EP0949915A1 - Nitrone free radical trap treatment of dementia associated with aids virus (hiv-1) infection - Google Patents
Nitrone free radical trap treatment of dementia associated with aids virus (hiv-1) infectionInfo
- Publication number
- EP0949915A1 EP0949915A1 EP97920402A EP97920402A EP0949915A1 EP 0949915 A1 EP0949915 A1 EP 0949915A1 EP 97920402 A EP97920402 A EP 97920402A EP 97920402 A EP97920402 A EP 97920402A EP 0949915 A1 EP0949915 A1 EP 0949915A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- pbn
- nitrone
- active agent
- adc
- free radical
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- 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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
-
- 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
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
-
- 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
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
-
- 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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
Abstract
Nitrone-based free radical traps are disclosed to have activity as therapeutic and prophylactic agents in the treatment of neuronal damage associated with HIV-1 virus infection, referred to in advanced stages as dementia associated with AIDS infection or AIDS Dementia Complex (ADC).
Description
NITRONE FREE RADICAL TRAP TREATMENT OF DEMENTIA ASSOCIATED WITH AIDS VIRUS (HIV-1) INFECTION
FIELD OF THE INVENTION This invention relates to the treatment of dementia associated with AIDS virus (HIV-1) infection. More particularly it concerns compositions and methods for prophylactically or therapeutically treating this condition.
BACKGROUND INFORMATION
This Background Information section is divided into two parts. The first provides information on the condition being treated by this invention, the dementia associated with AIDS virus infection. The second provides information concerning nitrone-based free radical trapping compounds and their use as medicaments, such compounds being the active agents employed in the methods and compositions of this invention.
AIDS Dementia Complex (ADC*!
Acquired Immune Deficiency syndrome (AIDS) is often accompanied by neurological complications at later states of the disease. Approximately one third of adults and one half of children with AIDS eventually have these complications. These neurological conditions involve a complex set of cognitive, motor and behavioral dysfunctions which have been grouped under the names "AIDS Dementia Complex" (ADC) or more properly "HIV-associated dementia". As many as 50% of infected children have neurological deficits manifested as delayed developmental milestones. Neurological diseases associated with HIV infection include
myelopathy, peripheral neuropathy and myopathy. The neuropathological alterations that accompany HIV infection in the CNS include myelin pallor, increased astrogliosis, neuronal loss, and loss of dendritic arborization as well as a decrease in the presynaptic area. Resulting neurologic dysfunction can impair daily function, work productivity and in severe cases mandate expensive institutional care. Although early losses in mental capacity are not considered full-blown dementia, they nevertheless reflect neuronal damage associated with HIV-1. At present there are no effective therapies for AIDS-dementia or HIV-associated dementia. The medicaments described herein minimize the neuronal damage and prevent the progression of neuronal damage thus allowing extended functional capabilities of the affected individuals and hence considerable savings to society.
In the United States alone, over 1 million individuals are infected with HIV and approximately one third of this group have AIDS. Thus, the potential target population for an anti-ADC therapeutic treatment is currently greater than 100,000 patients/year and the target population which would acutely benefit from a prophylactic ADC treatment some ten times that. The need for treatments of ADC is expected to grow as more effective therapies allow persons with AIDS to live longer.
There is no known cure for AIDS available at the present time and in the absence of an effective treatment to completely eliminate the virus from afflicted individuals it is unlikely that any completely effective treatment for ADC is available. Zidovudine (AZT) has been used extensively to treat the AIDS infection. Although there is now doubt as to the long term effectiveness of this treatment because of high mutational frequency of the virus there is no
doubt that AZT has been effective in treating ADC on a short-term basis. The neurological symptoms associated with ADC have been treated with certain drugs. For instance, the psychosis associated with ADC has been treated with haloperidol and thioridazine. Molindone has been used for psychotic and delirious ADC patients. Methylphenidate has been used for treatment of depression associated with ADC. Electro-convulsive therapy has been used for HIV-induced stupor. All of these treatments serve to ameliorate symptoms of ADC. None treat ADC itself.
The envelope glycoprotein of HIV, gpl20, has been implicated in the pathogenesis of ADC. This protein, which is shed abundantly by infected cells, has been found to be neurotoxic to neurons in culture at extremely low concentrations, to impair learning, to induce cytokines, and to reduce cerebral glucose utilization. Hill et al. (.Hill, J.M. , Mervis, R.R. , Avidor, R. , Moody, T.W. and Brenneman, D.E. (1993) Brain Res . , 603:222-233.) have shown that in neonatal rats, administration of gpl20 causes morphological damage to the brain as well as retardation of the development of complex motor behaviors.
No approved treatments are available for ADC. Use of calcium channel antagonists and NMDA antagonists have been proposed as possible therapies by Lipton. Numerous calcium channel antagonists are available on the market, e.g., nimodipine, but NMDA antagonists are still being studied clinically by many companies, primarily for acute use in stroke or chronic use of epilepsy and Parkinson's disease. Amantadine, which is on the market as an anti-viral, is now known to possess NMDA antagonist properties. A closer cogener of amantadine, memantidine, is on the market in Europe and has been proposed by Lipton as a possible candidate for treatment of HIV dementia. Another agent which is
available for testing is nitroglycerin. Under certain circumstances, the NO generated from the nitroglycerin can protect neurons from overstimulation of the NMDA receptors with the resulting calcium and glutamate excitotoxicity. However, cardiovascular effects and the extremely erratic pharmacokinetics of nitroglycerin make this approach seem problematic.
