WO2002000221A1 - Fonction cerebrale amelioree par stimulation gaba-ergique - Google Patents

Fonction cerebrale amelioree par stimulation gaba-ergique Download PDF

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WO2002000221A1
WO2002000221A1 PCT/US2001/019719 US0119719W WO0200221A1 WO 2002000221 A1 WO2002000221 A1 WO 2002000221A1 US 0119719 W US0119719 W US 0119719W WO 0200221 A1 WO0200221 A1 WO 0200221A1
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gaba
function
age
cortical
visual
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WO2002000221B1 (fr
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Audie G. Leventhal
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University Of Utah Research Foundation
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Priority to AU6860901A priority patent/AU6860901A/xx
Priority to EP01946582A priority patent/EP1303280A4/fr
Priority to US10/311,821 priority patent/US20040023952A1/en
Publication of WO2002000221A1 publication Critical patent/WO2002000221A1/fr
Publication of WO2002000221B1 publication Critical patent/WO2002000221B1/fr
Priority to AU2006203432A priority patent/AU2006203432A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine

Definitions

  • This invention concerns treatments for improving age-related cortical function of a subject.
  • BACKGROUND Cognition is the ability of a subject to use information about and from the environment in an adaptive way.
  • cognitive and other cortical functions such as auditory discrimination, somatosensory function, motor function, and language abilities
  • This decline is a common cause of incapacity, morbidity and even death in elderly animals and humans.
  • These problems are expected to become more widespread as life span increases, and more individuals live into senescence.
  • One of the great medical and social challenges of the coming decades is to develop approaches to deal with this often incapacitating problem.
  • GABA A Gamma-ammobutyric acid
  • GABA is regarded as one of the major inhibitory amino acid transmitters in the mammalian brain. Widely (although unequally) distributed through the mammalian brain, GABA is believed to be a transmitter at approximately 30% of the synapses in the brain. GABA mediates many of its actions tlirough a complex of proteins (GABA receptors) localized both on cell bodies and nerve endings. Postsynaptic responses to GABA are mediated through alterations in chloride conductance that generally, although not invariably, lead to hyperpolarization of the cell. Drugs that interact at the GABAa receptor can possess a spectrum of pharmacological activities depending on their abilities to modify the action of GABA.
  • GABA agonist is a benzodiazepine receptor agonist, such as diazepam or chlordiazepoxide.
  • 1,4-benzodiazepines such as diazepam are among the most widely used drugs in the world as anxiolytics, muscle relaxants, and anticonvulsants. A number of these compounds are extremely potent drugs; such potency indicates a site of action with a high affinity and specificity for individual receptors.
  • Compounds which have activity opposite to benzodiazepines are called inverse agonists, and compounds blocking both types of activity have been termed antagonists.
  • the GABA receptor subunits are categorized as alpha, beta, gamma, delta and epsilon, and they provide a molecular explanation for the GABA receptor heterogeneity, and distinctive regional pharmacology.
  • the gamma subunit appears to enable drugs like benzodiazepines to modify the GABA responses.
  • these compounds are capable of producing a spectrum of activities, such as sedation, anxiolysis, anticonvulsant activity, or wakefulness, seizures, or anxiety. It is generally accepted that GABA agonists provide cortical inhibition which impairs cognitive and other cortical activities, and are to be avoided in situations wherein optimal higher cortical functions (such as thinking and visual perceptual) are required.
  • GABA inverse agonists which block the cortical inhibitory action mediated by GABA receptors, have been proposed as treatments for cognitive disorders, such as Alzheimer's disease (see e.g. WO 99/06401, which is incorporated by reference).
  • cognitive disorders such as Alzheimer's disease (see e.g. WO 99/06401, which is incorporated by reference).
  • human visual function declines with age. This decline has usually been attributed to abnormalities in the optical properties of the eye, such as cataracts (opacities in the crystalline lens of the eye) or retinal degeneration (for example of the type that is seen in age related macular degeneration).
  • visual research and care for the elderly primarily involves addressing these problems, for example by extraction of cataracts and treatment of choroidal neovascularization that precedes macular degeneration.
  • Certain disclosed embodiments include treating a subject having age-related decreases in cortical function by administering to the subject a therapeutically effective amount of a GABA-ergic agonist.
  • the age-related decrease in cortical function is a decrease in cognitive function or visual function (such as a decrease in orientation and direction selectivity).
  • the GABA-ergic agonist is a GABA- A, GABA-B, or GABA-C receptor agonist, such as a benzodiazepine receptor agonist, and in particular a member of the class of drugs known as benzodiazepines.
  • Other examples of the GABA-ergic agonist include GABA, muscimol, baclofen, CaCa, valproic acid, a barbiturate, gabapentin, tigabine, or vigabatrin.
  • the method includes determining, prior to treating the subject, whether the subject has an age-related decrease in GABA-ergic activity, such as an age-related decrease in visual orientation and direction selectivity, auditory frequency discrimination and/or sound localization, somatosensory function (such as a decrease in an ability to detect quality, intensity or position of sensation), motor function (such as a control of voluntary movements), and/or language ability (such as a decrease in speech comprehension and/or generation, such as sentence formation).
