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DIAGNOSIS AND TREATMENT OE
NEURO-COGNITIVE DISORDERS ASSOCIATED
W I TA SYSTEri I C I MM<JNOLOG I CAL MALEUNCT I ON
Cross-Reference to Related A lications
This application is a continuation-in-part of earlier
application Serial No. 07/421,596 Filed October 16, 1989.
This invention relates to procedures for diagnosing the
presence of neuro-cognitive disorders for the type
associated with systemic immunological malfunction and
distinguishing same from primary psychological or
neuropsychiatric disorders presenting similar clinical
symptoms. The invention also includes diagnosing dementias
of the type associated with immunological derangements.
Therapeutic intervention for such conditions with dsRNAs is
also described.
Summary of the Invention
Central nervous system (CNS) manifestations of memory
loss, reduction :in intelligence quotient (IQ) and loss of
cognitive skills may be associated with disturbances in
2'-5'A pathway metabolism in peripheral blood mononuclear
cells (PBMC). These disturbances may signal underlying and
clinically undetected release of lympholcines which, in turn,
contribute to a clouded sensorium. Also, often at play as
undetected retroviral infections and/or herpes infections.
The occult viral infection also contributes to the brain
pathology both directly and indirectly (by triggering
inappropriate and chronic lymphokine production). The dual
disease manifestations are diagnosed in a facile manner by
2
examination of 2'-5'A pathway components and interventive
therapy with dsRNAs which biologically neutralize the
lymphokine toxicity while reducing the intrinsic viral
burden.
I have found that certain dementias are the result of
sustained immunological activation following an unresolved
virus infection, which perpetuates the memory loss. I have
also observed malaise and depression. Elevated serum levels
of various lymphokines, such as IL-2, IL-6, TNF and
interferon which are associated with bodily defenses, and
these molecules (alone or collectively) appear to contribute
to the symptomatology.
The ill-defined etiology of dementias (Alzheimer's
disease, etc.), has contributed heretofore to a difficulty
of discovering effective treatment for this disorder.
Therefore, T developed a new strategy: first to define an
abnormal cellular phenotype in dementia patients which may
result in significantly impaired control over various
immunological derangements including viral infections,
without regard to the specific nature of the inciting
organisms, and second, to determine if the abnormal phenotpe
might be found in certain intracellular enzymatic pathways
which confer an immunomodulatory antiviral state on human
cells, including brain tissue. Such pathways are known and
include the dsRNA dependent 2-5A synthetase/RNase L pathway
and the dsRNA dependent protein ltinase pathway, These
enzymatic pathways are activated or deactivated by a variety
of animal viruses, as well as by by-products of viral
infection, such as interferon or dsRNA (double stranded
RNA). The pathways, when properly regulated, elicit
cellular resistance to a wide variety of pathogens because
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the resultant biochemical products can interfere with the
synthesis and stability of pathogen components. I refer
here to these enzymatic pathways as "antiviral" pathways,
but it should be noted that they have also been implicated
in the differentiation of immune cells. In particular,
these pathways may participate in the activation of T cells,
B cells and monocytes or their precursor cells.
I previously noted abnormalities in the 2-5A
synthetase/RNase L pathway in vivo in another chronic virus
infection of human (ref. 1). For example, acquired immune
deficiency syndrome (AIDS) caused by human immunodeficiency
virus (HIV) is accompanied by abnormally high levels of
latent 2-5A synthetase, reduced levels of bioactive 2-5A and
low to undetectable levels of RNase L (ref. 1). This
phenotype is consistent with a relatively inactive antiviral
pathway owing to lack of appropriate activation of 2-SA and
consequent absence of the product of 2-5A synthetase, 2-5A.
In my present study of dementia patients, herpes virus
was also identified with monoclonal antibodies in some
patients discussed below, while the remainder were monitored
with sera containing antibodies against several
herpeviruses. I will therefore refer to certain of the
dementia patients as "virus positive by culture", rather
than as HHV-6 positive. Novel retroviruses were also
isolated from these and other patients with similar
pathology of the CNS.
---------_-__
Detailed Disclosure of the Invention
I have discovered and hereby disclose that a
hyperactive or an aberrance in the 2-5 A synthetase/RNase L
4
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pathway exists in individuals having any chronic dementia
associated with metabolic disttarbances, defects in
neuro-cognition or chronic cerebral dysfunction (CCD). This
derangement is often accompanied by other biochemical and
gross tissue anomolies such as cerebral lesions revealed by
imaging as well as cognitive deficits such as loss of IQ and
impaired attention and abstraction ability. Further, the
natural killer cell function and NK cell phenotype are often
unusual in CCD patients.
These disorders are effectively treated with dsRNA
therapy which normalizes and favorably adjusts the
2-5 A synthetase/RNase L pathway and restores a dsRNA
deficit in the patient. Improvement is seen on both the
cellular and functional levels with memory and IQ values
restored to near or at previous levels while exercise
tolerance and oxygen consumption are increased. Patients
having chronic cerebral dysfunction were assessed using
various techniques and procedures as explained below.