Neuropathology associated with ADC has been postulated to be mediated by nitric oxide (NO) , among other factors. Dawson et al. (Dawson, V.L., Dawson, T.M., Uhl, G.R., and Synder, S.H. (1993) Proc. Natl . Acad . Sci . USA, 90:3256-3259) demonstrated that in a rat cortex neuronal culture, gpl20-mediated killing of neurons was due to generation of NO. The evidence supporting this conclusion was that neuronal killing was prevented by L-arginine depletion as well as by addition of nitroarginine, conditions expected to inhibit NO production by nitric oxide synthase (NOS) . Mollace et al (Mollace, V., Colasanti, M. , Persichini, T., Bagetta, G., Lauro, G.M. and Nistico, G. (1993) Biochem . Biophys . Res . Commun . , 194:439-445.) demonstrated that gpl20 addition to human T67 astrocytoma cell caused large increases in NO formation, which was found to be due to the activity of the inducible isoform of the enzyme, iNOS. Antibodies to gpl20 were shown to prevent iNOS induction and NO formation.
Nitrone-Based Free Radical Trapping Compounds as Medicaments This invention's approach to mitigating ADC employs nitrones and nitrone-related analogues as the active agent, in particular nitrone-based free radical trapping compounds. The best known and most widely studied nitrone is α-phenyl-t-butyl nitrone (PBN) .
Another well known nitrone is α-(4-pyridinyl-l-oxide)N- tert-butyl nitrone (POBN) .
Before any pharmaceutical use was made of nitrones, they were used as analytical tools capable of reacting with highly reactive radicals to yield free radical adducts that are much less reactive. In many cases, the free radical/NRT adduct complex is stable enough to allow in vivo isolation and quantitation using electron spin resonance (ESR) . The use of nitrones as therapeutics in neurological diseases is reported, for example, in United States Patent Nos. 5,025,032, 5,036,097; 5,405,874; 5,475,032; 5,488,145; 5,508,305; 5,578,617; and published PCT patent applications US91/05552 and WO 92/22290. Conditions proposed to be treated with nitrones in the past have included stroke and other ischemia events, as well as age-related disorders such as short term memory loss. Nitrones have been shown, when administered chronically, to reverse the age-associated increase in oxidatively damaged protein and the age-associated decrease in the activity of the oxidative-sensitive enzyme, glutamine synthetase (GS) in the brain. Accompanying the nitrone-mediated changes in oxidized protein and GS activity is improvement in the performance of animals in behavioral tests measuring short-term spatial memory.
STATEMENT OF THE INVENTION It has now been found that nitrone-based free radical trapping compounds (NRTs) have activity in the treatment of AIDS Dementia Complex (ADC) .
The present invention in one embodiment comprises a pharmaceutical composition comprising an effective AIDS dementia complex-treating amount of a nitrone- based free radical trapping compound in a pharmaceutically acceptable carrier.
The present invention in another embodiment comprises a method for treating a patient afflicted with AIDS dementia complex. This method involves administering an effective AIDS dementia-treating therapeutic dose of a nitrone-based free radical trapping compound to a patient in need of such therapy.
The present invention also comprises a method for prophylaxis for a patient likely to become afflicted with AIDS dementia complex. This method involves administering an effective AIDS dementia-preventing prophylactic dose of a nitrone-based free radical trapping compound to a patient identified as being in need of such prophylaxis.
BRIEF DESCRIPTION OF THE DRAWING The invention will be further described with reference being made to the drawing in which the sole figure is a series of spectral traces illustrating the effect of nitrone-based free radical traps in interfering with the formation of nitric oxide (NO) in the brain. NO is a species associated with the ADC disease state.
DETAILED DESCRIPTION OF THE INVENTION The Nitrones
The method of treatment of this invention employs one or more nitrone-based free radical trapping compounds as its active agent. PBN (α-phenyl butyl nirone) is the most studied nitrone and on that basis is a preferred compound for use in this invention. The art-taught related nitrone materials, 4-pyridinyl-l- oxide)N-tert-butyl nitrone (POBN) and derivatives thereof, also find application here. Derivatives of these materials may also be used, including by way of example but not being limited to hydroxy derivatives, especially 2-, 3- or 4-hydroxyphenyl t-butyl nitrone
and phenyl (mono-, di- or trihydroxy) tert-butyl nitrone; PBN esters, especially esters which release 2- , 3-, or 4-hydroxyphenyl t-butyl nitrone such as acetoxy derivative; 2-, 3-, or 4-carboxyphenyl t-butyl nitrone; phenyl hydroxybutyl nitrone; alkoxyl derivatives, especially alkoxyl derivatives which release 2-, 3-, or 4-hydroxyphenyl t-butyl nitrone, for example, the 2-, 3-, or 4-methoxyphenyl derivatives of PBN; and acetamide derivatives such as those related to 2-, 3-, or 4-aminophenyl t-butyl nitrone; diphenyl nitrone (PPN) and the analogous diphenyl nitrone derivatives; N-tert-butyl-α-(4-nitro-phenyl) nitrone; and N-tert-butyl-α-(2-sulfophenyl) nitrone. Corresponding POBN derivatives may also be employed. It will be appreciated that some of these materials can exist as pharmaceutically acceptable salts as well as the compounds described above. In the case of salts the materials will be ionized and accompanied by a pharmaceutically acceptable anion or cation as appropriate. Most commonly, a cation is a monovalent material such as sodium, potassium or ammonium, but it can also be a multivalent cation in combination with a pharmaceutically acceptable monovalent anion, for example calcium with a chloride, bromide, iodide, hydroxyl, nitrate, sulfonate, acetate, tartrate, oxalate, succinate, palmoate or the like anion; magnesium with such anions; zinc with such anions or the like.
