WO1992021340A1

WO1992021340A1 - A method of treating infectious encephalitis of viral or bacterial, or parasitic pathogenesis

Info

Publication number: WO1992021340A1
Application number: PCT/US1992/004454
Authority: WO
Inventors: Edward W. Bernton; Frank C. Tortella
Original assignee: United States Of America, As Represented By The Secretary Of The Army
Priority date: 1991-06-05
Filing date: 1992-05-27
Publication date: 1992-12-10
Also published as: AU2159792A

Abstract

This invention is directed to pharmaceutical compositions and a methodology for their use in the pharmacologic treatment of infectious and para-infectious encephalitis and encephalopathy from diverse causes, to prevent or minimize neuronal damage end and central nervous system dysfunction.

Description

A Method Of Treating Infectious Encephalitis of Viral or Bacterial, or Parasitic Pathogenesis

I. Government Interest

The invention described herein may be manufactured, licensed and used by or for governmental purposes without the payment of any royalties to us thereon.

II. Technical Field of Invention This invention relates to the use of effective therapeutic agent(s)

for the treatment of mammals suffering from infectious and para-infectious encephalitis and encephalopathy, thereby preventing or minimizing neuronal damage and central nervous system dysfunction.

III. Background of the Invention The NMDA-binding excitatory amino acid receptor complex,

mediates excitatory neural signalling by endogenous ligands such as glutamic

acid, aspartic acid, and quinolinic acid. The generic receptor binds NMDA (n-methyl aspartic acid) whereas various receptor subtypes are defined by their preferential binding and excitation by quisqualate, kainic acid, and glutamic

acid. The predominant endogenous ligand is probably glutamic acid. Excessive or prolonged accumulation of excitatory amino acids acts via this receptor to mediate irreversible toxicity to neurons. This neuronotoxicity has been implicated by many recent studies as a common pathway of brain injury in organophosphate or nerve agent poisoning, seizures, or ischemic injury

following stroke. In general the pathologic changes within the brain due to viral

encephalitides are minimal, and discordant with the degree of CNS dysfunction or physiologic impairment. This statement also applies to the encephalopathic changes which frequently complicate Acquired Immunodeficiency Syndrome (AIDS). Mycoplasma infection by the M. fermentens strain, may play a role as a co-pathogen in AIDS, as suggested by Lo and Montagnier, and M. fermentens has been identified in brain, as well as liver, spleen and kidney

specimens from patients with AIDS. In this case as well, the histologic picture is remarkably bland, and in infectious burden of mycoplasma extremely low, given the degree of physiologic dysfunction. Two years ago, we identified a neuronotoxic factor in the supematants of HIV-infected human monocytes, which was cytopathic for

cultured primary rat fetal neurons. On further characterization, this toxic

factor proved to be low level mycoplasma contamination of the HTV viral stocks and subsequently infected monocytes. Of note, these supematants were not cytopathic for astroglial cells in the rat brain cultures, nor for any other non-neural cell line to which they were added, suggesting a possible cell and pathogen specific interaction between neuron and mycoplasma. Recently

Giulian et al published in Science. Vol. 250, December 12, 1990, at pages 1593-1596, that culture supematants from HIV-infected monocytic cell lines

were toxic to neurons in culture. Based on our extensive experiences, which ultimately showed HIV-infected monocyte supematants had no neuronal

toxicity unless co-infected with mycoplasma, we suspected the results of Giulian et al were attributable to mycoplasma. Giulian et al also noted that pretreatment of neurons with MK-801, as antagonist of the NMDA-receptor

complex, which can protect against excitatory amino acid neuronotoxicity, protected against the cytopathic effects of the monocyte supematants.