  • an age-related decrease in GABA-ergic activity such as an age-related decrease in visual orientation and direction selectivity, auditory frequency discrimination and/or sound localization, somatosensory function (such as a decrease in an ability to detect quality, intensity or position of sensation), motor function (such as a control of voluntary movements), and/or language ability (such as a decrease in speech comprehension and/or generation, such as sentence formation).
  • the electrophysiological changes in these visual functions can be used as a diagnostic marker for more widespread cortical loss of GABA-ergic activity that can
  • the assay involves administering a test agent, and measuring a change in a neuron in a specific area of the brain that is associated with age-related decline.
  • GABA-ergic agents that increase orientation bias, direction bias, or the signal to noise ratio, or decrease spontaneous baseline frequencies, are then selected for further testing in cognitive and visual function studies.
  • a decreased spontaneous baseline frequency or an increased signal to noise ratio in many other areas of the brain can be used to select for GABA- ergic agents that will improve cortical function by decreasing the spontaneous cortical activity that masks efficient neurotransmission in the aging brain.
  • FIG. 1 shows photomicrographs of whole-mounted, HRP-labeled retinae and single intracellularly dye-filled ganglion cells.
  • the ganglion cell density within and surrounding the foveal pit appeals qualitatively normal in the old monkey (a) compared to the young monkey (b)(scale bars, 500 ⁇ m).
  • the ratio of A (P ⁇ or parasol) and B (p ⁇ or midget) ganglion cells also appears normal in old monkey (c), in which the photomicrograph is taken 4 mm from the center of the foveal pit (scale bar 50 ⁇ m).
  • Type B cells have characteristically smaller soma and dendritic fields relative to A cells.
  • FIG. 2 is a series of graphs showing orientation and direction biases in young and old macaque VI cells which were exposed to either driftmg sinusoidal gratings or drifting luminance bar stimuli.
  • Orientation biases of 0.1, 0.3 and 0.5 correspond to maximum-to-minimum response ratios of 1.5:1, 3:7:1 and 10.8:1. respectively.
  • An orientation bias of 0.1 or greater indicates significance at thejc > 0.005 level (Rayleigh test).
  • FIG. 3 shows tuning curves and corresponding polar plots obtained from four old monkey cells. Responses are shown to drifting luminance bars (a, b) and sinusoidal gratings (c, d) of systematically varied orientation and direction. The responses of two selective and two nonselective cells are provided for comparison. Orientation biases for each plot are 0.307 (a), 0.042 (b), 0.505 (c) and 0.081 (d). Direction biases are 0.065 (a), 0.018 (b), 0.118 (c) and 0.023 (d). The orientations of the driving gratings and bars are orthogonal to the directions indicated. Each point in the polar plots represents the response for the stimulus moving in the indicated direction.
  • FIG. 4 is a series of graphs which illustrates the relationship between orientation biases and peak visual evoked response of young monkeys (a, c, e) and old monkeys (b, d, f).
  • FIGS. 4(a) and 4(b) shows the relationship between orientation biases and peak visual evoked response (baseline subtracted) of young (a) and old (b) monkey VI cells to drifting bar stimuli.
  • FIGS. 4(c) and (d) show the relationship between orientation bias and peak FFTI response of different young (c) and old (d) monkey VI cells to drifting sinusoidal gratings.
  • FIGS. 4(e) and (f) show the relationship between peak visual evoked response and baseline activity of young (e) and old (f) monkey VI cells.
  • Cells shown in (a) and (b) are identical to those shown in (e) and (f) respectively.
  • Selective and nonselective cells in both age groups show a wide range of peak amplitudes.
  • FIGS. 5-9 are a series of bar graphs which illustrate the peak response (in spikes/second) in old monkeys and young monkeys, as well as in old monkeys which are given GABA, muscimol, or bicuculline.
  • FIG. 10A-F shows tuning curves and corresponding polar plots for monkeys that received treatment with GABA, a GABA agonist (muscimol) and a GABA antagonist (bicuculline).
  • a cell includes a plurality of such cells and reference to “the cell” includes reference to one or more cells, and so forth.
  • An “agent” includes conventional chemical pharmaceutical compounds, as well as polypeptides, peptidomimetics, biological agents, antibodies or other molecules with a desired function.
  • an “animal” is a living multicellular vertebrate organism, a category which includes, for example, mammals and birds.
  • a “mammal” includes both human and non- human mammals.
  • subject includes both human and veterinary subjects.
  • Cortical function refers to function of the cortex of the brain, as measured either functionally by neurological testing, or electrophysiologically, for example by a decreased signal to noise ratio.
  • GABA-ergic agent is an agent that exerts a GABA-like effect, and include GABA-agonists and agents that have effects like GABA-agonists.
  • a “therapeutically effective amount” is a quantity of an agent sufficient to achieve a desired effect in a subject being treated.
  • a therapeutically effective amount of a GABA-ergic agent is the amount necessary to improve cortical functioning, for example as measured by an improvement in cognition, somatosensory, visual or auditory function.
