WO1993013419A1

WO1993013419A1 - Method of diagnosing combined cognitive/debilatory disorders

Info

Publication number: WO1993013419A1
Application number: PCT/US1992/011180
Authority: WO
Inventors: William A. Carter
Original assignee: Hem Pharmaceuticals Corp.
Priority date: 1991-12-23
Filing date: 1992-12-23
Publication date: 1993-07-08
Also published as: ZA929945B; AU3419793A; CN1074537A

Abstract

Presence of the cytokine interleukin-1 is used as a marker to diagnose debilitation, lethargy, loss of intellectual acuity, impaired overall performance, general inability to care for one's self and to diagnose the presence of Chronic Fatigue Syndrome and distinguish these conditions from psychological disorders.

Description

METHOD OF DIAGNOSING COMBINED

COGNITIVE/DEBILATORY DISORDERS

BACKGROUND OF THE INVENTION

This invention relates to procedures to diagnose severely debilitation conditions, cognitive disorders of diverse types especially, but not limited to, Chronic Fatigue Syndrome, in a patent using immunological testing procedures. The

diagnostic method is neither reliant upon nor subject to misinterpretations as with clinical observations which was the diagnostic standard of the art prior to the present invention.

Chronic Fatigue Syndrome (CSF), also known as Myalgic Encephalitis (ME), is a severely

debilitating disorder with painful symptoms

manifested in multiple organs. CSF patients have specific immunological dysfunctions characterized by overproduction of lymphokines and cytokines. As a result of severe debilitation, patients frequently report suicidal thoughts and acts.

The Center for Disease Control, Atlanta, GA, USA, has established criteria as a case definition for CFS(see Table 5). Clinical criteria currently used to diagnose CFS often prove to be unreliable, subjective and open to a considerable degree of interpretation. This invention provides for the first time a

biochemical test or assay and support for a

constellation of clinical symptoms for which

previously there was no test. Because many disorders can instigate one or more of the classic CFS symptoms, it is significant to identify for the first time the chemical substance associated with and/or causing these clinical symptoms - alone or in the aggregate.

Prior to this invention, biochemical tests and other diagnostic procedures were used to exclude the diagnosis of CAF, i.e., it was a disorder made by exclusionary testing, showing what disorders it was not, rather than what the disorder was).

The substance believed to cause, or to be an indication of such symptoms is IL-1, IL-1 has not been reported in individuals in the absence of immunological stimulation, thus the presence of IL-1 in severely debilitated patients, particularly with other performance and/or cognitive assessment criteria, serves as a marker for CSF and related severely debilitating conditions.

Interleukin-1 (IL-1) is the term for two polypeptides (IL-1α and IL-1β) that possess a wide spectrum of inflammatory, metabolic, physiologic, hematopoietic and immunologic properties. These two species of IL-1 are encoded by two separate genes, share 26% homology at the amino acid level and both bind to a single high affinity IL-1 receptor.

Although both forms of I1-1 are distinct gene products, they recognize the same cell surface receptors and share the various biologic

activities.

In general, significant amounts of IL-1 are not observed in health in most individuals.

However, there is a dramatic increase in IL-1 production, and hence blood level, by a variety of cells in response to stimuli including infection, microbial toxins notably retroviruses and human herpes virus, inflammatory agents, products of activated lymphocytes, complement, and clotting components.

Elevated IL-1 production has been reported in the following various human disease states, including:

Rheumatoid arthritis

Rheumatoid arthritis (synovial lining cells) HIV infection and AIDS

Bacterial infection

Respiratory distress syndrome (alveolar

macrophages)

Smokers (alveolar macrophages)

Coal miner pneumoconiosis (alveolar macrophages)

Alcoholic cirrhosis

Cuprophane hemodialysis

Cardiopulmonary bypass

Chronic hepatitis B

Thermal injury (burn) Reticulohistiocytosis

Sarcoidosis; tuberculosis

Obstructive jaundice

Pagets's disease and osteomalacia

Insulin dependent diabetes mellitus (newly

diagnosed)

Kawasaki's disease

Inflammatory bowel disease (mucosal mononuclear

cells)

