![Ulllted States Patent [19] [11] Patent Number: 5,053,419 Lipton [45] Date of Patent: Oct](http://data.docslib.org/img/755eca981f70eeb5b4352818e347b8ba-1.webp)
Ulllted States Patent [19] [11] Patent Number: 5,053,419 Lipton [45] Date of Patent: Oct. 1, 1991
[54] TREATMENT OF AIDS DEMENTIA, [56] _ References Cited MYELOPATHY AND BLINDNESS PUBLICATIONS [75] Inventor: Stuart A. Lipton, Newton, Mass. Bfennem?n ei 3L, 1988, Nature, 3351639 Price et al., 1988, Science, 239:586. [73] Assignee: The Children’s Medical Center H0 et al., 1989, Annals in Internal Medicine, 111:400. Corporation, Boston, Mass. Pomerantz et al., 1987, New Eng. J. Med., 317:1643. [21] AppL No‘: 502 296 Primary Examiner-Stanley J. Friedman 22 F] d M 30 1990 [57] ABSTRACT [ 1 1c ' an ’ A method of reducing death of CNS neurons in a human patient infected with a human immunode? Related US. Application Data ciency virus, involving administering to the patient a [63] Continuation-impart of Ser. No. 331,872, Mar. 31, compound Capable of rcducing the gplzo'responsive ‘1939, abandoned‘ rise in free Ca+ + ion concentration in the CNS neurons of the patient, in a concentration effective to cause such [51] Int. Cl.5 ...... A61K 31/44 reduction. [52] US. Cl...... , ...... 514/356 [58] Field of Search ...... 514/356 10 Claims, 6 Drawing Sheets US. Patent Oct. 1, 1991 Sheet 1 of 6 5,053,419
Surviving retinal ganglion cells
-e rgp120-3B *- gp120-RF2
-~q.4a-.
-1 3 ~12 40 log concentration (M)
Surviving retinal ganglion cells E] solitary Q clustered
- F|G.2 US. Patent 0¢t.1,1991' - sheet 2 of 6 5,053,419
[JA\\\\\\\\1 ? 3-01 ° 200 pM gp120 I ' 20 pM gp120 25; = 2pM gp120 1 J 2.0 l [Ca 2"1, (pM) 1.5% 1.0% 0.5
[Ca2+], (PM)
0 - 260 400 660 Concentration (pM) FIG.3B US. Patent 0a. 1,1991 Sheet 3 of 6 5,053,419
1.5- _"F_
1.0
y [ca2*], (PM) -1\‘ 05- \ Wm§ m Control gp120‘ gp120+ gp120+ pre post immune immune
FIG.4
3.0 - ° 200 pM gp120 I I 200 pM gp120 + 100 nM nimodipine 2:5 -
2.0 :
1.0:
0.5 " US. Patent 0a. 1, 1991 Sheet 4 of 6 5,053,419
[1 solitary @ clustered
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1 T Cont-fol.///////////////////////////// gp120-3B nlfedipineT%/////////////////¢ gp120+ (1 0 FM) nlfedipineTW//////////////////% w///////////////// control 'gp120-RFr2' nifedlplne ‘ 0+ (10 PM) nl e ipine
FIG.5B US. Patent 0a. 1, 1991 Sheet 5 of 6 5,053,419 11111 8
r .r t l’ gp120-3Br%////////% nif edipine 9 20+ (10PM) nie i ine FIG.6A
ed
2D% 4
. Fm.. AM n 8 g + (10 PM) nife i ine U.S. Patent Oct. 1, 1991 Sheet 6 of 6 5,053,419
200pA VH=-40mV \ 50ms VC=-1OmV cell #481 #481
“PM -40 -30 -20 -10 10 20 30 —¢ 1 : 1 i 1 Wm")
plus nifedipine 400
-200 q FIG.8B gp120-RF2 I -3oo
-5OO - 5,053,419 1 2
TREATMENT OF AIDS DEMENTIA, Drawings MYELOPATHY AND BLINDNESS FIG. 1 is a dose response curve showing retinal gan glion cell death at different gpl20 concentrations; BACKGROUND OF THE INVENTION 5 FIG. 2 is a graph of retinal cell survival in the pres This application is a continuation in part of U.S.S.N. ence of recombinant gpl20 and antiserum; FIG. 3(a) is a graph of kinetics of intracellular free 331,872, ?led Mar. 31, 1989, now abandoned. This invention relates to the treatment of central Ca2+concentration ([Ca2+]i) in a retinal ganglion cell in response to various doses of gpl20, and (b) is a steady nervous system disorders caused by infection with state dose response graph of gpl20 concentration versus human immunode?ciency virus type I (HIV-I). [Ca2+]; HIV-I infection in humans causes general immuno FIG. 4 is a bar graph of gpl20 promoted rise in suppression and involves other disorders, such as blind [Ca2+]in the presence of gpl2O alone or gpl20 that has ness, myelopathy, or a dementing neurological disorder, been immunoprecipitated with gplZO antiserum; i.e., the AIDS dementia complex, the latter of which is 15 FIG. 5 is a graph of retinal cell survival in the pres a common and important cause of morbidity in patients ence of gpl20 and/or lOuM nifedipine; in advanced stages of infection. HIV-1 infection has FIG. 6 is a graph of retinal cell survival in the pres been documented in various areas of the CNS, including ence of gpl20 and/or lpMM nifedipine or nimodipine; the cerebral crotex, spinal cord, and retina. Price et al. FIG. 7 is a graph of intracellular [Ca2+]in the pres (1988, Science 239:586) and Ho et a1. (1989, Annals in ence of gpl20 and the Ca2+channel antagonist nimodi Internal Medicine 111:400) review the clinical, epidemi pine; ological, and pathological aspects of the AIDS demen FIG. 8(a) shows current flowing through calcium tia complex, and suggest that the