Severe Chronic Dementia Stud
Fifteen patients having severe chronic dementia were
given 200-400 mg Ampligen~, a mismatched double-stranded RNA
with minimal side effects, twice a week for up to 24 weeks.
Blood levels of 30-50 ug/ml Ampligen~ were attained. Levels
of 10 Ng/ml were maintained for one hour or more in most
patients. All 15 patients presented with abnormal antiviral
2-5A synthetase/RNase L pathway components in peripheral
blood mononuclear cells. The components of this pathway
returned toward more normal values during Ampligen~
treatment. For example, bioactive 2'-5' oligoadenylage
concentrations were 3-665 times normal (average = 242)
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before treatment and declined in 13 patients to normal by 12
weeks. In 13 of 14 patients tested, evidence for reduction
of culturable virus during therapy was also obtained and in
11 patients IiHV-6 (human herpesvirus 6) and/or novel
retroviruses was detected with monoclonal antibodies. The
15 patients displayed a highly-significant increase in
performance status, as judged by improved Karnofsky scores,
and most showed sustained improvements with regard to
memory, IQ and other psychometric tests. No clinical
significant toxicities were seen. Though mild flu-like
symptoms can be observed, mismatched dsRNA therapy was well
tolerated with no patient requiring dosage reduction.
In the majority of dementia patients, the abnormalitiy
took the form of reduced levels of latent 2-5A synthetase
and elevated levels of bioactive 2-5A and activated RNase
L. This phenotype, which was markedly different from that
following HIV infection, suggested hyperactivity of the
- antiviral pathway resulting from chronic overstimulation of
the 2-5A synthetase. I describe herein clinical and
virological improvements in dementia patients having certain
of these 2-5A synthetase/RNase L antiviral pathway
abnormalities during treatment with Ampligen~, a prototype
dsRNA.
Patients - Patients had pretherapy Karnofsky
performance scores of 60 or below. Patients exhibited
persistent (or relapsing) debilitating dementia severe
enough to reduce or impair average daily activity below 50%
of their premorbid activity level for a period of at least
24 months, with no other clinical conditions that might have
produced similar symptoms. An extensive medical workup
excluded other possible causes including brain tumors,
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vascular abnormalities, situational depressions or other
primary psychiatric disorders, as well as infectious
diseases of non-viral origin. All patients gave evidence of
dementia by lower than normal scores on the Wechsler Memory
Scale and lower than expected full scale IQ scores (Wechsler
Adult Intelligence Scale - Revised, WAIS-R), base on the
patient's academic background and/or past performance on
similar tests. In addition, these individuals exhibited
greater than a two standard deviation range in scores of
individual tasks within the WAIS-R test. Typically patients
displayed an abnormal magnetic resonance imaging (MRI) scan
of the brain on 1.4 Tesla MRI scanning.
Drug Regimens - Patients initially received 200 mg
doses of Ampligen~ (HEM Research, Rockville, MD, USA);
administered intravenously twice a week for various periods
of time (see Table 1), after which the dose was escalated to
400 mg twice weekly.
2-5A Pathway Measurements and Virus Culture - These
measurements were performed on blinded samples in central
laboratories. The 2-SA synthetase/RNase L antiviral pathway
was studied as follows. 2-SA synthetase was purified from
PBMC extracts by poly(I)~poly(C) agarose affinity
chromatography (ref. 2). Only latent 2-5A synthetase was
purified by this procedure, since enzyme occupied by dsRNA
did not bind. The percent conversion of ATP to 2-SA,
measured by thin layer chromatography, was then used to
quantitate the activity of affinity-purified, latent 2-5A
synthetase. Activated RNase L in PBMC extracts was
determined by its ability to cleave ribosomal RNA (.rRNA) to
specific cleavage products, measured by electrophoresis
(ref. 3). These assays were quantitated by densitometer
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scanning and were expressed as % of 18S rRNA
cleaved/dilution of the extract. 2-5A was recovered from
PBMC extracts by extraction with ethanol. Bioactive 2-5A
was then estimated by its ability to activate purified
latent RNase L to degrade poly(U)[32ppCpJ to acid soluble
products.
Virus culture is described generally (ref. 4) for
herpes viruses. Measurements involved preparing peripheral
blood mononuclear cells from patients and culturing them for
up to 30 days, periodically examining cultures for the
presence of giant cells characteristic of herpesvirus
infection. The presence of virus in giant cells was
confirmed in some patients by immunofluorescence, using sera
from patients with relatively high titers of antibodies
against human herpevirus-6 (HHV-6). HHV-6 specific
monoclonal antibodies were also used. The number of giant
cells obtained after culture was related to virus load _in
viyo. To quantify virus load, cultures showing > 10% of
cells to be giant cells were scored 2+, those showing
between 0% and 10% were scored 1+, and those showing no
giant cells were scored 0. Decreases in virus load were
also evaluated by monitoring the frequency of negative
cultures after Ampligen~ treatment.