Luteal phase; strenuous exercise (1,2)

The present invention relies more on detection of pathological amounts of IL-1 alpha in patient sera and other biological fluids and therein provides a level of diagnosis specificity since in the other disorders delineated above, the typical abnormality is in IL-1 beta production. The production and biologic activities of IL-1 contribute to the pathogenesis of certain diseases. IL-1 contributes to pathogenesis of a variety of diseases by virtue of a range of

biological functions. The reported biological effects of IL-1 include:

T cell activation

IL-2 release

NK activation

Neutiophilia

Neuro peptide release

Eosinophil degranulation

Hypotension, shock, death

Beta islet cell toxicity

Hyperlipidemia

Synthesis of collagenase and collagen and

osteoblast activation

Synovial cell activation

In the present invention, I establish the first insight between the clinical manifestations of the CSF disorder and the relative presence, or absence, of IL-1, especially IL-1 alpha.

When IL-1 prepared by recombinant DNA techniques is exogenously administered to patients, the effects reported in Table 1 have been

reported. In this table, the IL-1 dose is

indicated as ng per kg of the patient's body weight and N.D. indicates the minimum dosage causing the indicated effect has not yet been determined. For comparison, under the heading "CSF" a correlation of the same or similar symptoms as seen in Chronic Fatigue Syndrome patients is given where + = yes, - = no and N.D. is no data.

When IL-1 is present in humans, systemic symptoms include fever, myalgia, headache, lassitude and even sleepiness. Other biological effects include fever, chills, rigors, headache, increased muscle proteolysis, nausea, vomiting, increased heart rate, histamine release, depressed myocardial function, pulmonary congestion, meningeal irritation and brain parenchymal lesions.

IL-1 is but one of several lymphokines which when upset starts a cascade effect of the immune system in which the immune system eventually attacks the host itself. The presence of IL-1 may lead to the production of IL-2 setting in motion a complex, multi-step lymphokine "cascade" - as many as 11 interleukens are known to date. IL-1 acts as a trigger and plays a pivotal role in causing this lymphokine cascade. Separately, I have confirmed that the immune system is upregulated by these abnormal levels of IL-1 alpha, especially the

"intracellular immunity" characterized by the 2-5 oligoadenylate pathway and biochemical components of this pathway.

A lymphokine cascade is essential and appropriate if turned on for short periods of time, i.e., during influenza. It is inappropriate and destructive of the immune system and other tissues if chronically stimulated such as I have now delineated in untreated CFS patients. Discovery of specific processes in the immune cascade which are deranged in CFS shows event though viruses can contribute to IL-1 production, the present invention shows virus load, including both retroviruses or herpes viruses, and IL-1 are independent variables with respect to the effect on low patient performance scores.

BRIEF DESCRIPTION OF THE DRAWINGS

Table 5 is a chart listing the Center for Disease Control's case definition for Chronic Fatigue Syndrome also known as Myalgic Encephalitis; and

Figure 1 is a bar graph showing the conversion of patients meeting tho CDC defining criteria for CFS without elevated II.-1 levels to elevated IL-1 levels over a 24 week period. After the conversion to positivity, the patients manifested progressively more symptoms.

DETAILED DESCRIPTION OF THE INVENTION

IL-1 is a potential toxic substance (a function of dose and duration of dose) which causes

various physical manifestations and damage to the body. In addition, it causes confusion and

diminished intellectual competency. The inventor has now discovered IL-1 to be a surrogate marker for disease causing agent(s) and related this marker to the disease conditions. The invention provides the first reliable, immunologically-based, tool to identify those patients falling within the CDC clinical symptom-based definition of CFS who are severely debilitated and enables the clinician to initiate appropriate therapy and avoid inappropriate treatment measures, such as hospitalization in a psychiatric institute. The invention also enables the clinician to distinguish between CSF and related severely debilitating conditions from psychological disorders.