A more exhaustive list of suitable nitrone-based free radical trap (NRT) materials is set forth in published patent applications WO91/05552 and WO92/22290 and U.S. Patent Nos. 5,025,032, 5,036,097; 5,405,874; 5,475,032; 5,488,145; 5,508,305; and 5,578,617, which disclosures are incorporated herein by reference. Mixtures of two or more of these materials may be employed, if desired.
PBN and exemplary derivatives thereof which may be employed in the present invention include those defined by the formula: :
wherein:
X is phenyl, imidazolyl, phenothiazinyl,
where R = independently (can vary within the molecule) halogen, alkenyl, oxyalkenyl, oxyalkyl, OH, NH2, NHZ,
NZ2, NO, -SO3H, -OSO3H, -S(alkyl) , -S(alkenyl), haloalkyl (including CF3) , Z,
Z,
A A
II II
-C-OZ, or -0-C-Z, where A is 0 or S; Z is a C1-6 straight or branched alkyl or cyclic group; and n is a whole integer from 1 to 5 (preferably 1 to 3) ; and Y is a tert-butyl group that can be hydroxylated or acetylated on one or more positions; phenyl; or the moiety
wherein R and n are as defined above, and
Z is a Cj to C5 straight or branched alkyl group.
Other spin-trapping agents can also be used, such as α-(4-pyridyl 1-oxide) -N-tertbutylnitrone (POBN), and spin-trapping derivatives thereof. Derivatives are made using standard techniques, for example, for substitution of the methyl groups. The general formula
for POBN and exemplary derivatives thereof is the following:
O T
wherein
Y is a tert-butyl group that can be hydroxylated or acetylated on one or more positions; phenyl; or
wherein
W is
0 0 0
II I! II
-NH-C-Z, -C-CH3, -C-OZ, or Z; and (0R)n, wherein R is H,
0 O"
Z-C-, Z, or -CH=N+
n is a whole number from 1 to 4 , or
Z is a Cx to C5 straight or branched alkyl group.
It has also been found that the following additional nitrone-based compound is suitable for use in connection with practice of the present invention:
Pharmaceutical Compositions
The nitrone-based free radical trapping compound (including its salts) may be formulated into pharmaceutical compositions suitable for oral or parenteral, e.g. intravenous or intramuscular injection, administration.
The compositions for oral administration can take the form of liquid solutions or suspensions, powders, tablets, capsules or the like. In such compositions, the nitrone or its salt is usually a minor component (0.1 to 50% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form. A liquid form may include a suitable aqueous or nonaqueous vehicle with buffers, suspending dispensing agents, colorants, flavors and the like.
A solid form may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch
or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, sugar, methyl salicylate, or orange flavoring.
In the case of injectable compositions, they are commonly based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. Again the active nitrone is typically a minor component, often being from about 0.05 to 10% by weight with the remainder being the injectable carrier and the like.
These components for orally administrable or injectable compositions are merely representative.
Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences. 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated by reference.
One can also administer the compounds of the invention in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can be found in the referenced materials in Remington's Pharmaceutical Sciences.
Conditions Treated and Treatment Regimens
The conditions treated with the nitrone-based free radical trapping compound-containing compositions generally include ADC and the various symptoms which fall within the ADC definition. The nitrone-based formulations can be administered to achieve a therapeutic effect and slow or counteract the progression of ADC. Alternatively, the formulations can be administered prophylactically to patients not yet exhibiting ADC but exposed to the HIV-1 virus. The nitrone-containing composition is administered in a manner designed to get the drug into the patient's bloodstream and across the blood-brain barrier into the patient's brain. One excellent mode for accomplishing this is intravenous administration. Intravenous dose levels for treating these conditions range from about 0.01 mg/kg/hour to about 10 mg/kg/hour, all for from about 1 to about 120 hours and especially 1 to 96 hours. A preloading bolus of from about 10 to about 500 mg may also be administered to achieve adequate steady state levels. Other forms of parenteral administration, such as intramuscular injection can be used, as well. In this case, similar dose levels are employed. While parenteral administration is attractive from a drug delivery point of view, it should be recognized that the course of AIDS infection can stretch over many months or even years. As a result, oral dosing may be preferred for patient convenience and tolerance. With oral dosing, one to three oral doses per day, each from about 0.02 to about 50 mg/kg are called for with preferred doses being from about 0.04 to about 10 mg/kg. These same dosing levels and regimens would be used for prophylactic treatment as well. In any treatment regimen, the health care professional should assess the patient's condition and
determine whether or not the patient would benefit from nitrone treatment. Some degree of experimentation to determine an optimal doing level and pattern may be necessary. A positive dose-response relationship has been observed. As such and bearing in mind the severity of the side effects and the advantages of providing maximum possible protection or amelioration, it may be desired in some settings to administer large amounts of nitrone such as those described above.
The invention will be further described by the following examples. These are provided to illustrate the invention and are not to be construed as limiting its scope.