  • a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in neurons of the CNS) that has been shown to achieve improvements in cognition using direct neuronal administration (as described in Examples 1-2 and 8).
  • target tissue concentrations for example, in neurons of the CNS
  • direct neuronal administration as described in Examples 1-2 and 8
  • Studies of visual perception indicate that aged humans show decreased visual acuity, binocular summation, contrast sensitivity, motion sensitivity and wavelength sensitivity.
  • Extracellular single-neuron recording techniques were used to examine the stimulus selectivities of VI cells in very old rhesus monkeys showing normal optics and retinogeniculate projections.
  • a total of 187 neurons were studied in 4 young rhesus monkeys (Maraca mulatto.), and 254 neurons in 4 old rhesus monkeys.
  • Subjects for physiological experiments were abbreviated as OM1-4 (for old monkey 1-4) and YMl-4 (for young monkey 1-4).
  • Retinal data were obtained from one additional young macaque (YM5, Macacafascicularis). Multiple ophthalmological exams were conducted to ensure optical and retinal health of all subjects prior to testing.
  • Subjects for this study were four young adult (7-9 year old) and four very old (28-30 year old) female rhesus monkeys (Macaca mullata).
  • a life-span analysis of rhesus macaques housed at this center found that. only 25% reached the age of 25, and only 6% reached the age of 30 or older.
  • the 28-30 year old monkeys were considered old, whereas the 7-9 year old monkeys were at an age considered sexually mature.
  • Retinal control data for Fig. 1 are provided from one additional young female Macaca facsicularis, which was used in previous studies.
  • Onset latency data from YM4 are published. Monkeys were examined ophthalmoscopically, and no apparent optical or retinal problems were detected that would impair visual function.
  • halothane 5%; Halocarton Laboratories, River Edge, New Jersey
  • Intravenous and tracheal cannulae were inserted, the animals were placed in a stereotaxic apparatus, and pressure points and incisions were infiltrated with a long-acting anesthetic (2% lidocaine HCI, Copley Pharmaccuticak, Canton, Massachusetts).
  • a mixture of D-tubocurarine (0.4 mg per kg per hour) and gallamine triethiodide (7 mg per kg per hour) was infused intravenously to induce and maintain paralysis. Animals were ventilated, and anesthesia was maintained with a mixture of nitrous oxide (75%), oxygen (25%) and halothane (0.25-1.0%) as needed.
  • the level of anesthesia was adjusted so that vital signs were comparable in old and young animals.
  • a small burr hole was made above the striate cortex (VI), and filled with a 4% solution of agar in saline and sealed with wax.
  • the eyes were protected from desiccation with contact lenses; spectacle lenses and artificial pupils were used when needed to focus the eyes on a tangent screen positioned 228 cm from the retina.
  • the locations of the optic discs and foveae were determined repeatedly during the course of each recording session. No visible deterioration in optics occurred during the experimental period (2-5 days).
  • Extracellular action potentials of isolated cortical cells were recorded with microelectrodes having impedances of 3-5 M ⁇ .
  • the electrode was advanced using a hydraulic microdrive (David Kopf instruments, Tojunga, California) to precisely position it. The position of the electrode was confirmed by determining the receptive filed positions of the cortical cells at the recording site. Al VI cells studied had receptive fields between about 3 and 7 degrees from the fovea.
  • each drifting stimulus presented was orthogonal to its direction of motion. (The orientation is 90° less than the direction.)
  • Five to twenty presentations of moving bars, spots, or sinusoidal gratings at each of 24- 36 randomly generated orientations or directions from 0° to 360° were used to compile the tuning curves for the cells studied.
  • the responses of the cells were studied at a variety of spatial frequencies (cycles per degree) when gratings were used.
  • Each cell's optimal size and temporal frequency/velocity was chosen for the drifting stimulus, h general, each cell provided quantitative orientation bias (OB) and direction bias (DB) values in response to 2-6 different stimulus sets, and some in response to as many as 12.
  • OB orientation bias
  • DB direction bias
  • the maximum OB obtained for each cell was included in the data set, along with the maximal DB obtained from either the same stimulus presentation or from a different stimulus set, where the preferred direction was similar but the orientation bias was sub- maximal. All of the orientation and direction biases were taken from either driftmg bar stimulation (OBs, 212 of 441 neurons; DBs, 200 of 441) or drifting sinusoidal grating stimulation (OBs 229 neurons; DBs, 241).
  • the luminance of the stimuli used was 837 cd per m 2 for white spots and bars, and 0.91 cd per m 2 for black spots and bars.
  • the contrast for bar and spot stimuli was defined as the ratio of the luminance of the spot or bar to its background.
  • the contrast for sinusoidal gratings was defined as the ratio of the luminance of the center of the light and dark cycles of the gratings. In both cases the contrast was kept at 80% [(8.37 - 0.91 cd per m 2 )/(8.37 + 0.91 cd per m 2 )].
  • the responses of the cells to the drifting visual stimuli presented were stored electronically for later analysis.
  • the responses to the sinusoidal gratings were fast Fourier transformed (FFT) and defined as the peak-to-peak value of the fundamental Fourier component (FI) of the poststimulus time histogram integrated over a time equaling the stimulus modulation period (FFTI spikes per s).