mechanism underlying channels in the presence of nifedipine and gpl20, and the neurological dysfunction may be indirect tissue (b) is a graph of the current-voltage relationship. damage by either viral- or cellular-derived toxic sub 25 stances released by infected cells. SELECTION OF CA+ +CHANNEL Pomerantz et al. (1987, New Eng. J. Med. 271:1643) ANTAGONIST document the presence of HIV type I infection of the Any suitable antagonist, generally, of neuronal volt retina in two patients with AIDS. Brenneman et a1. age~dependent Ca+ +channels may be used to reduce or (1988, Nature 3352639) found gpl20, the coat‘protein of prevent AIDS related vision loss, myelopathy, or de HIV, killed hippocampal neurons. mentia. Preferred calcium channel antagonists include, SUMMARY OF THE INVENTION but are not limited to, the following drugs, of which the most preferred are those that are capable of crossing the The invention features a method of reducing death of blood brain barrier, for example, nimodipine (Miles CNS neurons in a human patient infected with a human 35 Pharmaceuticals, West Haven, CT) Smith Kline drug immunode?ciency virus, by administering to the patient no. 9512 (Smith Kline, French Beecham, Philadelphia, a compound capable of reducing the gpl20 responsive PA), and diproteverine (Smith, Kline, French-Bee rise in intracellular free Ca+ +ion concentration in CNS cham) . Less preferred antagonists are those that are less neurons of the patient, in a concentration effective to CNS permeable, for example, verapamil (Calan, G.D. cause such reduction. Searle & Co., Chicago, Ill.; Isoptin, Knoll, Whippany, Preferably, the blood of the infected patient contains NJ), nitrendipine, diltiazem (Cardizem, Marion, Kansas antibodies to HIV 1; most preferably, the patient mani City, MO), and nifedipine, U.S. Pat. No. 3,485,847, fests symptoms of AIDS related complex or of acquired hereby incorporated by reference (Procardia, L P?zer, immune de?ciency syndrome. The method may also NY, NY; Adalat, Miles). Other Ca2+channe1 antago include administration of a second compound that is 45 nists which may be useful are mioflazine, flunarizine, capable of reducing the gpl20 responsive rise in intra bepridil, lidoflazine, CERM-l96, R 58735, R-56865, cellular free Ca++ion concentration. The compounds Ranolazine, Nisoldipine, Nicardipine, PNZOO-llO, most preferred are calcium channel antagonists capable Felodipine, Amlodipine, R-(—)-202-79l, and R-(+) of crossing the blood brain barrier; these may be admin Bay K-8644 (Miles, Bayer), whose chemical formulae istered orally or intravenously; of these compounds, are described in Boddeke et al., Trends in Pharmaco nimodipine is currently most preferred. Calcium chan logic Sciences (1989) 10:397 and Triggle et al., Trends nel antagonists that do not freely cross the blood brain in pharmacologic Sciences (1989) 10:370. barrier are less preferred; these may be administered For any given calcium channel antagonist, effective intrathecally to the brain and/ or spinal cord, or intravit ness in preventing neurological disorders associated really to the retina; most preferred among these antago 55 with HIV-1 (or other HIV) infection is determined by nists are nifedipine, verapamil, nitrendipine, diltiazem, screening the drug using one or more of the following nicardipine, flunarizine, and diproteverine; oral or in assays of neuronal cell function; i.e., neuronal cell traveous routes of administration may be effective. death, detection of intracellular free Ca2+ion concen The invention can reduce dementia, myelopathy, or tration in neurons, and detection of current flow vision loss associated with infection by a human im through Ca2+channels An effective antagonist will munode?ciency virus. cause a decrease in HIV-l-associated neuronal cell Other features and advantages of the invention will death, and will prevent the rise in intracellular Ca2+ion be apparent from the following description of the pre concentration that occurs in the presence of gpl20. In ferred embodiments thereof, and from the claims. addition, an effective antagonist will decrease Ca+ rion 65 in?ux through neuronal calcium channels to a degree DESCRIPTION OF THE PREFERRED suf?cient to reduce neuronal cell death, while not com EMBODIMENTS pletely blocking Ca++ion in?ux, an event which itself The drawings will ?rst briefly be described. might kill neuronal cells. The antagonist may be com 5,053,419 3 4 pounded into a pharmaceutical preparation, using phar signal sequence (Berman et al., 1985, Science 227:1490) maceutical compounds well-known in the