Preparation of Pharmaceutical Compositions
Example 1 PBN is admixed as a dry powder with a dry gelatin binder in an approximate 1:10 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 240-270 mg tablets (20-30 mg of active nitrone) in a tablet press. These tablets may be administered to a patient suffering from ADC or and ADC-related condition on a daily, twice daily or thrice daily regimen.
Example 2
A PBN ester is admixed as a dry powder with a starch diluent in an approximate 1:4 weight ratio. The mixture is filled into 250 mg capsules (50 mg of active nitrone-based free radical trapping compound.) These capsules may be administered to a patient suffering from a ADC-associated condition on a daily, twice daily or thrice daily regimen.
Example 3 The nitrone, POBN, is admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 450-900 mg tablets (150-300 mg of active nitrone-based free radical trap) in a tablet press. These tablets may be administered to a patient suffering from a HIV infection on a daily, twice daily or thrice daily regimen.
Example 4 PBN is dissolved in a buffered sterile saline injectable aqueous medium to a concentration of approximately 5 mg/ml. 50 mis of this liquid material may be administered to a patient suffering from ADC on a daily, twice daily or thrice daily regimen.
It will be appreciated that any of the nitrone compounds described herein may be employed in any of these representative formulations, and that any of these formulations may be administered in any manner so as to treat or protect against ADC.
Biological Testing These tests utilized two neural cell culture systems for determining the efficacy of nitrone-based free radical trapping compounds in reversing neurotoxicity which mimics that observed with ADC or HIV dementia. In both assays, human neural cell cultures were used either as a bilayer (neurons on an astrocyte layer) or a three dimensional model (brain cell aggregates). TNF-α (100 pg/ml) was used as the neurotoxin and the length of incubation was 72 hours. A considerable body of evidence supports the notion that TNF-α is one of the neurotoxins responsible for
HIV Dementia. Brain concentrations of TNF-α are elevated in deep grey matter from AIDS patients with mold HIV Dementia. The distribution of messenger RNA expressing TNF-α in the brain follows a similar pattern. Gelbard et al. have shown that HIV-1 infected monocytes in culture with astroglial cells produce concentrations (>200 pg/ml) of TNF-α sufficient to cause neurotoxicity. TNF-α is reported to cause its neurotoxicity by inducing apoptosis. Recently, it was shown that gpl20 exerts toxic effects through induction of IL-6 and TNF-α.
Brain Aggregate Procedure
Brain cell aggregates were prepared from second trimester abortion tissue. Briefly, human brain tissue between 16 and 18 weeks gestation are gently dissociated through nylon screens to obtain single cells. Approximately 4X107 cells within 4 ml DME supplemented with 0.6% dextrose, 50 mg/ml gentamicin and 10% FCS are distributed into 25 ml DeLong flasks. Aggregates are constantly rotated and incubated at 37°C in an atmosphere of 10% C02. After 2-3 days, aggregates are transferred to 50 ml flasks and 5 ml of DME supplemented with 15% FCS (exchange medium) added. Each flask contains several thousand aggregates that can be sampled over time. Five ml of medium is exchanged every other day in culture. After 10-12 days in culture samples are taken for histology and trypan blue exclusion is performed to determine viability. Samples are screened for HIV, Hepatitis A, B, C and mycoplasma. Aggregates remain viable for approximately 40 days in culture. Brain cell aggregates are differentiated at the time of sampling in that they express neural cell markers for identification. Brain cell aggregates contain all the cells of the CNS (approximately 40% neurons, 40% astrocytes, 10%
oligodendrocytes and myelin and 10% microglia) . Neural cell apoptosis/death was measured by DNA fragmentation Elisa technique according to manufacturer's directions (Boehringher Mannheim) at a concentration of 10 micromolar.
Neural Cell Bilayer Procedure
Brain aggregates were prepared as described above. Several aggregates are placed in each well of a multi- well chamber slide (Nunc) coated with Cell TAK (collaborative Research) at a concentration of 20 ug/ml. Cells migrate from the brain aggregates within 3 days. Astrocytes form a monolayer with neurons on top and rare microglia (<1%) /oligodentrocytes (<1%) . These cultures are confluent within 1 week. Monolayers can be maintained for up to three weeks.
Characterization of cell types is determined by using immunohistochemistry and the antibodies neuron specific enolase (NSE, Dako) for neurons and glial fibrillary acidic protein (GFAP, Dako) for the identification of astrocytes. Confocal microscopy was used to visualize and identify neurons and astrocytes by size and shape. Neuronal viability was determined by exposing chambers with and without different treatments to AO and ethidium bromide (EtBr) . Neurons and total cell counts were determined by AO staining with visual confirmation by phase microscopy. Enumeration of cell viability by computerized software was performed at the time of microscopy. In addition, a visual printout of the fields observed always accompanied the data. The cell viability (programmed cell death/PCD) data from the above tests is summarized at Tables 1-2 below:
Table 1
Brain Sample Treatment PCD Evaluation
A Control 0±0.359
A TNF-α 1.1810.0759
A TNF-α+Compound 1 0.7722±0.09
A TNF-α+Compound 2 0.50610.107
A TNF-α+Compound 3 0.8410.074
Table 2
Brain Sample Treatment PCD Evaluation
B Control 010.68
B TNF-α 1.1610.088
B TNF-α+Compound 1 0.615+0.03
B TNF-α+Compound 2 0.688+0.041
B TNF-α+Compound 3 0.56810.049
In the above tests, the following compounds were employed: Compound 1 = PBN
Compound 2 = N,N-tert butyl-α-(2-sulfophenyl) nitrone
Compound 3 = Compound of formula III
In order to further confirm the effectiveness of the nitrone compounds as agents for treating ADC, a series of in vivo biological tests were carried out.