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dsRNA Blood Levels - Ampligen~ blood levels were
determined as described (ref. S). Heparinized blood
(100 ~1) was mixed with 162 u1 of 6 M GuSCN/.1M EDTA, pH 8
and incubated with a 3H labelled poly(C) probe. After
hybridization, the unpaired probe was eliminated with RDlase
and hybridized probe was precipitated and collected by
filtration. The amount of hybridized probe was converted to
an equivalent amount of Ampligen~ by reference to a standard
curve consisting of known amounts of Ampligen~ added to
heparinized blood and processed as described above. To
measure elimination of Ampligen~9 from blood, blood was drawn
at various times after the end of the infusion from the arm
opposite that used for infusion.
Neuropsycholoaical Testina - Each patient completed a
battery of cognitive/neuropsychological function tests,
including the Wais-R full scale IQ test, as well as the
Halstead-Reitan Neuropsychological tests, and the Immediate
Verbal, Delayed Verbal, Immediate Visual and Delayed Visual
components of the Wechsler Memory Scale. Wechsler Memory
scores reported here are Verbal score x 2 plus visual
scores. To avoid a potential "training effect", alternative
forms of the Wechsler Memory Scalse were introduced over
time.
Treadmill Exercise Test - Certain patients, though not
all, with dementia, experience profound inability to perform
routine exercise. Therapeutic improvements in CNS function
were often associated with more normal activities of daily
living including increased exercise. Treadmill exercise
tolerance testing was conducted according to the Modified
Bruce Protocol, with aerobic fitness being estimated
according to standard equations by expired gas exchange with
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02 and C02 electrodes. 02 consumption was measured at the
anaerobic threshold, in order to eliminate motivation as a
factor.
The first dementia patient to receive Ampligen~ was
chronically ill with progressive debilitation and thus was
felt to have a limited life expectancy. Specifically,
though she was formerly a professional golfer, she had been
unable to care for herself for approximately five hears, was
unable to walk 25 feet without assistance, and had
experienced increasingly frequent seizures. She was unable
to read or even follow television due to lack of short term
memory. She demonstrated NK dysfunction, virus culture
positivity, an abnormal MRI scan, markedly impaired
psychometric testing and low exercise tolerance (Table 1).
She exhibited reduction of circulating virus within 8 weeks
of starting therapy. Her Karnofsky score improved from 30
pretherapy to 80 after receiving Ampligen~ for 24 weeks.
Clinical improvements were seen in the areas of psychometric
testing and exercise tolerance (Table 1). She exhibited
reduction of circulating virus within eight weeks of
starting therapy. Her Karnofsky score improved from 30
pretherapy to 80 after receiving Ampligen~ for 24 weeks.
Clinical improvements were seen in the areas of psychometric
testing and exercise tolerance (Table 1 and Table 3). The
patient how has a sustained improvement and is leading a
normal life, fully self-sufficient, having been on Ampligen~
for 18 consecutive months.
Immunomodulatorv Virological Claanges - Including
patient 00, all 15 patients presented with a perturbed
antiviral pathway within their peripheral blood mononuclear
cells. Specifically, all 15 patients had altered levels of
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bioactive 2-5A in blood mononuclear cells. This was
corroborated by altered RNase L activity in 13 patients.
Twelve patients also had altered levels of latent 2-5A
synthetase in peripheral blood cells at the onset of
therapy. In all, 11 cases had a pretherapy antiviral
pathway phenotype characterized by: (a) low levels of latent
2-5A synthetase, accompanied by (b) elevated amounts of
bioactive 2-SA, and (c) abnormally high activity of RNase
L. I term this the "profile 1 phenotype", a phenotype which
is consistent with chronic stimulation of the pathway due to
extracellular lymphokines. Two patients, patients O1 and
06, presented with elevated levels of latent 2-5A synthetase
accompanied by elevated amounts of bioactive 2-5A and normal
or elevated RNase L levels ("profile 2 phenotype").
Elevated levels specific of later 2-5A synthetase have been
reported with infection by Epstein-Barr Virus (EBV), and
could indicate a coactive infection in patients O1 and 06.
EVB reactivation in patients O1 and 06 was supported by
serological evidence.
During Ampligen~ treatment, the 2-5A pathway returned
toward a normal phenotype. In 13 patients, bioactive 2-5A
levels had fallen to normal by 12 weeks and had declined in
the remaining two, while RNase L activities had been
significantly reduced to only 1-2 times normal (Table 2).
In most patients, latent 2-5A synthetase levels rose toward
normal by 12 weeks (Table 2), but declined again over time.
Many patients were positive for virus culture at baseline.