  • FFT fast Fourier transformed
  • FI fundamental Fourier component
  • the responses were defined as the peak response (in Hz) of the post-stimulus time histogram.
  • baseline values were obtained during a 0.5-0.67 second blank stimulus period. All baseline values below 1 spike per second were set equal to 1 spike per s for peak-to-baseline analyses. This modification reduced skewing of the data and provided a more conservative estimation of aging differences because many young monkey cells would have peak-to-baseline ratios well above 100 before modification.
  • Orientation and direction selectivity were calculated for each cell using the statistical methods disclosed in Leventhal et al., J Neurosci. 15:1808-1818, 1995. Briefly, the responses of each call to the different stimulus orientations and directions were stored as a series of vectors. The vectors were added and divided by the sum of the absolute values of the vectors. The angle of the resultant vector gave the preferred orientation and direction of the cell. The length of the resultant vector, termed the orientation or direction bias, provided a quantitative measure of the orientation or direction sensitivity of the cell.
  • a bias of 0.1 is significant at thep ⁇ 0.005 level (Raleigh test) and orientation biases of 0.1, 0.3 and 0.5 correspond to maximum to minimum response ratios of 1.5:1, 3.7:1 and 10.8:1, respectively. Hence number higher than 0.1 indicate better selectivity bias.
  • retinae were transferred to an injection chamber and superfused with oxygenated Ames medium (Sigma) at a flow rate of about 4 ml per minute at room temperature. Cells were visualized with 0.5% acridine orange (Sigma). Under visual control, cells were penetrated electrically or mechanically with an injection electrode containing 4% Lucifer yellow (CH dilithium salt, Sigma) and 3% biocytin (Molecular Probes, Eugene, Oregon). A small biphasic current pulse (up to 2 nA hyperpolarizing and 0.5 nA depolarizing for 1-3 min) was applied to inject the dye. After completion of injections, retinae were fixed for about 12 hours in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C.
  • Orientation and direction biases were calculated for the 187 young (YMl-4) and 254 old (OM1-4) macaque VI cell using drifting bar and sinusoidal grating stimuli.
  • the mean or median DB for each individual young monkey was compared with that for the old monkey population (Table 1) and was significantly greater in each case (p ⁇ 0.01). Also, with one exception, the mean (t-test) or median (Mann- Whitney rank-sum test) direction bias for individual old monlceys was significantly less than that for any individual young monkey (Fig. 2, Table 1). A separate analysis compared the average direction biases for young monkeys versus old monkeys and also showed a significant aging effect (Mann- Whitney test, p ⁇ 0.05).
  • a reduction in orientation and direction biases could result from either an increased responsiveness to previously non-optimal orientations and directions, or from a reduced responsiveness to the previously optimal orientations and directions, or both.
  • the peak responses of young and old monkey cells to the drifting stimuli were used to compile tuning curves. If a substantial number of old monkey cells lost selectivity via reduced responses to the optimal stimulus alone, then the average peak response would be reduced in old compared with young monlceys. If old monkey cells instead lost selectivity via increased response to previously non-optimal stimuli, then the average peak response would be retained or increased in old monkeys. The latter result was obtained (Figs. 3 and 4). Old monkey cells demonstrated increased peak responses to drifting luminance bars (Fig.
  • the baseline response levels of neurons in old and young animals was also examined. VI cells in old monkeys had a significant increase in spontaneous activity when compared with young animals (Fig. 4e and f; p ⁇ 0.001). A separate analysis compared the average baseline responses for young versus old monkeys and also showed a significant aging effect (Mann- Whitney test, ? ⁇ 0.05). Taken together, the increases in peak and baseline activity in old compared with young animals resulted in decreased peak-to-baseline (signal to noise) ratios in old animals (4.63; 7.8 + 9.5; median; mean + s.d.) compared with young (17.6; 27.2 + 27.2; p ⁇ 0.001). A separate analysis compared the average peak-to-baseline ratios for young versus old monkeys and also showed a significant aging effect (Mann- Whitney test, ⁇ 0.05).
  • the maximum visually evoked responses of the VI cortical cells were measured in untreated old monkeys, untreated young monkeys, and old monkeys treated with GABA, the GABA agonist muscimol, and the GABA antagonist bicuculline.
  • Bicuculline reduces GABA mediated inhibition, and further increases peak response (FIG. 5) and spontaneous activity (FIG. 6).
  • GABA and the powerful GABA agonist muscimol increase GABA mediated inhibition and reduce the peak responses and spontaneous activity of cortical cells to the normal levels seen in young monlceys.
  • Extracellular action potentials of isolated cortical cells, LGNd cells and optic tract fibers were recorded with 3-5 MQ tungsten microelectrodes or microcapillary glass electrodes containing 4M NaCI.
  • the electrode was advanced using a hydraulic microdrive (Kopf) or a piezoelectric microdrive (Burleigh Instruments) and was moved 50 to 75 ⁇ m between units to reduce sampling bias.