We have demonstrated that a variety of drugs, inhibiting either

release of excitatory amino acids or their actions at their receptors, block the neuro-cytopathic effect of mycoplasma infection when added to cultures of rat brain cortex. Furthermore, a similar neuronotoxicity is seen following the addition of bacterial endotoxin. since both mycoplasma and endotoxin stimulate cells to release immenu cytokines, several recombinant cytokines were screened for neurocytopathic effects on addition to cultured neurons. One of these, tumor necrosis factoralpha (TNF), was found to reproduce the

neurocytopathic actions of mycoplasma or endotoxin addition. This effect was

blocked by addition to cultures of MK-801, a prototypic NMDA-receptor antagonist, which blocks the toxic effects of mycoplasma or endotoxin in these cultures. Neutralizing antibody to TNF, when added to brain cultures, was subsequently found to decrease neuronal cell damage following mycoplasma infection, implying that TNF does, at lease in part mediate the neuronotoxic effect. TNF release is a frequent tissue response to viral, parasitic, or bacterial infections, and brain tissue contains abundant microglia, immune accessory cells which can produce TNF. This data therefore suggests that TNF production leads to excitatory amino acid mobilization and that these events mediate a final common mechanism of neuronal damage following infectious and immunologic stimuli. Furthermore, drugs which protect neurons from amino acid excitoxicity when this system is triggered in vitro by mycoplasma or endotoxin should likewise protect neurons and preserve central nervous system function in diverse infections resulting in encephalitis (brain infection or inflammation) and/or in encephalopathy (brain disfunction).

IV. Summary of the Invention It is therefore an object of the present invention to provide pharmaceutical compositions and a methodology for their use in the pharmacologic treatment of infectious and para-infectious encephalitis and encephalopathy from diverse causes, to prevent or minimize neuronal damage and central nervous system dysfunction. The method consists of treatment of

the patient with antagonist drugs specifically blocking the functioning of the neuronal excitatory amino acid receptor, the main type of which is known as the N-methyl aspartate-binding receptor, or of treatment of the patients with other drugs which block amino acid excitotoxicity by inhibiting release of endogenous excitatory amino acids.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and

scope of the invention will become apparent to those skilled in the art from this detailed description.

V. Brief Description of the Drawings Figure 1 shows electron photomicrographs of neurons from rat fetal brain cultures exposed to culture fluids from monocytes either: A. uninfected with mycoplasma

B. infected with mycoplasma hominis, showing spherical mycoplasma organisms as noted by arrow and extensive cytoplasmic vacuolization and neurofilament disruption within the neuron. Figure 2 shows neuronal cell cultures at 20 days were exposed to 1000 colony forming units/ml mycoplasma. Cultures were photographed before mycoplasma infection and at daily intervals thereafter. AT 72 hours,

cultures were fixed with 5% paraformaldehyde and stained with cresyl violet. Arrows identify the same cell before and after mycoplasma infection. (A) Neuronal cell culture prior to addition of mycoplasma; (B) same sulture as (a) 18 hours after addition of M. hominis shows rounding and swelling of some neurons and complete disintegration of others with perikarial swelling and loss of dendritic processes. (C) Neuronal cell culture prior to addition of mycoplasma shows phase bright neurons and lacy fields of dendritic networks; (D) same sulture as (C) 18 hours after addition of M. fermentans, strain incognitans, shows perikarial swelling, cytoplasmic vacuolization and complete loss of the neuritic field. (E) Vacuolated and disintegrating neurons in same culture as (C) 36 hours after addition of M. fermentans. (F) Replicate

culture of (E) treated with 20 uM MK-801 15 minutes before and continuously after addition of mycoplasma. (G) Cresyl violet-stained neurons 72 hours after addition of M. fermentans show dark cytoplasmic granules within disintegrated neuronal bodies. Arrowhead points to an intact bipolar neuron. (H) Replicate culture of (G) treated with 20 uM MK-801 15 minutes before

and ocntinuously after addition of mycoplasma. Top of field shows a clump of

viable cells outside the plane of focus. Intact neurons with lacy dendritic processes are clearly visible. Morphologic changes in neurons 18 to 48 hours after mycoplasma infection closely approximate those induced by toxic concentrations of glutamic or aspartic acid. In each instance, 5-20% of neurons (see arrowhead) are bipolar, spindle-shaped, and apparently resistant to toxic lysis. By 72 hours after mycoplasma infection, marked proliferation of glial cells in noted. Glial cell proliferation is not seen following exposure to neurotoxic concentrations of glutamic or aspartic acid.