Virus titers were reduced during Ampligen~ treatment in most
. _ patients. In addition, patients 02, 03, 04, 06 and 10
became negative on three successive cultures after 28-40
weeks of Ampligen~. The antiviral pathway profile
associated with low levels of latent 2-5A synthetase
11
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(profile 1) was accompanied by more rapid reduction in virus
load during therapy; however virus load decreased in 10 of
the 11 patients tested during 24 weeks of therapy regardless
of the phenotype at study entry (Table 1). The ability of
dsRNA to reduce simultaneously the level of different virus
agents (retroviruses and DNA viruses) present in the
subjects contributes to the magnitude of response.
Performance Status and Exercise Tolerance - Patients
entering the study were significantly incapacitated, with an
average Karnofsky score of 47. The average score improved
to 67 after 12 weeks of therapy and to 79 in the 11 patients
evaluated at 24 weeks (Table 3).
Certain patients participated in treadmill testing
before and during Ampligen~ therapy. The length of time the
treadmill exercise could be performed and the maximum 02
consumption during exercise testing were determined. By 24
weeks in the group as a whole, treadmill duration increased
by 20% and maximum OZ consumption during exercise doubled.
Twelve of fifteen patients increased 02 consumption by more
than 50% (Table 3). One patient (O1) improved by only 10%
by 24 weeks and one patient (09), for whom only eight week
data were available, declined 15%. Since 02 consumption
measurements were taken generously at the anaerobic
threshold, improvements cannot be attributed primarily to
increased motivation, as by "lifting" a situational
depression.
Neurophvsioloaical Testina - Twelve of the 15 patients
entered the study with impaired short term memory, as
evidenced by the Wechsler Memory Scale. Wechsler scores
improved 63% in these patients during the study. The
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largest gains occurred in the verbal tasks as opposed to
visual tasks, probably because the group started with lower
verbal scores. All patients having reached 24 weeks
improved to within normal limits in the visual portion of
the Wechsler Scale. The improvements in the delayed tasks
of the Wechsler Memory Scale suggest an improvement in
interference memory, a deficit increasingly noticed in early
dementias.
Gains in IQ were also noted. Full scale IQ increased
by 12%, from 106 to 119. Performance IQ increased 19% while
verbal IQ increased 10%. Considering patients treated for
24 weeks, increases in full scale IQ appeared to be more
pronounced during the third 8 weeks (8%) than during the
first or second (2 and 0%, respectively), suggesting that IQ
gains may occur more slowly than improvements in immunology,
performance, or short term memory.
A~licaen~ Blood Levpt~ and Adverse Events - Ampligen~
blood levels were determined by molecular hybridization.
Immediately following infusion of 400 mg, blood levels
averaged 43 Ng/ml, ranging from 31-75 ~g/ml (Table 2).
Blood levels approximately half as high were obtained by
infusion of a 200 mg dose. In these and other patients the
apparent elimination rate varied; however, blood levels of
20 Ng/ml were typically maintained for at least 1 hour in
the majority of patients.
No clinically significant side effects were aeon in any
of the patients during the study. One patient developed
iron deficiency anemia secondary t~ multiple phlebotomies
and required the institution of iron supplementations. The
majority of patients reported increase in flu-like symptoms,
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including myalgias and headaches. There was not evidence of
alternation of coagulation, blood chemistry, white blood
cell count, liver and renal function, or other abnormalities
throughout the course of this study.
Patients displayed signs of clinical improvement,
performances and improved cerebral function. The earliest
improvements were related to normalization of the 2-5A
synthetase antiviral pathway and NKiil+ cell numbers as well
as reductions in apparent virus load in peripheral blood
cells. In one patient (05) who received Ampligen~ for only
8 weeks, virus titers did not fall during the immediate
study period. This patient also exhibited delayed
correction of the antiviral pathway abnormality. Patient O1
exhibited improvement in Karnofsky score during the first 24
weeks, deteriorating in other categories. This patient did
not have the same 2-5A synthetase abnormality which
characterized 13 of the 14 other patients. Patient O1
improved after 24 weeks. At 32 weeks, his IQ was 138 and
his C02 uptake during treadmill testing was 1.56 1/min. Two
patients who voluntarily discontinued Ampligen~ have now
deteriorated as noted by falling Karnofsky scores and
decrease in exercise tolerance and neurocognition.
The patients studied here presented with significantly
altered levels of bioactive 2-5A and changes in latent 2-5A
synthetase and activated RNase L which suggest altered
metabolism of their intraccellular enzyme pathways. I have
observed such a "hyperactive" phenotype to be caused by
by-products of virus infection, namely by chronic
overproduction of inciting cytokines. Since only a small
proportion of blood mononuclear cells are likely to be
directly virus infected in these patients, hyperactivation
14 ;3 a'. .~~' ,~ ;:? f .
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of the pathway by viral infection itself is not likely. In
any event, altered metabolism of the 2-5A synthetase/RNase L
pathways is a useful marker for identifying patients with
chronic dementias unrelated to primary psychiatric or
cerebrovascular disturbances.