  • Visual stimuli were generated on a Tektronix 608 display driven by a Picasso image synthesizer and specially designed texture/motion generator (Innisfree).
  • the Picasso and texture/motion generator are controlled by computer (software package developed by Cambridge Electronics Design, LTD.).
  • the system is able to randomly generate a broad spectrum of visual stimuli under computer control, collect the data, and perform on-line statistical analyses.
  • the oscilloscope display can be moved to any point in the animal's visual field while at the same time maintaining a fixed distance between the display and the animal's retina.
  • cells subserving all eccentricities can be observed without distortion.
  • the responses of the cells to the visual stimuli presented are stored in the computer for later analysis.
  • the responses to the sinusoidal gratings are defined as the amplitude of the fundamental Fourier component of the post stimulus time histogram.
  • the responses are defined as the peak response of the post stimulus time histogram with the total analysis time of 150-300 m/sec depending on the velocity of the drifting stimulus.
  • Orientation and direction preferences and sensitivities are calculated for each cell using the statistical methods described elsewhere in detail (Batschelet, 1981; Leventhal et al, 1995; Thompson et al., 1989, 1994a,b; Wdrgotter et al., 1990; Zar, 1974).
  • a variety of visual stimuli can be generated by programming the graphics card (Stealth64 2001, Diamond Corp., New York). Visual patterns are displayed on a 5" VGA monitor (Kristal Corp., St. Charles, EL) and imaged with a first-surface mirror (Edmund Scientific. Barrington, NJ) and lens on the film plane of the microscope's camera port. This ensures that when the electrode tip is in focus in the eyepieces the visual stimulus is also focused on the retina.
  • the sensitivity of cells to spatial frequency is determined by testing the responses of cells to high contrast sinusoidal gratings of various spatial frequencies.
  • the sensitivity of cells to stimulus contrast is determined by systematically testing the cells' responses to sinusoidal gratings of optimal spatial and temporal frequency.
  • High contrast sinusoidal gratings of different temporal frequencies and optimal spatial frequencies are employed to determine temporal sensitivity.
  • drugs were delivered through multibarreled micro- electrodes which had been positioned in the cortex as described in Example 1.
  • the multibarrel electrode had an impedance of 5 M Ohm, and contained 0.1-0.5 M solutions of drug, which were administered by passing a current of 15-50 n amp for 1-3 minutes.
  • Three barrels of the microelectrode held the drugs to be administered, and one barrel was filled with 4M NaCl in order to record the action potentials of the cells.
  • Administration of one drug at a time was accomplished by passing current through the appropriate barrel. Holding current of 10 n amp is applied through the other barrels simultaneously in order to prevent leakage of the other drugs. The observed effects were seen three to five minutes after drug administration. The drug effects wear off five to ten minutes after drug administration ceases, and the cells in old animals revert back to their abnormal condition after drug administration ceases and GABA inhibition decreases. Intravenous drug application also improves cortical function in a similar way.
  • FIG. 7 shows the signal-to-noise ratios of cortical cells in old monkeys, young monlceys, and old monlceys treated with GABA and GABA agonists and antagonists.
  • the signal-to-noise ratio of the cortical cells is the ratio of the response of the cell to appropriate stimuli, divided by the cell's spontaneous discharge rate. High signal-to- noise ratios allow cortical cells to function accurately and reliably. Low signal-to-noise ratios result in diminished cortical function throughout cerebral cortex.
  • FIG. 7 shows that signal-to-noise ratios are abnomially low in old monkeys compared to young monkeys.
  • the GABA antagonist bicuculline does not improve signal-to-noise ratios in old animals.
  • microelectrode of this Example in any of these brain areas, and confirm the effect of the drug by direct administration into that area of the brain.
  • the drug can be given orally or by intravenous administration, and the effect recorded in the precisely positioned electrode.
  • FIGS. 8 and 9 show the orientation (FIG. 8) and direction (FIG. 9) selectivity of cells in area VI of old-world monkeys.
  • Orientation and direction selective cells mediate the ability to perceive the shapes and directions of motion of objects.
  • a reduction in the number of selective cells adversely affects visual perception.
  • the ability to perform tasks such as driving a car are also be affected, because shape and direction discrimination are crucial in order to navigate through traffic.
  • old monkeys exhibit a reduction in orientation and direction selective cells compared to young monkeys.
  • the GABA antagonist bicuculline results in a further decrease in the number of selective cells.
  • GABA and GABA agonists are capable of increasing the orientation and direction selective responses of cortical cells.
  • FIG. 10 shows the tuning curves and corresponding polar plots obtained for two representative cells in old world monkeys that received treatment with GABA, a GABA agonist (muscimol) and a GABA antagonists (bicuculline).
  • GABA GABA
  • a GABA agonist muscimol
  • a GABA antagonists bicuculline
  • FIG. 3 shows the same as in FIG. 3, in which the peak responses [MR], orientation biases [OB], and direction biases [DB] are shown for each condition.
  • a typical cortical cell showing a lack of orientation and direction sensitivity is shown in (A).
  • Three minutes following GABA application (C) this cell exhibited strong orientation and moderate direction selectivity. The cell's peak response decreased as did its spontaneous activity.