Figure 3 shows 100 x magnification photomicrogrphas of cresyl-violet stained neuronal cultures 3 days following addition of A. inactive vehicle, B. 1/0000 dilution of mycoplasma fermentans, C. protective 2.5 microMolar addition of MK-801, D. non-protective 0.5 micromolar addition of MD-801, E. protective 20 micromolar addition of 7-chlorokyurenic acid, F. protective 20 micromolar addition of 20 micromolar DTG, G. protective 20 micromolar addition of carbetopentane.

Figure 4 shows 100 x magnification phase-contrast photomicrogφhas of living neuronal cultures 48 hours following addition of A and B. nothing, C. bacterial endotoxin, 500 ng./ml., D. bacterial endotoxin and 5 micromolar each dextromethoφhan and MK-801, E. 1000 units/ml or recombinant mouse tumor necrosis factor alpha (TNG), F. 1000 units/ml of

TNF and 5 micromolar each dextromethoφhan and MK-801, G. 300 units/ml

of TNF, H. 300 units/ml of TNF and 5 micromolar each dextromethoφhan and MK-801.

VI. Detailed Description of Preferred Embodiments We propose that infection of the brain matter causes a metabolic response or release of tissue factors which either compromise glial re-uptake of the excitatory amino acid glutamate, and/or stimulate its excessive secretion.

Other excitatory amino acids may possibly also be present in excessive concentrations, such as quinolinic acid, which accumulates due to the increased activity of indoleamine 2,3 -dioxygenase induced by immune cytokines such as gamma-interferon. Ischemia due to microvascular compromise, as in cerebral malaria, also would increase concentrations of glutamic acid in the neuronal

micro-environment. The damage, reversasible or irreversible, resulting to neurons via excitotoxicity, would mediate the CNS dysfunction and damage characterizing these infectious diseases. Accordingly, this invention in its preferred embodiment, consists of the use of MK-801, or any other potent NMDA-receptor antagonist, for the treatment of acute infectious encephalitis, at an effective dose to prevent excitotoxic damage to neurons. This would improve the physiologic and clinical manifestations of the infectious disease, minimizing both permanent CNS damage and death as clinical outcomes. It is further proposed that encephalopathy in patients with severe bacterial infection or sepsis, in whom cerebrospinal fluid concentrations of the excitatory amino acid quinolinic acid are increased due to activation of the immune system, may be treated with MK-801, of any other potent NMDA-receptor antagonist to

restore more normal CNS function. More precisely, this invention relates to a method for the treatment of a mammal (human or nonhuman) suffering from encephalitis or encephalopathy caused by a pathogen comprising administering to said mammal a therapeutically-effective amount of a pharmaceutical composition comprising MK-801, dextromethoφhan, carbetopentane, 7-chlorokyurenic

acid, caramiphen, dithiolguanidine (DTG), excitatory aminoacid-induced or NMDA-induced neuronal excitotoxicity antagonists and a neuroprotective kappa opioid agonist, such as CI-972, or mixtures thereof, thereby preventing or minimizing neuronal damage and/or central nervous system (CNS) dysfunction. The encephalopathy or encephalitis can be infectious or parainfectious. The pathogens which are the causative agents of the encephalitis or encephalopathy are selected from the group consisting essentially of a virus, bacterium and parasite, or a combination thereof. An illustrative group of viral pathogens include arboviruses, Japanese B, St. Louis, Venezula, vaccinia, variola, herpes simplex vims type 1, herpes simplex vims type 2, cytomegalovirus, enterovirus, varicello-zoster vims, Epstein-Barr vims, rubella vims and rubeola vims. Representative bacteria

include Haemophilus influenzae type b, streptococcus, E. Coli, gram-negative bacterium and Mycoplasma fermentans. Parasitic pathogens include African or American trypanosomes and P. falciparum. The practice of this invention is applicable in those instances where the central nervous system (CNS) dysfunction results following the onset