After Ampligen~ therapy, bioactive 2-SA levels returned
to normal as did levels of activated RNAse L. This result
is secondary restoration of more normal cytokine production
or changes in cytokine utilization of blood cells. Also the
activity of the 2-SA synthetase/RNase L pathway is down
regulated by other dsRNA activated protein kinase pathway.
Improvements in clinical symptoms accompanied
virological improvement. Improvement in Karnofsky scores,
Wechsler memory test, performance and IQ scores were noted
at the earliest times analyzed and improvements continued
for the duration of the study. The more dramatic gains in
Karnofsky scores appear to have been made after doses were
elevated to 400 mg of Ampligen~. The average Karnofsky
score at entrance, 47, described patients incapable of
working and requiring considerable assistance in carrying
out daily activities at home. The Karnofsky score at 80 the
plateau during the study period, described patients able to
carry out normal activities at work and home with effort,
but without assistance. Wechsler memory test showed the
greatest gains relatively early (16 weeks), whereas IQ test
scores tended to improve later.
In several areas, improvements observed during
Ampligen~ therapy were statistically significant during the
~4 weeks. Reduction in biaactive 2-SA and activated RNase L
were significant at the p<0.0001 level. Improvements in
r, a r? .
15 ;~ ~~'~,'°a
parameters associated with reduction of dementia associated
fatigue were highly significant. Improved performance on
the treadmill was significant at the p<0.01 level at 16
weeks and p<0.001 level at 24 weeks. Improvements in
Karnofsky scores were significant at the p<0.001 level at 16
weeks and p<0.0001 at 24 weeks. Improvements in the
Wechsler memory testing were significant at 16 and 24 weeks
(p<0.02). Full scale IQ increases were also significant.
Most patients displayed the same dramatic improvement
in lifestyle as was seen with the first case. Many were
unable to care for themselves and, in fact, were unable to
remember or to ambulate without assistance prior to
therapy. By 24 weeks, many that stayed on therapy were
Leading a more normal lifestyle. Improvements occurred
while patients were receiving Ampligen~. Furthermore, two
patients who have discontinued Ampligen~ for at least two
months have deteriorated neurocognitively.
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TAHI.E 1 - PJ~TIENT' 00
T~st
Pre Postc
Treadmill
duration 1.5 min 9 min
02 consumption 1.15 units 2.36 units
Karnofskya 30 80
Wechsler memoryb 0.5 2.75
Full Scale IQ 88 134
Halstead-Reitanb
hand tapping 0 3
Trails A 0 3
Trails B 0 3
Wais-R
information 11 16
digit span 8 16
vocabulary 12 16
arithmetic 5 14
similarities 9 12
block design 5 10
digit symbol g
a
- reconstructed from clinical charts
b
- 0 = severe, 1 = moderate impairment, mild
2
impairment, 3 = within normal limits
c
- 10 months of therapy
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?ABUE 2 - VIROLOGY AiD Al09LICEH1 BLOOD LE(S
pt wka a! Blood Antiviral pathuay
200 rg Ampligen
H goal 2-5A 2-5A RHaae L Virus
synth. culture
re ost re oat re oat re oat
00 8 -- 0.15 7.20 165 33 6 1 _- -_
01 15 38 3.51 2.40 665 6 1 2 2 1
02 14 52 A.45 0.30 67 1 19 1 2 Oc
03 15 47 0.30 0.60 665 1 40 1 2 0.5c
04 24 -- 0.20 0.40 50 1 1 2 2 0,5c
05 10 36 0.25 1.1 200 1 13 1 1 2
06 10 42 1.57 0.9 85 1 20 2 2 0.5c
07 9 31 0.25 0.5 335 1 20 1 2 0.5
08 9 45 0.35 0.9 16 1 19 1 2 1.5
09 9 48 0.10 1.1 665 1 30 1 2 1.5c
10 9 52 0.25 0.6 165 1 30 0.5 2 0.5
11 2 35 0.50 0.56 335 1 30 0.2 1.5 0.5
12 2 75 0.40 1.37 3 1 3O 0.3 1 0.5
13 2 " 0.45 0.83 50 1 10 1 2 1
14 2 0.10 0.28 167 1 30 2 2 1.5
a
Z Fraction of noraal average, post t 12 uks. SiAilar results were obtained at
24 wka.
b
Calculated as described in methods, virus, culture values were on sverape of 2
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at 20 and 24 Nmeka during treatment Ipoatl, patients 11-14 ~~poat~~ values
repaesent the leaf tuo determinations, lakan betNOen 8 and 16 ueeka.
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19
Chronic Cerebral Dysfunction Study
I also studied group of patients suffering from an
organic brain syndrome characterized by cerebral dysfunction
were studied, some for more than 15 months. Following an
infectious-like episode, they developed a novel
post--infectious immune dysfunction characterized by
progressive mental deterioration, associated with memory
lapses, occasional seizures and loss of higher mental
abilities (the so-called cognitive functions). Magnetic
Resonance Imaging (MRI) of the brain, performed on patients
complaining of cognitive problems, showed a variety of
abnormalities of high signal intensities, including large
patchy areas consistent with edema or demyelination.