The presence of IL-1 in a patient sample such as blood or other biological fluid, in

conjunction with meeting specific medical diagnostic criteria, indicates the presence of a more severely debilitating and progressively deteriorating

condition such as CSF. The lower limit of

quantitation for this assay is 10pg/ml although lower IL-1α levels are detectable. Since IL-1α has not been reported in individuals in the absence of immunological stimulation, the presence of IL-1α in any amount was considered to be a positive. Conveniently, concentrations of 10pg/ml or greater may be used.

The procedures of this invention thus enables the clinician to distinguish those patients having chronic fatigue syndrome from those having a serious and debilitating psychological disorder. The incidence of neuropsychologic complaints is a recognized feature of CSF given in the case

definition of Figure 1. Following the CDC

guidelines for CSF and using structured psychiatric interview, investigators report a significantly higher frequency of depression in patients with CSF with about half of the CSF patents experiencing at least one major depressive episode before onset of the fatigue syndrome (3). Gold et al found that patients with CFS had significantly more lifetime episodes of major depression (73% versus 22%) and current major depression (42% versus 0%) than controls (4). Citing the higher incidence of concurrent and past psychiatric illness in CSF patents compared with other medical patients, some investigators suggest an underlying psychopathology in CFS (5) leading to the use of primarily

psychotherapeutic procedures and psychoactive medications that are often inappropriate to the situation. Similarly, patents experiencing profound psychological complaints and disorders are often misdiagnosed (CSF is not considered) and

psychotherapeutic procedures and psychoactive medications adopted in stead of more appropriate therapies designed to effectively treat the CSF patient.

Provided is a novel means of determining the presence of a debilitating organic/medical condition and/or cognitive disorder associated with an occult viral infection by ascertaining the presence or absence of IL-1. When prepared by recombinant DNA technology and injection into the human body, IL-1 is associated with a variety of symptoms similar to those individuals manifesting chronic fatigue disorders. These symptoms cause morbidity among the range of characteristic symptoms such as lack of capacity for sustained physical activity or loss of the ability to think clearly.

Elevated levels of IL-1 in a patient may be caused by viral or bacterial pathogens or can be elevated by an external pathogenic event. The acute effects of increased IL-1 levels, as for instance by injection of IL-1, are different in physiological consequences from the chronic effects. For purposes of clarity, acute means within hours or up to several days after IL-1 administration, subacute refers to a period of from approximately 1 to 6 months after IL-1 administration and, if the symptoms persist for six months or longer, the elevated IL-1 levels are deemed to be chronic and the appropriate, predictable symptoms can result within the context of the present invention.

Provided for the first time is a biochemical clarification and explanation for the greater level of patient debilitation as quantified, for example by KPS and ADL scores. The invention explains the number and relative severity of

physical findings and clinical observations

including but not limited to pharyngitis, enlarged lymph node, achy joints and muscles that are seen in a group of people who typically meet the criteria for chronic fatigue syndrome.

The relative amount of IL-1 present in the patient's blood serum is correlatable to the extent of the patient's debilitation - that is, the higher the relative amount, the greater the degree of debilitation and higher the incidence of physical findings when examined by the physician.

Of particular value is the high degree of correlation of elevated IL 1 levels to decreased KPS or physical performance scores. The patient

typically has a general feeling of malaise, feels "bad" and has poor physical performance.

Studies also indicate various viruses, notably herpes virus and retroviruses, are capable of eliciting production of IL-1 during human cell multiplication cycles in cell culture.

As reported, elevated IL-1 levels cause divergent derangement of multiple aspects and components of the immune system. In the patients studied, many of them had increased IL-1 levels and they were compared to a matched group of patients who also met certain diagnostic criteria but had low, or non-detectable, IL-1 alpha levels. The invention includes a novel elucidation of the prospective clinical courses in the two groups as a function of the IL-1 level.

In addition, an increase in the level of one or more of the following lymphokines was also reported: IL-1α, IL-4, IL-6, TNFα, TNFβ, GM-CSF, sIL2R, sCD8, and Neopterin. Not all patients had increased levels of all of these lymphokines but many patients were observed to have increased IL-1 levels. The invention also includes identification of a specificity of the elevated IL-1 level in CFS as compared to IL-4, IL-6, TNF, SCD-8 and neopterin levels.