of cerebral malaria, arbovims-caused encephalitis, herpes vims hominis

encephalitus, vaccinia encephalitus, variola encephalitus, cytomegalovirus encephalitis, enterovirus encephalitis, measles encephalitis, mycoplasma pneumonia meningitis, Reyes syndrome, and encephalopathy or encephalitis associated with acute severe bacterial infection or sepsis, or acquired Immuno-deficiency Syndrome (AIDS). The causative pathogenic agent or pathogen can be a combination of a vims and bacterium, such as a HTV-1 vims .and Mycoplasma fermentans. It is also contemplated that the encephalitis associated with acute severe bacterial infection can be acute meningio-encephalitis. The pharmaceutical compositions of matter useful, in accordance with this invention, for the treatment of a mammal (human or nonhuman) suffering from encephalitis or encephalopathy caused by a pathogen comprise a therapeutically-effective amount of an active ingredient selected from the group consisting essentially of MK-801, dextromethoφhan, carbetopentane, 7-chlorokyurenic acid, caramiphen, a neuronprotective kappa opioid agonist such as CI-972, dithiolguanidine (DTG), excitatory aminoacid-induced or NMDA-induced neuronal excitotoxicity antagonists, or mixtures thereof. These pharmaceutical composition can be administered intravenously, intramuscularly, or orally. Especially preferred active ingredients are MK-801, dextromethoφhan, caramiphen, and carbetopentane.

Examples The herein offered examples provide methods for illustrating, without any implied limitation, the practice of this invention in the treatment of infectious and para-infectious encephalitis and encephalopathy. The representative experiments have been chosen to illustrate the effectiveness of therapeutic agents such as MK-801 dextromethoφhan, caramiphen and carbetopentane. All temperatures not otherwise indicated are in degrees Celcius (°C) and parts or percentages are given by weight. Experimental Data: EXAMPLE 1 Pure culture of Mycoplasma fermentans, strain incognitus, titer approximately 10⁸ per ml were obtained from Dr. S.C. Lo and added at dilution of 1/1000, 1/10000, and 1/100,000 to 22 day old cultures of fetal rat neurons. Control wells received sham innocula. Other wells were identically innoculated 30 minutes after pre-treatment by addition of 10 microM MK-8-1.

cytopathic effects were recorded by 30 X phase contrast photomicroscopy 24 and 48 hours later. M. fermentans caused a dose-dependent and selective initial

rounding, swelling, vacuolization, and loss of neuritic processes in approximately 70% of the neuronal population, followed by complete lysis. Pre-treatment with MK 801 completely prevented these effects.

Further studies showed that recovery of the mycoplasma into SP-4 culture media was identical from neuronal culture wells with and without MK-801 present, and that addition of MK-801 at 1, 10, and 20 micromolar concentrations to SP-4 media did not inhibit the growth of M. fermentans. Thus the effects of MK-801 in the neuronal culture reflected a neuroprotective action and not an unanticipated anti-mycoplasma or antibiotic action.

EXAMPLE 2

In a subsequent experiment, primary rat neuronal cultures (21 days in culture) were innoculated with a 1/10,000 dilution of pure culture of Mycoplasma fermentans (for approximately 10,000 organisms/ml final infectious dose in culture). Various wells were pre-treated at either 20 or 2.0 micromolar concentrations with: 1.) MK-801, 2.) carbetopentane, 3.) dextromethoφhan, 4.) caramiphen; 5.) 7-chlorokyurenic acid (a glycine binding site antagonist which blocks activation of the NMDA receptor complex), 6.) DTG (dithioguanidine, a sigma opiate ligand of high specificity), and 7). CI-972, a kappa opiate agonist with protective effects against in vivo and in vitro NMDA neurotoxicity. Sixty hours after innoculation, neuronal