Peripheral blood cells showed enzymatic deficiencies in a
novel antiviral pathway termed the double-stranded (ds) RNA
linked 2-5 A/RNase L pathway. Treatment with an exogenous
source of dsRNA caused improvement in cognitive function,
performance, MRI and loss of the 2-5 A pathway deficit.
Chronic cerebral dysfunction (CCD) may occur in
virtually any age group and is often associated with a post
infectious disease episode thought to be viral in nature.
Chronic cerebral dysfunction can occur following
mononucleosis (ref. 6), brucellosis (ref. 7) or herpes-type
6 infections as well as other factors.
Loss of Coanitive functions - As with the study reported
above, a variety of neuropaychologic instruments are
available and I have used them to study and define a subset
of patients with chronic cerebral dysfunctions. A typical
test battery used has included the Wechsler Adult
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Intelligence Scale Revised (WAIS-R), the Wechsler Memory
Scale, Immediate and Delayed (WMSj, the Minnesota
Multiphasic Personality Inventory (MMPI) and similar tests.
Patients with CCD typically have test scores which are 2
standard deviations or more below the mean; some patients
have an average of all scores which are one standard
deviations or more below the mean and an occasional patient
has multiple scores which are at least 4 standard deviations
below the mean. Often I also observed "abstraction ability"
and their performance on "set switching tasks" to also be
impaired. Memory impairment seems to be secondary to a
difficulty with attention and abstraction. Several patients
were unable to perform such simple tasks as to balance their
check books or place a key to the front door into the lock
in the right configuration. These measurable and consistent
cognitive deficits are unlikely to be caused by malingering
or purely psychologic reasons since they often are
associated with a downhill course in which tlae patient
eventually becomes bed-ridden and almost moribund. I have
also discovered a novel biochemical defect associated with
this disease.
21
Magnetic Resonance Imaging of the Brain - Most of the
patients from whom MRI scans were obtained complained of
cognitive problems. The MRI scans for this study were
generally performed using a 1.5 T superconducting Signs
Magnetic Resonance Imager manufactured by the General
Electric Company.
77% of the patients examined had abnormal MR scans:
primarily punctuate areas of high signal intensity, and
sometimes larger patchy areas of high signal intensity,
consistent with focal edema and/or demyelination. The
subcortical white matter was,affected most often, but
lesions were seen in the deep white matter also.
There was a correlation between the anatomic area and
the clinical presentation: for example, one. patient with
ataxia had lesions involving the cerebellum, nine patients
with visual symptoms had lesions involving the occipital
cortex, one patient with paresis had a lesion involving the
contralateral internal capsule. In the several instances
where MRI scans were repeated, lesions persisted until I
intervened with dsRNA therapy.
NK (Natural Killer) Cell Phenotype and Function An
Immunoloaical Derangement - The NK cell phenotype and
function are often unusual Phenotype: The usually
predominant NKH1 + T3 subpopulation (a subtyping of
lymphocytes determined with monoclonal antibodies) can be
diminished, but was normal in control subjects including
patients with situational depressions of a transient nature
which are often improved by mood elevating drugs, change in
environment, exercise, etc. Also, NK cell function
w (cytolytic activity against various target cell lines) was
~y Jib ,'~ a fv ;
$.j :.~ ", d '.': ~.
22
generously reduced in patients but usually not in the
control subjects. This defective cycolytic activity was
particularly prominent after stimulation with interleukin-2,
and when the target cell line was an (Epstein-Barr virus)
EBV-infected cell line (LAZ 388). I have now performed the
first serial studies on such patients and observed that the
phenotypic and functional abnormalities in NK cells changed
as the patients' clinical course improved following
treatment with a source of exogenous dsRNA.
Antiyiral Defenses and Virologic Studies - I have also
observed that the majority of these patients are infected
with herpes-6, a relatively new class of herpes virus only
recently described (ref. 4, discussed above), and never
before associated with loss of mental capabilities. Earlier
studies found the virus in cancer patients. The virus has
been variously called HBLV (human B cell lymphocyte virus)
or HHV-6 (human herpes virus-6). The main features of this
virus are its icosahedral symmetry with about 162 capsomers
and a lipid membrane. Infected cells develop into large
refractive cells and some patients may have up to 20-30% or
more of their circulating lymphocyte cells (a certain type
of lymphocyte of bone marrow or thymus lineage) infected.
Following dsRNA therapy for several months, the percentage
of infected cells falls dramatically and is associated with
clinical improvement in cognitive abilities. I have also
found EB (Epstein-Barr) virus in many of these patients as
well as a novel retrovirus similar (though different
genetically) to HIV, a retrovirus causing acquired immune
deficiency or AIDs.