  • GABA application was then discontinued and bicuculline application was begun (E). Within five minutes the cell lost its orientation and direction sensitivity and its peak response and spontaneous activity increased dramatically.
  • Example 8 The procedures described in Example 8 for studying cortical cells in old animals before, during, and after the administration of various drugs also provides new assays for finding agents that improve cortical function in the elderly.
  • This testing can be used in all regions of cerebral cortex and will allow screening for drugs that will improve function of cortical areas involved in visual, auditory, somatosensory, motor, memory, language, analytical thought, language, and cognition.
  • a battery of different tests can be applied to assess function in old animals and animals in which the various drugs and doses of drugs thought to affect GABA mediated inhibition are delivered.
  • the electrodes can be placed in the specified locations of the cortex using the procedures described in Examples 1 and 2 for single neuron recording. Drugs being screened can be administered through the measuring microelectrodes as described in Example 8.
  • function is assessed by testing for one or more of peak response, spontaneous activity, orientation selectivity, direction selectivity, signal-to- noise ratio, contrast sensitivity, and spatial frequency sensitivity. If testing spatial sensitivity, for example, a frequency of 40 cycles per second at arms length distance would be considered normal, while a frequency of 15 cycles per second would be considered low (and is a frequency that can be seen in older animals).
  • the effect of administering GABA-ergic agents can be measured by determining whether the frequency after administration of the agent increases toward the "normal" value (such as 40 cycles per second).
  • somatosensory cortex function is assessed by the ability of cells to signal qualitatively different stimuli (temperature, pain, vibration, pressure) that are presented to the test subject.
  • stimuli temperature, pain, vibration, pressure
  • the ability of cells to signal the intensity of different stimuli i.e. how hot, how hard, how fast the vibration can also be determined.
  • microelectrodes can be positioned in multiple cortical areas, such as visual cortex, somatosensory cortex, and or auditory cortex, and responses simultaneously measured from each of these areas. Single or multiple recordings can be performed while administering one or more test agents through the microelectrodes, or while systemically administering the one or more test agents.
  • Drugs that result in significant improvement at the single cell level are selected for further testing.
  • An example of such further testing is to administer them orally or by injection to old and young awake, behaving monkeys that are trained to perform a variety of different sensory and motor tasks. These tasks can include visual discrimination (for example discriminating lines of different orientation and direction), auditory discrimination (for example discriminating different frequencies), somatosensory discrimination (for example trained to respond to differences in pressure, temperature, vibration), motor tasks (such as rapidly assembling blocks), cognitive discrimination (such as choosing a unique shape in a complicated background, for example "find Waldo").
  • visual discrimination for example discriminating lines of different orientation and direction
  • auditory discrimination for example discriminating different frequencies
  • somatosensory discrimination for example trained to respond to differences in pressure, temperature, vibration
  • motor tasks such as rapidly assembling blocks
  • cognitive discrimination such as choosing a unique shape in a complicated background, for example "find Waldo"
  • Monlceys are restrained in a primate chair, and their heads positioned so that they must look straight ahead. They view two Tektronix 608 oscilloscopes simultaneously. Stimuli are generated in the same fashion and at the same distance as during single unit recordings. Thus, behavioral results will be directly comparable to physiological ones. Monkeys are trained to touch a touch pad to signal a correct choice, and correct choices are rewarded by administering food.
  • Orientation discrimination is studied by first training the monkey to discriminate between a matched condition (two high contrast, high spatial frequency gratings where both are horizontal), and a non-matched condition (two identical gratings where one grating is vertical and the other is horizontal). Gratings are flashed on for two seconds, and the animal has a total of three seconds from stimulus onset to respond. This timing is altered as needed to assure that old animals can easily complete the task. An equal number of matched and non-matched trials may be randomly interleaved. The animal is rewarded for responding to the non-matching condition, and for not responding to the matching condition. Once this task is learned (i.e.
  • the orientation of the vertical grating is changed and the difference between the two gratings in the non-matching condition is decreased in 5 -degree increments with training before each increment.
  • the increments are reduced to one degree and testing continues until threshold (the disappearance of the ability to distinguish between the two conditions) is determined.
  • Direction sensitivity is studied similarly with moving spots. Animals are first trained to discriminate between a matching condition (two high contrast, one degree spots moving horizontally in the same direction) and a non-matching condition (one spot moving vertically and the other moving horizontally). The direction difference is then decreased as described above, until the ability to discriminate between the directions is lost.
  • Spatial frequency sensitivity is studied as above with flashing gratings where the non-matched condition is one sinusoidal grating of one cycle/degree, and a blank screen of equal size and overall luminance and the matched condition is two blanks screens. After saturation the spatial frequency of the grating will be increased in one- cycle/degree increments until peak performance deteriorates. Then the increments will be decreased to 0.1 cycles/degree until threshold is reached. A value of 40 cycles per degree is usually normal, whereas 15 cycles per second is a lower spatial frequency sensitivity. Hence an increase of spatial frequency sensitivity (for example from 15 toward 45 cyles per degree) would be an indication that an agent has improved sensitivity.