cultures were examined by phase photomicroscopy. Control wells (no

mycoplasma) showed many viable neurons comparable to the pre-innoculation population. Wells with mycoplasma and vehicle all showed lysis or loss of approximately 80% of the neuronal population. All of the above dmgs were completely neuroprotective at 20 micromolar and many were protective at 2 micromolar. EXAMPLE 3 Subsequently additional experiments were conducted to examine the mechanism of mycoplasma-induced neuronotoxicity. It was found that killed mycoplasma which are non-infective were able to cause neuronal death. This suggests the killing of neurons was mediated by a host response to the

immunologic stimulus of mycoplasma cell constituents. Bacterial endotoxin, which potently evokes host immune responses involving the secretion of Interleukin-1, Interleukin-6, and Tumor-necrosis factor, which have been

shown to be induced in mycoplasma infected cultures of monocytes, was added to the neuronal cultures. Bacterial endotoxin was also neuronotoxic, at concentrations of 25 ng/ml and above, and most importantly, this toxicity was

also blocked completely by MK-801 at 10 um concentration. A variety of

mammalian cytokines induced by LPS were screened for neuronotoxicity, including gamma-interferon, IL-2, IL-1, IL-6, TGF-beta, M-CSF, and TNF alpha. Only addition of TNF alpha to cultures was found to cause neuronal destruction similar to that seen following addition of LPS or Mycoplasma.

Again, MK-801 was found to block these effects of TNF alpha on cultured neurons, suggesting that infectious neuronotoxicity may be mediated by TNF

alpha as a common mechanism, operating through the mobilization of excitatory amino acids or the activation of the NMDA-binding recxeptor. This

data suggests that in all acute infectious encephalitides the neuronal damage may be mediated via TNF and the NMDA receptor and pharmacologically blocked with agents modulating the NMDA-receptor.

EXAMPLE 4 A thirty year old soldier stationed in Germany is admitted to the hospital with fever, stiff neck, and mild delirium. He was hiking in the woods

the previous weekend and received several tick bites. A spinal tap shows elevated protein and a moderate lymphocytosis. Tick-bom encephalitis is suspected. His delirium worsens. Treatment is given with MK 801, (effective dose 100-500 ug/kg sc. every 4 to 8 hours), and his CNS symptoms clear over the ensuing 8 hours.

EXAMPLE 5 A first grade student is brought to a hospital emergency room during a summer outbreak of sporadic St. Louis Equine encephalitis, following onset of fever and a seizure. Spinal tap and EEG are consistent with viral encephalitis, he becomes stuporous on his second hospital day. Treatment is instituted with dextromethoφhan, (effective adult dose 100 to 200 mg every 6 hours orally) at half the adult dose, via a nasogastric tube.

EXAMPLE 6 Following a viral illness which was treated by the mother with aspirin, an 8 year old girl develops nausea, vomitting, and confused behavior.

At hospital admission, lever function test are abnormal, the spinal tap shows elevated opening pressure, CT scan is normal, and physical exam shows papilledema. The diagnosis of Reyes syndrome is made. Supportive measures to decrease intracerebral pressure are instituted. As the patient becomes comatose, treatment is initiated with CI - 977 at an effective dose of 0.5 to 3.0 mg/kg, intravenously, at 4 hour intervals.

EXAMPLE 7 A 67 year old male was alcoholic cirrohsis, portal hypertension, and ascites becomes slightly febrile and quite disoriented. A tap of his abdominal ascites shows bacteria and white cells, and the diagnosis of spontaneous bacterial peritonitis with encephalipathy due to liver disease and

sepsis. He is treated with antibiotics for his bacterial infeciton and with caramiphen, (effective dose 1 to 4 mg/kg) orally, for his encephalopathy. Since there exist no specific antiviral treatments for most of the encephalitides of viral etiology, many of which are highly lethal or result in

permanent adverse central nervous system sequellae, this invention will limit

morbidity and mortality in such infections. This is a particularly unique and

clinically useful application of this invention. An in vitro neuronal culture model for adenovirus-induced neuronotoxicity is being developed as a secondary in vitro dmg screen, and we are developing an arbovims screen which could be used at research facilities with level III biologic containment.