Importantly, IL-1 in elevated quantities stimulates the production of tumor necrosis factor (TNF), a lethal cytokine which can cause brain damage and loss of other quality of life and/or basic functions. Elevated TNF levels, in turn, are implicated in cachexia (weight loss) which is often secondary to the presence of many types of solid tumors. The ability to ameliorate such weight loss leads to an improvement of quality of life for patients having tumors associated with elevated IL-1 and TNF levels. As examples, elevated TNF levels are present during disease levels including HIV and malaria. Several patients reported in this study have been observed to have reduced or impaired cerebral blood flow as determined by SPEC scanning, again a secondary manifestation of abnormal cytokine levels, especially IL-1.

The examples that follow will serve to further illustrate but not limit the invention.

EXAMPLES OF THE INVENTION

Ninety-one patients participated in a six-month study of which 38 were diagnosed as having elevated levels of IL-1 in addition to one or several clinical symptoms such as fever chills, rigors, headache, etc. as specified in Table 1, below.

Clinical symptoms of the patients beginning the study included:

Fever

Painful cervical or axillary lymph nodes Generalized muscle weakness

Prolonged fatigue

Myalgia or muscle discomfort

Migratory Arthralgia

Generalized headaches

Sleep disturbances

Neuropsychological complaints

Patients were assessed for their symptoms, physical findings (noted with an *) as well as general level of debilitation. The incidence of CFS-related symptoms as defined by the the CDC defining criteria is reported below as a percentage of the patients examined. In Table 2, the *

indicates a finding made on physical examination. The balance of complaints are reported to the physician as occurring historically.

Two highly regarded, reliable tests were used to determine the patient's condition and performance. Karnofsky Performance Scores and Modified Barthel's Activities of Daily Living were used to assess patient status and quality of their life. Each patient was evaluated and assigned a patient Karnofsky Performance Score (KPS) based upon an investigator's (medial doctor) clinical

observations and questioning of the patient or his or her spouse, companion, or custodian who was needed to care for the patient's daily need in light of the patient's severe chronic debilitation.

The KPS is a global evaluation of the patient's ability to conduct activities of daily living and is sensitive to the patient's status in chronic disease states, especially where the

patient's functional status declines, such as those associated with severe debilitation, e.g., AIDS, various neoplastic diseases, etc. (6, 7, 8). On a scale of 100 to 0 with 100 being a normal healthy individual with no evidence of disease and no complaints and 0 being death, those patients having a score of 50 or lower on the Karnofsky Performance Scale, correlate well with the incidence of positive IL-1s.

Second, to compliment the investigators global evaluation, the patient or caretaker's determination of the patient's ability to perform a broad range of specific activities routinely

encountered in daily living were recorded using a modified Barthel's Activities of Daily Living (ADL) index. The original ADL test was designed to evaluate the abilities and progress of patients with neuromuscular or muscular skeletal disorders to care independently for themselves evaluated over a series of ten categories or modules of basic self-care activities such as eating, dressing, grooming, personal toilet and functional mobility (9, 10, 11). The score assigned is based upon the patient's actual, not potential, ability to perform specific activities.

The KPS and the ADL data classify performance status and quality of life numerically. Each method independently allows evaluation of individual patient progress over time as well as interpatient comparisons. While related evaluations and assessments result, there is a correlation between KPS and ADL results. The KPS reflects a global performance estimate independent of the patient's own estimate of their own activity

levels. The correlation reflects the fact that patient responses on the ADL checklist (see below) were not the primary factor in determining their KPS scores.

The ADL is comprised of 13 individual "modules" and factor analysis (a type of statistical test) was performed to analyze the relative effect of the 13 individual ADL modules and the total ADL score. The ADL score is the average for scores in 13 modules multiplied by 20. Since there are 5 levels of performance within the component modules, the maximum score is 100 which reflects an

asymptomatic individual who can fully perform all 83 discrete activities of daily living. A 10 unit ADL score increase reflects an average increase of 0.5 unit of ability in each 13 activity modules. It also represents significant improvement in patient's ability to perform independently the routine

activities of daily living. Similarly, a 15 unit

ADL score increase represents a further 50%

improvement in patient's ability to function

independently.