Specimens from several patients I have studied indicate
the intriguing possibility of "pseudotyping" as a means of
23
spreading this disease. By pseudotyping, I refer, for
example, to the coat of a herpes type 6 virus surrounding
the genetic material for a retrovirus. This would give rise
to a xiovel form of contagion where the infectious agent
spreads epidemiologically through the population like a
herpes virus but the genetic information being spread is
actually that of a retrovirus. I have utilized a variety of
genomic probes (ref. 8), coupled with serological and
morphological considerations (ref. 9), to put forward this
novel mechanism of biological spread leading ultimately to
mental deterioration and morbidity.
I have also studied the 2-5 A/RNase L pathway in
peripheral blood lymphocytes of 10 patients having CCD with
both MRI lesions and neurological deficits associated with a
decreased intelligence quotient. In the first phase of the
study, I looked at the antiviral defenses of patients with
only intelligence quotients (IQs) equal or less than 105
whose prior occupation strongly suggested a higher premorbid
IQ; in many patients I was able to locate tangible evidence
revealing a higher IQ either months or years before the
present cerebral illness.
Eight out of ten patients had a hyperactive or aberrant
RNase L which caused an unnatural (unexpected) cleavage of
'' ribosomal RNA in a typical polyacrylamide gel (PAGE) assay.
Also, these eight patients had below normal levels of 2-5A
synthetase before therapy. These patients responded most
dramatically to dsRNA therapy in terms of improvement in IQ,
MRI, etc., whereas the two patients without the biochemical
defect had marginal responses.
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Diagnosis is conveniently conducted from a sample of
the patients blood to analyze the peripheral blood cells for
enzymatically deficient 2-5'A using the procedures described
by Carter et al in The Lancet, (ref. 1). The aberrance once
noted is compared to the test results of otherwise healthy
individuals, and is corrected by the exogenous
administration of dsRNA, preferably a mismatched dsRNA, to
improve the patient's clinical condition and restore
cognitive abilities. During and at the conclusion of
therapy, the patient is followed to ascertain his/her
improvement on a cellular level, which usually precedes the
clinical improvement by several weeks, and to determine the
amount of dsRNA, if any, needed to maintain a normal 2-5'A
synthetase/RNase L pathway and restore/maintain the
patient's cognitive skills.
Diagnostic Procedures -
The in yiyo concentration of 2'-5'A molecules in normal
individuals and subjects with chronic cerebral dysfunction
is assessed as follows: Ethanol-soluble fractions of patient
samples (Ficoll-Hypaque-purified peripheral blood
lymphocytes) were analyzed for their 2-'S'A content in
2'-5'A core-cellulose assays (affinity chromatography) with
32
poly U-( PJ-pCp. In this assay, the ability of
2'-5'A-activated RNase L to hydrolyze poly(U) is used to
determine the concentration of functional 2'-5'A.
Reference values were established by testing 15 normal
subjects with no recent history of viral infections as
evidenced by lack of fever, absence of constitutional
symptoms, rashes, etc. Concentrations of their lymphocyte
2'-5'A levels were determined using calibration curves
obtained with authentic 2'-5'A molecules.
CA 02060489 2001-12-07
26
The dsRNA may be a complex of a polyinosinate and a
polycytidylate containing a proportion of uracil bases or
guanidine bases, e.g., from 1 in 5 to 1 in 30 such bases
(poly~I ~ poly(C4-29x>U or G)).
The dsRNA may be of the general formula
rIn~r(C11-14'U)n or rIn~r(C12,U)n. Other suitable examples
of dsRNA are discussed below.
By "mismatched dsRNA" are meant those in which hydrogen
bonding (base stacking) between the counterpart strands is
relatively intact, i.e., is interrupted on average less than
one base pair in every 29 consecutive base pair residues.
The term "mismatched dsRNA" should be understood
accordingly.
The mismatched dsRNAs preferred for use in the present
invention are based on copolynucleotides selected from poly
(Cn,U) and poly (Cn,G) in which n is an integer having a
value of from 4 to 29 and are mismatched analogs of
complexes of polyriboinosinic and polyribocytidilic acids,
formed by modifying rIn~rCn to incorporate unpaired bases
(uracil or guanidine) along the polyribocytidylate (rC )
n
strand. Alternatively, the dsRNA may be derived from
poly(I)~poly(C) dsRNA by modifying the ribosyl backbone of
polyriboinosinic acid (rIn), e.g., by including 2'-O-methyl
ribosyl residues, The mismatched complexes may be complexed
with an RNA-stabilizing polymer such as lysine and
cellulose. These mismatched analogs of rIn~rCn, preferred
ones of which are of the general formula rIn~(C11-14'U)n or
rInl~r(C29,G)n, are described by Carter and Ts'o in U.B.