art; the exact to allow the envelope protein to be constituitively se formulation of the anagonist compound depends upon creted by the CH0 cell line. Production in a mamma the route of administration. lian cell ensured that the envelope protein was glycosy lated. This envelope glycoprotein, rgpl20-3B, was puri ASSAYS FOR NEURONAL CELL FUNCTION ?ed by immunoaf?nity chromatography to 5 parts in a AND DEATH million (99.995%) pure based on estimates from poly An antagonist may be tested for utility in the method acrylamide gel electrophoresis and Western blotting. of the invention using any type of neuronal cell from the The preparations of gpl20 (at low concentrations) were central nervous system, using the following assays, as highly labile in that they had to be freshly thawed (with long as the cell can be isolated intact using conventional refreezing avoided) in order to display activity. techniques. Retinal cultures were used in the following Cell survival was assayed after one day in culture. assays (but hippocampal cortex neurons have also been Incubations lasted 20-24 h at 37° C. in an atmosphere of used, e.g., in assays of neuronal death and intracellular 5% CO2/95% air. Ganglion cells could be unequivo calcium), because they can be produced from postnatal cally identified by the continued presence of the ?uores mammals, are well-characterized, and contain a central cent blue dye. The ability of retinal ganglion cells to neuron, the retinal ganglion cell, that can be unequivo take up and cleave ?uorescein diacetate to ?uorescein cally identi?ed with ?uorescent labels. A substantial was used as an index of their viability as described in portion of retinal ganglion cells in culture display both detail in Hahn et al., 1988, supra. Dye uptake and cleav functional synaptic activity and bear many, if not all, of age correlates well with normal electrophysiological the neurotransmitter receptors found in the intact re properties assayed with patch electrodes. tina. To perform the viability test, the cell-culture medium was exchanged for physiological saline containing GP120 INCREASES NEURONAL CELL DEATH 0.0005% ?uorescein diacetate for 15-45 5, and then cells IN VITRO 25 were rinsed in saline. Retinal ganglion cells that did not Neuronal cell death was assayed by incubating retinal contain the ?uorescein dye (and thus were not living) ganglion cells in vitro with puri?ed native or recombi often remained visible under both phase contrast and nant gplZO and scoring live cells. The ability of the UV ?uorescence optics, the latter because of the contin Ca2+channel antagonist to reduce neuronal cell death ued presence of the marker dye granular blue; other was determined by scoring live cells which had been dead retinal ganglion cells had disintegrated and only incubated with both gpl20 and the antagonist. debris remained. In contrast, the viable retinal ganglion Retinal ganglion cells from postnatal rats were identi cells displayed not only a blue color in the UV light but ?ed and their viability ascertained as follows. Under also a yellow-green fluorescence with ?lters appropri general anesthesia, the fluorescent dye granular blue ate for ?uorescein. Thus, the use of two exchangeable (Mackrornolekulare Chemic, Umstadt, FRG) was in fluorescence ?lter sets permitted the rapid determina jected as approximately a 2% (w/v) suspension in saline tion of viable ganglion cells in the cultures, which were into the superior colliculus of 4- to 7-day-o1d Long found as solitary neurons or lying among other cells in Evans rats (Charles River Laboratory, Wilmington, small clusters (usually in the ratio of approximately 1:10 MA). Two to 7 days later, the animals were killed by solitary to clustered). decapitation and enucleated, and the retinas quickly 40 FIG. 1 is a dose response curve for concentrations of removed. The retinas were dissociated and cultured in gpl20 ranging from 10*9 M to less than 10-13 M, and Eaqle‘s minimum essential medium (MEM, catalog shows that incubation of native puri?ed gp120 (RFZ) or #1090, Gibco Grand Island, NY), supplemented with recombinant gpl20 preparations with cultured retinal 0.7% (w/v) methylcellulose, 2 mM glutamine, 1 ug/ml cells resulted in the death of a signi?cant number of gentamicin, l6mM dextrose, and 5%(v/v) rat serum, as ganglion cells within 24 h. A signi?cant increase described in Lipton et al., 1987, J. Physiol. 385:361. The (P<0.0l) in cell death was observed at gpl20 concen cells were then plated onto 75 mm2 glass coverslips trations above 2>
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