The data that follow may be interpreted following the above general guidelines or the more precise references that follow, the disclosures of which are incorporated by reference.

Patients were evaluated to determine their

KPS and ADL values. The data in Table 3 were collected and averaged over three separate

determinations over a 6 week period.

Laboratory Testing--Plasma Interleukin 1α(IL-1α) concentrations were measured using a commercially available Elisa assay (IL-1 alpha ELISA). Briefly, patient plasma samples (50 ul) were incubated in sequence with mouse antibodies specific for human IL-1α, rabbit antibody to human IL-1α and enzyme-labeled goat anti-rabbit

immunoglobulin-g (conjugated with alkaline

phosphorylase). Between each incubation, unbound material was washed away and discarded. The bound specific anti-IL-1α antibody was quantitated by an enzymatic reaction resulting in a detectable color change using an Elisa reader. The measured

absorbance was proportional to the concentration of IL-1α in the test sample. A standard curve was obtained by plotting IL-1α concentrations of diluted standards versus absorbance. Patient samples and controls were processed concurrently with the standards, and sample concentrations were determined by comparison with the standard curve. The lower limit of quantitation for this assay is 10pg/ml although lower IL-1α levels are detectable. Since IL-1α has not been reported in individuals in the absence of immunological stimulation, the presence of IL-1α in any amount was considered to be a

positive. Conveniently, concentrations of 10pg/ml or greater may be used. Various other modes to measure IL-1, or its derivative products, also exist and they are also within the scope of this invention.

Ninety-one patients meeting the CDC- defining criteria for CFS were assessed for the presence of IL-1 in blood sera and elevated (10 pg/ml or more IL-1) and non-elevated IL-1 alpha values were compared to their KPS scores,

CDC-defining symptoms and physical findings. The data is as folloos:

Global performance of the 38 patients having elevated IL-lα protein level in blood sera was relatively low (median KPS 50; mean KPS 48.7) evidencing significant debilitation. An approximate 5 point difference in KPS score can reflect the difference In functional ability between someone needing custodial help "all of the time" versus someone needing custodial help "some of the time".

The natural history of patients meeting the CDC criteria for CFS without detectable IL-1 levels in their blood is if the condition is

untreated is to convert to IL-1 positivity. Figure 1 is a bar graph showing the cumulative number of IL-1 converted patients, that is, the number of patients at the beginning of the study having less than 10 pg/ml IL-1 detected in their blood serum over a period of 24 weeks which, untreated,

"converted" and were found to have IL-1 values in excess of 10 pg/ml. Patients with elevated IL-1 levels experienced a significant increase in KPS score over 24 weeks of observation.

Based on the progressive physiological deterioration secondary to elevated IL-1 alpha and the derivative biological changes, patient use of medication was compared to their respective baseline values. Consumption increased as the six month study progressed indicative of a progressive downhill clinical course. Towards the end of the study

(weeks 21-24), these patients were taking

significantly more medication of all types,

including significantly more medication specifically to relieve symptoms, to relieve CNS complaints, and to relieve pain, when compared to their consumption in weeks 1-4. These patterns of increased

consumption were significantly increased for the four medication categories.

Effects of exogenously administered IL-1 alpha on the central nervous system are widely reported in the medical literature and thus the presence of IL-1 in a patient may also be indicative of other conditions. As examples, the literature reports inflammatory edema induced by interactions between IL-1 and the neuropeptide calcitonin

gene-related peptide [see Buckley et al. J. Immunol. 146(10), p. 3424-30, May 15, 1991]. IL-1 has been reported [Quagliarello et al, J. Clin. Invest.

87(4), p. 1360-6, April 1991] as inducing meningitis and blood-brain barrier injury in the rat.

Cytokine levels in the cerebro-spinal fluid and serum of patients with multiple sclerosis have been reported [Maimone et al. J. Neuroimmunol. 32 (1) p. 67-74, April 1991].

IL-1 has been reported to induce myocardial depression in the heart [Hosenpud et al, J. Heart Transplant. 8(6) p. 460-4, Nov.-Dec. 1989] which agrees with the novel observation of depressed myocardial function observed by the inventor in patients having elevated IL-1 levels.