Material and Methods Used
Sodium N-methyl D-glucamine dithiocarbamate (MGD) and PBN were obtained from OMRF Spin Trap Source,
Oklahoma City, Oklahoma. gpl20 was obtained from Intracel Corporation, Cambridge, Massachusetts.
Treatment of Animals: Sprague-Dawley neonatal rats (sixteen siblings) were divided into four groups. Starting at day one after birth until day six, the neonates received 60 μl subcutaneous injections of the following treatments. Group 1: phosphate buffer-saline (PBS), Group 2: 5 ng gpl20 in PBS, Group 3: 5 ng gpl20 plus PBN (50 mg/kg) in PBS, and Group 4: PBN (50 mg/kg) in PBS. Rats were weighed daily and the amount of PBN injected was adjusted accordingly.
Behavioral Assessments: Time required to perform two developmental milestones were measured to determine the adverse effects of gpl20 administration on behavioral development as reported by Hill et al. and to determine the possible protective action of PBN on these parameters. Behavioral parameters studied were surface righting (animal placed head down on 45° inclined screen will turn around and climb up.) These two tests have been shown to be the most sensitive tests for assessment of the neurological disorder caused by gpl20 treatment. Furthermore, they can be examined early enough in the life of the animal (day 3 for surface righting and day 6 for negative geotaxis) that their determination will not interfere with NO trapping in the brain which was performed at the end of the first week of the life of the animal. Animals were tested for the time required for surface righting on day 3 and day 4 after birth, immediately prior to receiving the injections on those days, and on day 6 (2 hrs after the last injection that the animals received) as well as day 7 (20 hrs following the last injections) for the time required to perform negative geotaxis. The angle chosen for the setting used for negative geotaxis was decreased from 45° (the angle used by Hill et al) to 35° since under the experimental setting
employed, animals were not able to stay on the screen set at 45° and would slide down before being able to make an attempt to turn upward.
In Vivo NO Spin Trapping: Formation of NO in the brain of gpl20 treated rat pups was determined by NO spin trapping. At day 7 after birth, neonates treated as described were injected intraperitoneally with 200 μl of a sterile saline solution containing 100 mM MGD and 10 mM FeS04. Stock solutions of MGD (200 mM) and FeS04 (20 mM) were made separately in derated saline. Immediately prior to each injection, 100 μl of each solution was pulled into the same syringe and injection was performed. Rats were decapitated 30 min after administration of MGD-Fe and brains were immediately excised. A sagittal section immediately adjacent to the median plane was transferred onto the EPR tissue cell and the EPR spectrum was recorded using an ESP 300 E Bruker Electron Spin Resonance Spectrometer. The EPR settings were as follows: receiver gain: 5.00 x 105, modulation amplitude: 2.0 gauss, modulation frequency: 100 KHZ.
Results Obtained
Table 3 shows that gpl20 caused a significant decrease in the time elapsed before the neonates begin to crawl back upward as well as the time required before they righted themselves after being placed on their backs. PBN coadministration with the gpl20 prevented the gpl20-mediated damage. PBN treatment itself appeared not to have effected the response of the neonates in these tests. There were no differences in the weights of the neonates between the groups (data not shown) .
ADC is a neurological syndrome characterized by cognitive deficits and motor and behavioral dysfunction. The HIV-l envelope glycoprotein gpl20 has
been implicated in the development of ADC. This protein has been shown to be neurotoxic and to cause learning impairment and retardation of the development of complex motor behavior in rat neonates. Nitric oxide has been implicated in the gpl20-induced neurotoxicity. We now report in vivo evidence for the formation of nitric oxide in the CNS as a result of multiple subcutaneous injections of gpl20 to neonatal rats. NO was trapped in the brain of neonatal rats by N-methyl-D-glutamine dithiocarbamate-Fe and the NO content measured by electron paramagnetic resonance (EPR) spectroscopy. The nitrone-based spin trap PBN at 50 mg/kg was found to prevent gpl20-mediated nitric oxide formation and to also protect against gpl20- induced behavioral impairment.
Table 3
Effect of gp!20 on Neurological Development of Rat Neonates and PBN Protection
Time to Surface Negative Geotaxii -Right (Sec)* (Time in Sec)* Day 3 Day 4 Day 6 Day 7
Control 6.7 7.8 27.0 26.4 gpl20 20.0 18.6 78.2 58.3 gpl20+PBN 6.8 6.6 21.4 30.8
PBN only 7.2 4.9 28.2 19.7
* Negative geotaxis and surface-righting were conducted following the procedure of Hill et al. with slight modification. Each animal was given two trials, and the average of those two trials for four animals in the group represents the value presented for each group.
