ml diethyl ether (Fluka, New York) and evaporated. TLC extracts were assayed by specific radioimmunoassay (RIA). h separate experiments, steroids
• TLC tissue sample
• samples were treated as previously described (17). In brief, aliquots of homogenates were incubated with a final concentration of 30 mM (brain) or 10 mM
• Serum and CSF Levels of DHEA Serum was obtained from 11 patients, 5 with AD and 6 age-matched controls. Serum samples were split and treated with and without 10 mM FeSO 4 . Serum and CSF samples were extracted as described for the brain tissue samples, and purified by HPLC. DHEA levels were measured by specific radioimmunoassay. ⁇ -Amyloid Regulation of the Alternative Pathway: MGM-1 cells were plated on 96 well (-5,000 cells/well, for ROS measurements) or 24 well (-15,000 cells/well, for D measurements) plates (Corning Costar, Acton, MA). At the same time, 1 mg Ab (Ab
• MBP an oligodendrocyte marker
• GFAP an astrocyte marker
• oligodendrocytes These cells will be collectively referred to as oligodendrocytes.
• NHA cells from Clonetics are shipped immunopositive for GFAP and we
• the hNT-PF cells are derived from a Ntera2/Dl
• teratocarcinoma cell line by 5-6 weeks of differentiation by retinoic acid. These cells are
• adhesion molecules specific to neurons such as NCAM and N-cadherin (77).
• hNT-PF cells behave like neurons, with tetrodotoxin-sensitive
• Oligodendrocytes synthesize P de novo, whereas astrocytes and neurons do not
• a steroid-synthesizing cell is defined as a cell that is able to convert endogenous
• MGM-3 cell line synthesizes P de novo from a radiolabeled precursor.
• MGM-1, NHA and hNT-PF cells do not make P de novo
• ROS and D levels as compared to untreated cells (data not shown). ROS and D levels
• Human brain cells express mRNA and immunoreactivity for different components of the
• MGM-1 cells show a distinct
• MGM-3 cells express immunoreactivity
• MGM-3 does have
• oligodendrocytes, mRNA and protein expression corresponds with the ability of the cells
• Human astrocytes express mRNA for all four components (data not shown).
• NHA NHA
• NHA cells have the potential to synthesize P and D, but not
• progesterone Human neurons express mRNA for PBR, P450cl7 and 3 ⁇ -HSD, but not
• DHEA DHEA
• MGM-1 cell line has the ability to make D via the alternative pathway
• KG-l-C and MGM-3 are the cells that synthesized radiolabeled D de novo in
• FeSO4 treatment had a
• the hNT-PF cells were also incubated with increasing concentrations of FeSO4,
• a substrate can enhance the effects of FeSO4 in rat C6 cells (15). Human neurons were
• hNT-PF cells were also not affected by the SU 10603 treatment,
• Neuronal D may be completely derived from other sources.
• NHA cells responded more robustly to FeSO4 then did MGM-1 cells. This
• Treatment with ⁇ -Amyloid increases ROS and D levels in MGM-1 human glioma cells
• Vitamin E We found that treatment with Ab caused a significant increase in 2,7-DCF fluorescence (p ⁇ 0.01, Student's unpaired t test) versus control levels, indicating an
• Treatment with Ab also increased D levels significantly over control (p ⁇ 0.01, Fig. 8B).
• steroids found in the brain may be of both peripheral and local origin, nothing is known
• oligodendrocytes are the probable source of brain-synthesized P (62), and validates the
• steroid synthesizing cells designated as steroid synthesizing cells, and astrocytes and neurons as steroid
• neuroactive steroids which may play a
• Oligodendrocytes have mRNA and immunoreactivity for PBR, P450scc, P450cl7 and 3b-HSD. This confirms the ability of oligodendrocytes
• Astrocytes also have mRNA and immunoreactivity for PBR, P450scc and P450cl7,
• results indicate a possible role for astrocytes as steroid-metabolizing cells that may make
• neurons express mRNA for PBR, P450cl7 and 3b-HSD, but not for
• neurosteroids present in rat brain extracts that could react with compounds unrelated to peripheral steroid biosynthesis, and produce neurosteroids. They demonstrated that
• Oligodendrocytes and astrocytes can make D by an alternative pathway.
• oligodendrocytes and astrocytes. Astrocytes seem to produce more ROS in response to
• Fe ⁇ + than glioma cells which may indicate an increased sensitivity to FeSO4, and
• Oligodendrocytes that do not respond to Fe2+ can synthesize D de novo in a P450cl7-
• Vitamin E treatment of the 'glioma cell lines decreases MGM-3 ROS and D levels to
• P450cl7 it has been shown to inhibit other P450s, including P450scc (60).
• P450cl7 is not the mechanism of D formation in human brain cells.
• Alzheimer's disease plaques A ⁇ is toxic to neurons and causes an increase in cellular
• a ⁇ treatment increased cellular ROS in MGM-1 human glioma cells, and increased
• Vitamin E This is the first demonstration of a regulatory mechanism for D synthesis in
• proteoglycans (33) and growth factors (90) may be involved in plaque formation. It is
• binding sites are significantly increased in specific areas from AD brains, as well as
• components may be, like the activity of the alternative pathway, a sign of altered
• neurodegeneration or trauma in the brain. It may be that an increase in neurosteroid biosynthesis is a hallmark of an altered or toxic cellular environment.
• hypothalamus, hippocampus and cortex from AD patients and age-matched controls.
• ⁇ -amyloid can also induce D formation in human glioma cells, presumably by increasing levels of intracellular ROS, this increase in D in AD patients
• AD brain is probably due to an increased level of oxidative stress in the AD brain.
• a ⁇ -immunoreactivity present in control patients is a higher molecular weight
• AD patients have a relatively constant level of A ⁇ immunoreactivity, ranging
• Control patients also have detectable A ⁇ -immunoreactivity, but these
• ND patients showed a significant increase in D in the cortex, but no
• a ⁇ -immunoreactivity as detected by immunoblots, is increased in
• AD Alzheimer's disease
• hippocampus is either derived from peripheral sources and accumulated and stored in the
• AD patients show a significant increase in D only in the frontal cortex, indicating that
• the precursor of the alternative pathway is present there, but not in the hypothalamus or
• AD diagnosis and treatment is the inability of clinicians to determine the onset of the disease.
• this is done by a combination of MRI scans, to look for generalized shrinkage of the brain and cognitive tests to
• the alternative precursor is present in the blood or CSF indirectly by FeSO 4 treatment, one
• AD Alzheimer's disease
• RNA oxidation is a prominent feature of vulnerable neurons in Alzheimer's disease. J. Neuroscience 19: 1959-1964. A ⁇ oxidative damage to neurons.
• CCD-3693 An Orally Bioavailable Analog of the Endogenous Neuroactive Steroid, Pregnanolone, Demonstrates Potent Sedative Hypnotic Actions in the Rat. J. Pharmacol. Exper. Therap., 282, 420-429.
• PBR Peripheral-type Benzodiazepine Receptor
• HGF hepatocyte growth factor
• NTera 2 cells A Human Cell Line which Displays Characteristics Expected of a Human Committed Neuronal Progenitor Cell. J. Neurosci. Res., 35, 585-602.
• Presumptive neurons derived by differentiation of a human embryonal carcinoma cell line exhibit tetrodotoxin-sensitive sodium currents and the capacity for regenerative responses.

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