Three models, intracerebralventricular injection of suckling rats or 4-week old mice with adenovims, Japanese encephalitis vims, or with mycoplasma fermentans, all cause rapid lethal encephalitis. These will be used to screen the above-mentioned dmgs for in vivo potency and efficacy in preventing death due to CNS infection with these three pathogens.

Claims

We Claim: 1. A method for the treatment of a mammal (human or

nonhuman) suffering from encephalitis or encephalopathy caused by a pathogen

comprising administering to said mammal a therapeutically-effective amount of a pharmaceutical composition comprising MK-801, dextromethoφhan, carbetopentane, 7-chlorokyurenic acid, caramiphen, dithiolguanidine (DTG), excitatory aminoacid-induced or NMDA-induced neuronal excitotoxicity antagonists and a neuroprotective kappa opioid agonist, CI-972 or mixtures thereof, thereby preventing or minimizing neuronal damage and/or central nervous system (CNS) dysfunction.

2. The method in accordance with Claim 1 wherein the

encephalitis is infectious or parainfectious.

3. The method in accordance with Claim 1 wherein the

encephalitis is infectious or parainfectious.

4. The method in accordance with Claim 1 wherein the pathogen is selected from the group consisting essentially of a vims, bacterium and parasite, or a combination thereof.

5. The method in accordance with Claim 4 wherein the

pathogen is a vims.

6. The method in accordance with Claim 5 wherein the vims is selected from the group consisting essentially of an arbovims, Japanese B, St. Louis, Venezula, vaccinia, variola, herpes simplex vims type 1, herpes simplex vims type 2, cytomegalovirus, enterovirus, varicello-zoster vims, Epstein-Barr vims, rubella vims and rubeola vims.

7. The method in accordance with Claim 4 wherein the pathogen is a bacterium.

8. The method in accordance with Claim 7 wherein the bacteria is selected from the group consisting esentially of Haemophilus influenzae type b, streptococcus, E. Coli, gram-negative bacterium and Mycoplasma fermentans.

9. The method in accordance with Claim 4 wherein the

pathogen is a parasite.

10. The method in accordance with Claim 9 wherein the parasite is selected from the group consisting essentially of African or American trypanosomes and P. falciparum.

11. The method in accordance with Claim 1 wherein the central nervous system (CNS) dysfunction is selected from the group consisting

essentially of cerebral malaria, arbovims-caused encephalitis, herpes vims hominis encephalitus, vaccinia encephalitus, variola encephalitus, cytomegalovirus encephalitis, enterovirus encephalitis, measles encephalitis, mycoplasma pneumonia meningitis, Reyes syndrome, and encephalopathy or encephalitis associated with acute severe bacterial infection or sepsis, and acquired Immuno-deficiency Syndrome (AIDS).

12. The method in accordance with Claim 4 wherein the pathogen is a combination of a vims and bacterium.

13. The method in accordance with Claim 12 wherein the vims

is the HIV-1 vims and the bacterium is Mycoplasma fermentans.

14. The method in accordance with Claim 11 wherein the encephalitis associated with acute severe bacterial infection is acute

meningio-encephalitis.

15. The method in accordance with Claim 1 whereina the pharmaceutical composition is administered intravenously, intramuscularly, or orally.

16. A pharmaceutical composition of matter useful for the treatment of a mammal (human or nonhuman) suffering from encephalitis or

encephalopathy caused by a pathogen comprising a therapeutically-effective amount of an active ingredient selected from the group consisting essentially of MK-801, dextromethoφhan, carbetopentane, 7-chlorokyurenic acid, caramiphen, dithiolguanidine (DTG), and a neuronprotective kappa opioid

agonist, CI-972, excitatory aminoacid-induced or NMDA-induced neuronal excitotoxicity antagonists or mixtures thereof.

17. The composition of Claim 16 wherein the active ingredient is MK-801. 1 18. The composition of Claim 16 wherein the active ingredient

2 is dextromethoφhan.

3 19. The composition of Claim 16 wherein the active ingredient

4 is caramiphen.

5 20. The composition of Claim 16 wherein the active ingredient

6 is carbetopentane.