Fig. 1 illustrates a series of spectral traces which demonstrate the intervention of nitrone-based free radical traps in the production of nitric oxide generated by the action of gpl20. In Fig. 1, spectral traces illustrating nitric oxide generation in the rat neonate brain determined by in vivo EPR NO trapping. Trace A illustrates PBS (60 μL) administration for 6 consecutive days. Trace B illustrates gpl20 (60 μL, 83.3 ng/ml) administration for 6 consecutive days. Trace C illustrates gpl20 (60 μL, 83.3 ng/ml) plus PBN (50 mg/kg) administration for 6 consecutive days. The EPR spectrum in B exhibits three lines (indicated by arrows) with nitrogen hyperfine splitting of approximately 13 gauss at g = 2.04 which is consistent with the values obtained for the NO adduct of MGD-FE. Fig. 1 clearly shows that the gpl20 treatment caused the production of NO in the brain of the treated animals. The NO was trapped by MGD-Fe complex. The triplet signal in the middle spectrum is quite apparent. PBN treatment prevents NO formation in the gpl20-treated animals. Nitric oxide was not trapped in the PBN-treated controls or in the PBN-only treated animals (data not shown) . The amount of NO trapped in the brain of the gpl20-treated animals was estimated to be 5-10 μM based on past experiments. It should be noted that the spectra presented consists of only one animal each as shown in each case, but the other animals examined gave results consistent with those presented here. These results clearly demonstrate that systemic administration of gpl20 to neonatal rats (6 days) resulted in the formation of NO in the brain of these animals as shown by EPR spin trapping method. While several investigators have reported on gpl20-induced NO formation in cell culture, this is believed to be the
first report on the in vivo gpl20-mediated NO generation.
Most compounds used as traps for NO such as diethyldithiocaramate or MGD-Fe do not normally cross the blood-brain barrier and therefore are not suitable for in vivo spin trapping of NO in brain. However, due to the fact that in neonatal rats, the blood-brain barrier is not fully developed (open up to fourteen days after birth) MDG-Fe proved to be useful in trapping NO in the brains of these rats. The characteristic triplet signal of the trapped NO with a nitrogen hyperfine splitting of about 13 gauss and g *= 2.04, which is consistent with the values obtained for the NO adduct of MGD-Fe, was observed in the gpl20 treated rats. The PBS-treated control rat brains did not exhibit any signal due to trapped NO. There was no nitric oxide signal detected in the brains of the animals which were simultaneously treated with gpl20 and PBN. The source of NO (i.e., the constitutive versus inducible NOS) in the rat model used in this study, remains to be determined. However, Mollace et al have shown that NO formation caused by gpl20 in human cultured T67 astrocytoma cells is Ca2+-independent and therefore due to the inducible isoform of NOS, Miyajima and Kotake have shown that the increase in NO formation in the septic shock model is due to the induction of the inducible isoform of the enzyme, iNOS and that PBN treatment suppresses iNOS synthesis. The inventors have found high levels of iNOS mRNA following treatment of a rat mixed neuronal/glial cell culture with gpl20. Therefore, the NO detected in the brains of the gpl20 rat neonates is most likely due to iNOS induction, which is prevented by PBN coadministration. The dementia observed in individuals infected with ADC or HIV-1 is believed to be due to NO mediated killing of CNS neurons. The viral envelope protein
gpl20 has been shown to cause neural damage in a variety of neuronal preparations including rat cortical neurons and rat hippocampal cultures at very low concentrations (picomolar levels) . Glowa et al have demonstrated that intraventricular injections of gpl20 into rats result in behavioral deficits as well as dystrophic neurites in hippocampal pyramidal cells. Similarly, Hill et al have reported developmental retardation in neonatal rats follow systemic gpl20 administration. Expression of gpl20 in the CNS of transgenic mice by Toggas et al caused a spectrum of neuronal and glial changes resembling abnormalities in brain of HIV-1-infected humans. All these results indicate that gpl20 plays a key role in HIV-1-mediated nervous system impairment. ADC is associated with elevated brain cytokine levels. Induction of several cytokines, especially IL-1 and TNF-α by HIV-1 as well as gpl20 has been demonstrated in rat brain and human glial and monocyte cultures. Increased release of cytokines has been shown to result in the induction of iNOS through activation of nuclear factor-kappa B (NF- KB) . Reactive oxygen species (ROS) have been implicated in the activation of NF-KB and consequent induction of iNOS. The preventive action of PBN in the formation of NO in the model studied may be due to the ability of this compound to prevent the rapid rise in ROS which is associated with increased cytokine level and the subsequent iNOS induction.
Accompanying and possibly related to the rise in NO concentration in brain is a behavioral impairment in the gpl20-treated rats as indicated by assessment of two developmental milestones, negative geotaxis and surface righting. These two tests have been shown by Hill et al to be the most sensitive tests for assessment of the neurological damage caused by gpl20 treatment. In accordance with the findings of the
latter group, our results indicate that gpl20 administration causes neurological disorders in the model studied. The rats treated with gpl20 took almost three times longer to perform the surface righting task and 2-3 times longer for negative geotaxis (Table 3) . Interestingly, however, PBN coadministration prevented the gpl20 mediated behavioral retardation. The time required to perform the behavioral tasks were virtually identical between the control group and the group that simultaneously received gpl20 and PBN. This observation taken together with the fact that NO was not detected in the brain of the animals receiving gpl20+ PBN indicates that NO is closely associated with gpl20-mediated neurotoxicity which results in behavioral impairment. The protective action of PBN is believed due to its ability to prevent NO from being formed.
Claims
1. A method for treating AIDS Dementia Complex (ADC) in a patient suffering from AIDS Dementia complex comprising administering to a patient in need of such treatment as the active agent an effective amount of an AIDS Dementia Complex-treating nitrone-based free radical trapping compound.