Patents 4,130,641 and 4,024,222. The dsRNA described
,/ ~3 ~ 1j ~~
1~J ':I ~~ ~.Lr '.l Y~
therein generally are suitable for use according to the
present invention.
Other examples of mismatched dsRNA for use in the
invention include: -
Poly (I) . Poly (C4,U)
Poly (I) . Poly (C7,U)
poly (I) ~ poly (C ,U)
13
Poly (I) ~ Poly (C22'U)
poly (I) ~ Poly (C20'G)
Poly (I) ~ Poly (C29°G) and
Poly (I) ~ poly Cp23 c'p
Another class of dsRNAs suited to the practice of this
invention are short dsRNAs of defined structure, for example
oligonucleotides of the formula:
5'lock-(I)n-lock 3'
3'lock-(C)m-lock 5'
where m and n are each more than 5 and less than 100, I is
inosine monophosphate, C is cytidine monophosphate, and
where the locks in one strand are complementary to locks in
the opposite strand, or an oligonucleotide of the structure:
5'lock-[(I)xAjj-lock 3'
- 3'lock-[(C)yUjk-lock 3'
where x and y are each more than 5 and less than 25, j and k
each at least 1 and less than 10, I and C are as identified
above, A is a nucleotide which is not I, and U is a
nucleotide which base pairs with A.
CA 02060489 2001-12-07
28
Alternatively, the short oligonucleotide may have the
structure:
5'(I)n-hinge-(C)m3'
where n, m, I and C are as defined above.
These oligonucleotides may have substitutions in one
strand not complementary to nucleotides in the opposite
strand. Preferably these oligonucleotides are stabilized by
internal registers of complementary heteropolymer and
desirably the lock or hinge or both contain regions of
complementary heteropolymer. These oligonucleotides
desirably have single-stranded tails. These
oligonucleotides are described in more detail in U.S. patent
application Serial No. 07/181,385 filed April 14, 1988 to
Gillespie and Crater and in corresponding PCT/US89/02172;.
In addition, 2'-5'A concentration and molecular size
may be quantitated by high pressure liquid chromatography
(HPLC). Also, ribosomal RNA cleavage assays may be used to
assess biological functionality (activity) of the
2'-5'A-synthesized by the patient in vivo or to determine
the level of activated RNase L in patient samples.
Peripheral mononuclear blood cells are the preferred cells
for analysis.
Patients having chronic cerebral dysfunction are
treated with intravenous infusions of 200 to 700 mg of
rI~r(C11-14'U) as required, e.8., once a week to as often as
daily and 2'-5'A enzyme levels increase in association with
29
clinical improvement. The amount of dsRNA administered and
the frequency of administration will be guided by the 2'-5'A
synthetase levels measured in conjunction with the patient's
clinical improvement, particularly return of cognitive
functions. Amounts of dsRiJA administered will provide a
level of from 0.01 to 1,000 micrograms of dsRNA per
milliliter of the patient's systemic blood circulation
immediately following administration measured at a point
distal from the point of infusion.
s" ~''' ~ ' p 9
3 p ~, ij ~ ~~ t2 v.J
References
(1) Carter, W.A., Brodsky, I., Pelligrino, M.G. et al,
Clinical, Immunological and Virological Effects of
Ampligen, a Mismatched double-stranded RNA, in Patients
with AIDS or AIDS-Related Complex, 1987, The Lancet,
June 6: 1286-1292
(2) Kariko, K., Sobol, R.W., Suhadolnik, L. et al,
Phosphorothioate Analogues of 2',4'-oligoadenylate,
Enzymatically Synthesized 2'5'-oligoadenylate Dimer and
Trimer: Unequivocal Structural Assignment and
Activation of 2',5'-oligoadenylate Dependent Nuclease,
1987, Biochemistry 26: 7127-7135
(3) Wreschner, D.H., James T.C., Silverman, R.H. et al,
Ribosomal RNa Cleavage, Nuclease Activation and 2-5
(ppp(A2'p)n A) in Interferon-Treated Cells, 1981, Nue.
Ac. Res., 9: 1571-1581
(4) Salahuddin, S.Z., Ablashi, D.V., Markham, P.D. et al,
Isolation of a New Virus, HBLV, in Patients with
Lymphoproliferative Disorders, 1986, Science, 596-603
(5) Strauss, K.I, Strayer, D.R. and Gillespie, D.,
Detection of poly(I):poly(C12,U), Mismatched
double-stranded RNA Intravenous Infusion, 1990, J.
Pharmacy and Pharmacol
(6) DuBois, E.E, et al, Southern Med. Journal, Vol. 77, pp,
1376 (1984)
,,
~~a~ø~l~i=~A~3
31
(7) Imborlen, J.B. et al, Archives Internal Medicine, Vol.
103, pp. 406 (1959)
(9) Josephs, S.F. et al, Science, Vol. 234, pp. 601 (1986)
(9) Ablaahi, D.V. et al, Nature, Vol. 329, pp. 207 (1987),