  • Contrast sensitivity is studied as above, where the non-matching condition consists of one high contrast sinusoidal grating of one cycle/degree, and a blank field of equal size and luminance, and the matched condition is two blank screens. After learning has saturated, the contrast is then decreased (for example in increments of 0.1) until peak performance deteriorates. Increments are then decreased (for example in 0.01 increments) until threshold is reached.
  • the foregoing tasks were designed to be as stress free as possible to assure that old animals will have no problem learning them. In all cases the monkeys simply have to determine whether the two screens differ either in orientation, direction, contrast or spatial frequency.
  • monlceys used are prescreened to have normal optics and be in good health. These animals exhibit quite normal behavior. Both old and young animals can be tested successfully using this approach.
  • the apparatus is designed so that monkeys are trained by food reward to enter a primate chair, so that they push their head up and through an adjustable hole at the top of the compartment. Adjustable molded plastic baffles are attached at the sides and the back of the head to prevent large head movements.
  • the entire compartment rests on a mobile trolley, which is placed in front of the two visual display units. When the sliding front of the compartment is removed the animals can reach out and touch the monitor and retrieve a food reward.
  • the CANTAB battery and/or other tests of cognitive functioning can be applied to every subject in every step of cognitive decline. They can be applied to study the effects of various drugs in all stages of age-related decline.
  • test and treatment methods described in these examples are useful for a variety of age-associated disorders of cortical (for example cortical) decline in the elderly.
  • age-associated disorders of cortical decline extend on a continuum from no ⁇ nal age-related senescence to severe dementias associated with Alzheimer's disease and Parkinson's disease in an aging population.
  • GABA-ergic agents agents which increase GABA-mediated effects
  • agents which inhibit GABA aminotransferase such as vigabatrin
  • agents which inhibit GABA transferase and succinyl aldehyde such as valproate, valproic acid, and divalproex (or their pharmaceutically acceptable salts)
  • agents which facilitate GABA receptors such as topiramate
  • agents which block GABA uptake into presynaptic neurons, thus permitting more GABA to be available for binding such as tigabine and its pharmaceutically acceptable salts, such as tigabine hydrochlroide
  • agents which facilitate GABA-A mediated inhibition such as benzodiazepines, which enhance
  • GABA effects without directly activating GABA receptors, and/or which increase the frequency of chloride channel openings; agents which facilitate GABA-A mediated inhibition duration of GABA gated channel openings, such as barbiturates; GABA-A receptor binding agonists at the BZ1 (omega 1) receptor subtype, such as imidazopyradines; agents which facilitate GABA-B mediated inhibition, such as baclofen; and agents which facilitate GABA-C mediated inhibition, such as caca.
  • GABA agonists and GABA facilitators that are useful in the disclosed methods are shown in Table 2 (GABA Drugs).
  • GABA antagonists that can be used to offset undesired effects of the GABA agonists
  • Table 3 GABA Antagonists
  • the GABA-ergic drugs (which mediate GABA effects, and include GABA agonists) can be used in combination with a variety of other drugs.
  • the GABA-ergic drugs can be used in combination with GABA antagnoists, which are useful in reducing any side effects of treatment with GABA agonists such as a benzodiazepines.
  • the GABA-ergic drugs can be used in combination with already available cognition enhancing drugs, such as Cognex (tacrine hydrochloride).
  • two or more of the GABA-ergic drugs can be used in combination, for example drugs which mediate GABA-ergic activity by different mechanisms (for example one agent that facilitates GABA receptors and another agent that inhibits GABA-aminotransferase).
  • GABA-ergic agents that are found to enhance cortical function can be provided in a unit dosage form, for example in combination with a pharmaceutically suitable carrier.
  • Non-NMDA AMPA receptor antagonists such as CNQX (6- cyano-7-nitroqunoxaline-2,3-dione), DNQX and NBQX (see Pharmacol. Biochem. Behav. 51:153-158, 1995) are potential agents that can be tested in accordance with the techniques disclosed in this specification, and/or used to treat the age-related loss of GABA-ergic pathways.
  • AMPA receptor antagonists that are candidate agents include (S)-5-fluorowillardine; l-(quinoxalin-6-ylcarbonyl)piperidine (CX-516); (S)-2,3- dihydro-[3,4]cyclopentano-l,2,4-benzothiadiazine-l,l-dioxide.
  • Other candidate agents include those that block excitatory responses at the AMPA receptor, for example agents such as phenobarbital; and topiramate.
  • Agents described in this example are suitable for screening in the'present method, and at least some of them would be useful in the treatment of sensory, motor, and cognitive declines that accompany old age. They and their analogs can be screened for such uses with the techniques described in this specification.
  • the invention also contemplates various pharmaceutical and laboratory compositions that improve cortical function.
  • the agent When the agent is to be used as a pharmaceutical, the agent is placed in a form suitable for therapeutic administration.
  • the agent may, for example, be included in a pharmaceutically acceptable carrier such as excipients and additives or auxiliaries, and administered to a subject.
  • a pharmaceutically acceptable carrier such as excipients and additives or auxiliaries, and administered to a subject.
  • auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases.
  • Other pharmaceutically acceptable carriers include aqueous solutions, nontoxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., 1405-1412, 1461-1487, 1975, and The National Formulary JOV., 14th ed., Washington: American Pharmaceutical Association, 1975).
  • the pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman The Pharmacological Basis for Therapeutics, 7th ed.
  • the methods disclosed herein involve administering to a subject a therapeutically effective dose of a pharmaceutical composition containing the compounds of the present invention and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition containing the compounds of the present invention and a pharmaceutically acceptable carrier may be administered by any means known to the skilled artisan (for example, intravenous, subcutaneous, intra-peritoneal, topical, intra-nasal, or oral administration).
  • the pharmaceutical compositions are preferably prepared and administered in dose units.
  • Solid dose units are tablets, capsules and suppositories.
  • different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate.
  • the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit, or in several smaller dose units, and also by multiple administration of subdivided doses at specific intervals.
  • Initial dosage ranges can be selected to achieve an inhibitory concentration in target tissues that is similar to in vitro inhibitory tissue concentrations.
  • the dosage is ideally not so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex, and extent of the disease in the patient and can be determined by one skilled in the art.
  • the dosage can be adjusted for each individual in the event of any contraindications and can be readily ascertained without resort to undue experimentation.
  • the effectiveness of treatment can be determined by monitoring the subject's status on a neurocognitive test, such as the CANTAB battery.
  • any neurological function test can be used to assess cortical function, including repeating lists of items, reporting biographical information (such as one's own telephone number), or responses to questions about current events (such as the name of the President of the United States).
  • the pharmaceutical compositions according to the invention are generally administered intravenously, orally or parenterally, or as implants.
  • Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drag delivery, see Langer, Science, 249:1527-1533, 1990, which is incorporated herein by reference.
  • the pharmaceutical compositions maybe administered locally or systemically.
  • compositions of the invention include chemical compounds, peptides, and peptidomimetics.
  • the compounds When co-administered in combination with one or more other drags useful in the treatment of cortical decline, the compounds may be administered by either concurrent or sequential administration of the active agents.

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Abstract

Cette invention concerne des méthodes permettant de ralentir le déclin de la fonction corticale grâce à une augmentation de l'activité des trajets inhibiteurs, tels que les trajets liés à la fonction GABA-ergique, dans le système nerveux central. Dans certains exemples particuliers, des sujets atteints d'une baisse de la fonction corticale liée à l'âge sont traités au moyen de doses thérapeutiquement efficaces d'un agoniste GABA-ergique. Les méthodes selon l'invention permettent également de cribler des médicaments qui inhibent le déclin lié à l'âge de la fonction corticale, par exemple en exposant un sujet à un agent d'essai et en mesurant l'accroissement de l'activité inhibitrice corticale gaba-ergique.
PCT/US2001/019719 2000-06-23 2001-06-20 Fonction cerebrale amelioree par stimulation gaba-ergique WO2002000221A1 (fr)

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CA002413405A CA2413405A1 (fr) 2000-06-23 2001-06-20 Fonction cerebrale amelioree par stimulation gaba-ergique
AU2001268609A AU2001268609B2 (en) 2000-06-23 2001-06-20 Enhanced brain function by GABA-ergic stimulation
AU6860901A AU6860901A (en) 2000-06-23 2001-06-20 Enhanced brain function by gaba-ergic stimulation
EP01946582A EP1303280A4 (fr) 2000-06-23 2001-06-20 Fonction cerebrale amelioree par stimulation gaba-ergique
US10/311,821 US20040023952A1 (en) 2001-06-20 2001-06-20 Enhanced brain function by gaba-ergic stimulation
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US8652527B1 (en) 2013-03-13 2014-02-18 Upsher-Smith Laboratories, Inc Extended-release topiramate capsules
US9101545B2 (en) 2013-03-15 2015-08-11 Upsher-Smith Laboratories, Inc. Extended-release topiramate capsules

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Cited By (7)

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Publication number Priority date Publication date Assignee Title
WO2009016329A1 (fr) * 2007-07-31 2009-02-05 Cambridge Enterprise Limited Utilisation d'antagonistes du récepteur gabaa pour traiter un trouble cognitif dans des patients avec des états psychiatriques
US8652527B1 (en) 2013-03-13 2014-02-18 Upsher-Smith Laboratories, Inc Extended-release topiramate capsules
US8889190B2 (en) 2013-03-13 2014-11-18 Upsher-Smith Laboratories, Inc. Extended-release topiramate capsules
US10363224B2 (en) 2013-03-13 2019-07-30 Upsher-Smith Laboratories, Llc Extended-release topiramate capsules
US9101545B2 (en) 2013-03-15 2015-08-11 Upsher-Smith Laboratories, Inc. Extended-release topiramate capsules
US9555005B2 (en) 2013-03-15 2017-01-31 Upsher-Smith Laboratories, Inc. Extended-release topiramate capsules
US10172878B2 (en) 2013-03-15 2019-01-08 Upsher-Smith Laboratories, Llc Extended-release topiramate capsules

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