2. The method of claim 1 wherein the active agent is orally administered.
3. The method of claim 1 wherein the active agent is parenterally administered.
4. The method of claim 3 wherein the active agent is administered by injection.
5. The method of claim 1 wherein the active agent is selected from PBN, POBN and derivatives thereof.
6. The method of claim 5 wherein the active agent is α-phenyl butyl nitrone (PBN) .
7. The method of claim 1 wherein said active agent is selected from the group consisting of α-phenyl butyl nitrone (PBN); α-(4-pyridyl-l-oxide) -N-tert¬ butylnitrone (POBN) ; and derivatives thereof selected from the group consisting of hydroxy PBN and POBNs, POBN and PBN esters, acetoxy POBNs and PBN, alkyl POBNs and PBN, alkoxy POBNs and PBN, and phenyl POBNs and PBN.
8. The method of claim 2 wherein the active agent is administered in a dosage of between 0.01 mg/kg body weight/hour to 10 mg/kg body weight/hour for a period of time of from 1 to 120 hours.
9. The method of claim 1 wherein the active agent is α-(4-pyridyl-l-oxide) -N-tert-butylnitrone (POBN).
10. The method of claim 1 wherein said active agent is defined by the formula:
wherein:
X is phenyl, imidazolyl, phenothiazinyl, n
where R = independently (can vary within the molecule) halogen, alkenyl, oxyalkenyl, oxyalkyl, OH, NH2, NHZ,
NZ2, NO, -S03H, -OSO3H, -S(alkyl), -S(alkenyl), haloalkyl, Z,
<3~ A A A A
^^ II II II II
-C=N+ , -C-NHZ, -C-NZ2, -NHC-Z, -C-Z,
A A
II II
-C-OZ , or -o-c-z , where A is O or S; Z is a C1-6 straight or branched alkyl or cyclic group; and n is a whole integer from 1 to 5; and
Y is a tert-butyl group that can be hydroxylated or acetylated on one or more positions; phenyl; or the moiety
wherein R and n are as defined above, and
Z is a Cχ to C5 straight or branched alkyl group.
11. The method of claim 1 wherein said active agent is defined by the formula:
0 t
wherein
Y is a tert-butyl group that can be hydroxylated or acetylated on one or more positions; phenyl; or
wherein
W is
O O o II II If -NH-C-Z, -C-CH3, -C-OZ, or Z; and
R^H, (0R)n, wherein R is H,
0 ^,0"
II + ^
Z-C-, Z, or -CH=N+
n is a whole number from 1 to 4 , or
Z is a Ci to C5 straight or branched alkyl group.
12. The method of claim 1 wherein said active agent is defined by the formula:
H
CH-
13. A pharmaceutical composition for the treatment of ADC comprising an effective AIDS Dementia Complex (ADC) treating amount of a nitrone-based free radical trapping material in a pharmaceutically acceptable carrier.
14. Use of a nitrone-based free radical trapping compound and pharmaceutically acceptable derivatives and salts thereof in the manufacture of a medicament for the treatment of AIDS Dementia Complex (ADC) .
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US1570996P | 1996-04-17 | 1996-04-17 | |
| US15709P | 1996-04-17 | ||
| PCT/US1997/006253 WO1997038683A1 (en) | 1996-04-17 | 1997-04-17 | Nitrone free radical trap treatment of dementia associated with aids virus (hiv-1) infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| EP0949915A1 true EP0949915A1 (en) | 1999-10-20 |
| EP0949915A4 EP0949915A4 (en) | 2001-07-04 |
Family
ID=21773092
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP97920402A Withdrawn EP0949915A4 (en) | 1996-04-17 | 1997-04-17 | Nitrone free radical trap treatment of dementia associated with aids virus (hiv-1) infection |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0949915A4 (en) |
| AU (1) | AU725432B2 (en) |
| CA (1) | CA2251800A1 (en) |
| NZ (1) | NZ332315A (en) |
| WO (1) | WO1997038683A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991005552A1 (en) * | 1989-10-17 | 1991-05-02 | Oklahoma Medical Research Foundation | Method and compositions for inhibition of disorders associated with oxidative damage |
| US5185324A (en) * | 1988-12-14 | 1993-02-09 | Hoechst Aktiengesellschaft | Enzyme-inhibiting amino acid derivatives, a process for the preparation thereof, agents containing these, and the use thereof |
| US5314917A (en) * | 1991-03-22 | 1994-05-24 | E. B. Michaels Research Associates, Inc. | Method for inactivating enveloped viruses and sperm |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5488145A (en) * | 1993-12-23 | 1996-01-30 | Oklahoma Medical Research Foundation | 2,4-disulfonyl phenyl butyl nitrone, its salts, and their use as pharmaceutical free radical traps |
-
1997
- 1997-04-17 AU AU24606/97A patent/AU725432B2/en not_active Ceased
- 1997-04-17 EP EP97920402A patent/EP0949915A4/en not_active Withdrawn
- 1997-04-17 WO PCT/US1997/006253 patent/WO1997038683A1/en not_active Application Discontinuation
- 1997-04-17 CA CA002251800A patent/CA2251800A1/en not_active Abandoned
- 1997-04-17 NZ NZ332315A patent/NZ332315A/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5185324A (en) * | 1988-12-14 | 1993-02-09 | Hoechst Aktiengesellschaft | Enzyme-inhibiting amino acid derivatives, a process for the preparation thereof, agents containing these, and the use thereof |
| WO1991005552A1 (en) * | 1989-10-17 | 1991-05-02 | Oklahoma Medical Research Foundation | Method and compositions for inhibition of disorders associated with oxidative damage |