IL-1 has also been reported as enhancing tumor necrosis factor (TNF) which in turn increases all necrosis and accelerates hepatitis [Lin et al, Gastroenterol., 25(3), p. 339-42, June 1990]. TNF and IL-1 have been reported to inhibit Ia induction by interferon-gamma on endothelial cells from murine central nervous system microvessels [Tanaka et al, J. Neuroimmunol., 27(2-3) p. 209-15 May 1990] which agrees with the inventor's observations of patients with elevated IL-1 levels showing disruption or damage to cerebral blood flow as determined by SPEC scanning.

There is evidence that IL-1 accentuates the deposition of abnormal materials, i.e.

amyloid-like plagues, in animal brains. Such

plaques are specifically characteristic of

Alzheimer's disease [Blume et al, Neurobiol. Aging, 10(5) p. 406-8, Sept.-Oct. 1988]. Similarly, brain IL-1 has been reported to be elevated in Down

Syndrome and Alzheimer disease [Griffin et al. Proc. Natl. Acad. Sci. USA 86(19) p. 7611-5, October 1989].

The production of IL-1 by peripheral blood mononuclear cells in patients with chronic fatigue syndrome has been observed [Morte et al, J. Infect. Dis., 159(2), p. 362, Feb. 1989].

BIBLIOGRAPHY 1. Dinarello, C., Interleukin-l and

Interleukin-1 Antagonism. Blood, 77:

1627-52, 1991. 2. Dinarello, CA., et al, Interleukin-l and its relevance in patients treated with hemodialysis. Kidney Int., 33 (suppl 24): S21-S26, 1988. 3. Shafran, S.D., The chronic fatigue

syndrome. JAMA, 90:703-739 June 1991. 4. Gold, D., et al, Chronic fatigue. A

prospective clinical and virologic study. 264:48-53, JAMA 1990. 5. Krupp, L.B. et al, An overview of chronic fatigue syndrome, J Clin Psychiatry 52:10, 403-410, October 1991.

6. Capewell, S. and Sudlow, M.F., Performance and prognosis in patients with lung cancer. Thorax, 45:951-6, 1990. 7. McClellan, W.M. et al, Functional status and quality of life: predictors of early mortality among patients entering

treatment for end stage renal disease. J. Clin. Epidemiol., 44:83-9, 1991.

8. Wu, A.W., et al. Quality of life in a

placebo-controlled trial of Zidovudine in patients with AIDS and AIDS-related complex. J. Acquir. Immune Defic.

Syndrome., 3:683-90, 1990.

9. Mahoney, F.I. and Barthel, D.W.,

Functional evaluation: the Barthel Index, Maryland State Med J., 14:61-5, 1965.

10. Gresham, G.E. , et al, ADL status in

stroke: relative merits of three standard indexes. Arch. Phys. Med. Rehabil.

61:355-8, 1990.

11. Collin, C. et al, The Barthel ADL index: a reliability study. Int. Disabil. Studies, 10:61-3, 1988.

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing the presence of a combined neuroconitive/physical debilatory

disorder in a patient comprising determining the presence of IL-1 in a patient biological sample the presence of which is indicative of a combined cognitive/debilatory disorder.

2. The method according to claim 1 in which IL-1 alpha is detected in the patient's biological sample.

3. The method according to claim 12 in which the amount of IL-1 alpha is at least 10 pg per ml of the patient's blood or other biological fluid.

4. A method distinguishing a

psychological disorder from an underlying organic or medical disorder in a patient exhibiting a

diminished ability to perform the activities of daily living as assessed by the modified Barthel's Activities of Daily Living Index, exhibiting chronic debilitation or both, the method comprising

ascertaining the presence of lymphokines, including IL-1, in a patent biological sample, the presence indicating the absence of a primary psychological disorder as the major reason for debilitation and relative loss of congnitive function.

5. The method according to claim 4 in which IL-1 alpha is detected in the patient's biological sample.

6. The method according to claim 5 in which the amount of IL-1 is at least 10 pg per ml of the patient's blood.