| US5314917A (en) * | 1991-03-22 | 1994-05-24 | E. B. Michaels Research Associates, Inc. | Method for inactivating enveloped viruses and sperm |
Non-Patent Citations (5)
| Title |
|---|
| FLOYD R A ET AL: "The neurotoxic role of nitric oxide in AIDS dementia and stroke and the prevention of its formation by alpha-phenyl-tert-butylnitrone (PBN)." SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 22, no. 1-3, 1996, page 2141 XP000998212 26th Annual Meeting of the Society for Neuroscience;Washington, D.C., USA; November 16-21, 1996 ISSN: 0190-5295 * |
| See also references of WO9738683A1 * |
| TABATABAIE TAHEREH ET AL: "In vivo trapping of nitric oxide in the brain of neonatal rats treated with the HIV-1 envelope protein gp 120: Protective effects of alpha-phenyl-tert-butylnitrone." BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 221, no. 2, 1996, pages 386-390, XP000991161 ISSN: 0006-291X * |
| THOMAS, CRAIG E. ET AL: "In vitro and in vivo activity of a novel series of radical trapping agents in model systems of CNS oxidative damage" ANN. N. Y. ACAD. SCI. ( 1994 ), 738(NEUROBIOLOGY OF NO.BUL. AND.BUL.OH), 243-249 , 1994, XP000992143 * |
| YUE, TIAN LI ET AL: "Neuroprotective effects of phenyl-t-butyl-nitrone in gerbil global brain ischemia and in cultured rat cerebellar neurons" BRAIN RES. ( 1992 ), 574(1-2), 193-7 , 1992, XP000992139 * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU725432B2 (en) | 2000-10-12 |
| NZ332315A (en) | 2000-03-27 |
| CA2251800A1 (en) | 1997-10-23 |
| AU2460697A (en) | 1997-11-07 |
| WO1997038683A1 (en) | 1997-10-23 |
| EP0949915A4 (en) | 2001-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Potter et al. | Targeting the glutamatergic system for the treatment of HIV-associated neurocognitive disorders | |
| Viviani et al. | Glia increase degeneration of hippocampal neurons through release of tumor necrosis factor-α | |
| US5405874A (en) | PBN, DMPO, and POBN compositions and method of use thereof for inhibition of age-associated oxidation | |
| Dietrich et al. | CNS progenitor cells and oligodendrocytes are targets of chemotherapeutic agents in vitro and in vivo | |
| EP2114160B1 (en) | Antagonists of a non-selective cation channel in neural cells | |
| US20170259081A1 (en) | Methods for regulating cell mitosis by inhibiting serine/threonine phosphatase | |
| US20030078274A1 (en) | Method of reducing neuronal injury or apoptosis | |
| EP1658074B1 (en) | Inhibitors of 2-oxoglutarate dioxygenase as gamma globin inducers | |
| Kieburtz et al. | Excitotoxicity and dopaminergic dysfunction in the acquired immunodeficiency syndrome dementia complex: therapeutic implications | |
| SK154796A3 (en) | Use of rapamycin for the inhibition of neuronal cells necrosis | |
| Bjugstad et al. | Preventive actions of a synthetic antioxidant in a novel animal model of AIDS dementia | |
| WO1994027602A1 (en) | Use of metabotropic receptor agonists in progressive neurodegenerative deseases | |
| DE102008010362A1 (en) | Sgk1 as a therapeutic and diagnostic target for viral diseases | |
| US6034112A (en) | Use of flupirtine the prophylaxis and therapy of diseases associated with an impairment of the hematopoetic cell system | |
| AU725432B2 (en) | Nitrone free radical trap treatment of dementia associated with AIDS virus (HIV-1) infection | |
| TW201904579A (en) | Combination of MCL-1 inhibitor and blood cancer standard therapy, use thereof and pharmaceutical composition | |
| US11351229B2 (en) | Combination therapies for treating infantile spasms and other treatment resistant epilepsies | |
| US20070185045A1 (en) | Inhibitors of 2-oxoglutarate dioxygenase as gamma globin inducers | |
| AU720578B2 (en) | Benzamide treatment of dementia associated with AIDS virus (HIV-1) infection | |
| AU2004304716A1 (en) | Tellurium derivatives for prevention and treatment of neurodegenerative processes | |
| WO1995014465A2 (en) | Methods for amelioration of immunodeficiency virus induced neurodegeneration by covering nitric oxide or superoxide concentration | |
| MXPA98008661A (en) | Benzamide treatment of associated dementia the infection for the virus of the acquired immunodeficiency syndrome (human immunodeficiency virus) | |
| Gold | Laquinimod treatment in the R6/2 mouse model | |
| Mondal et al. | Low-dose maraviroc, an antiretroviral drug, attenuates the infiltration of T cells into the CNS and protects the nigrostriatum in hemiparkinsonian monkeys |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| 17P | Request for examination filed |
Effective date: 19981027 |
|
| AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
| AX | Request for extension of the european patent |
Free format text: AL PAYMENT 19981113;LT PAYMENT 19981113;LV PAYMENT 19981113;RO PAYMENT 19981113;SI PAYMENT 19981113 |
|
| RIC1 | Information provided on ipc code assigned before grant |
Free format text: 7A 61K 31/165 A, 7A 61K 31/135 B, 7A 61K 31/44 B |
|
| A4 | Supplementary search report drawn up and despatched |
Effective date: 20010514 |
|
| AK | Designated contracting states |
Kind code of ref document: A4 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
| 18D | Application deemed to be withdrawn |
Effective date: 20010802 |