CA2233987A1 - Materials and methods for treatment of plaquing diseases - Google Patents

Abstract

Methods and compositions are provided for alleviation of disease states involving plaque formation, such as are manifested in Alzheimer's Disease and other amyloid disorders, and arteriosclerotic disease. Methods for the treatment of herpes virus infections including chronic fatigue syndrome by administration of thimerosal are further provided by the invention.

Description

W O 98/05350 PCTrUS97/14005 MATERIALS AND ME'l~Ol~S FOR TREA'rMENT
OF PLAQUING DISEASES

- This is a contiml~tion-in-part of U.S. Application Serial No.
08/689,528 filed August 8, 1996 which is a co~ ;on-in-part of U.S.
Application Serial No. 08/249,175 filed May 25, 1994, which is a co"Li~luaLion-in-part of U.S. Application Serial No. 07/874,719, filed April 27,1992, which is a conli-~u~lion-in-part of U.S. Patent Application Serial No.
07/598,383, filed October 16, 1990, which is a co.~ n-in-part of U.S.
Application Serial No. 07/514,021, filed April 27, 1990.

~;LD OF 'l~l~; lNVENTION
The present invention relates to methods and materials for the treatment of diseases involving plaque fOIIIIdLiOn influrlin~ arteriosclerotic 15 diseases and hypertension and more specifically to materials and methods for the tre~tment of atherosclerosis. As a further aspect of the present invention, materials and methods are provided for the treatment of herpes virus infections including but not limited to Herpes simples types 1 and 2, Epstein-Barr virus, cytomegalovirus, Herpes zoster and further for treatment of chronic fatigue 20 syndrome BACKGROUND OF l~ INVE~ON
~ llul~r skeletal systems have three distinct l~lt~tr~lrtll~l fc~lul~;s, microtubules, interm~Ai~te f l~ment~ and microfil~mçnt~, all of 25 which are fibrous macromolecules associated with the cent~al nervous system (CNS~. Neuronal intermediate filaments, defined as neurofil~m~.n~
(co..~ amyloid beta protein constructs), are distinct from other intermeAi~te fil~mP.nts found in the cells of the centIal nervous system. R.D.
Goldman, A. Mil~t~ , J.A. Schloss and M.J. Yerna, Annu. Rev. Physiol., 41, p. 703-722 (1979~; R.J. Lasak, ~eurosci. Res. ProgramBull., 19, p. 7-32 W098/05350 PCTnUS97/14005 (1981); R.J. Lasek and M.T.. Shelanski, N'eurosci. Res. Program Bull., 19, p. 3-153 (1981); C.A. Maretta, ed., ~eurofil~ments (1983); M.L. Sh~J~nrl~i and R.K.H. Liem, J. Neurochem., 33, p. 5-13 (1979). Neurofilaments are composed of three proteins with molecular weights of 20û,000, 150,000 and 5 70,000 daltons. B.H. Toh, L.J. Gibbs, Jr., D.C. G~jd~ls~l~, J. Goudsmit and D. Dahl, Proc. ~atl. Acad. Sci. USA. An acdditional 62,000 dalton protein is also affiliated with the above-mentioned proteins. Such proteins are associated with slow axoplasmic transport. P.N. Hoffman and R.J. Lasek, J. Cell Biology, 66, p. 351-366 (1975).
.AI7h~imer's Disease, and other amyloid associated m~ liP.s including senile c~e~ , Down's syndrome, Pick's disease, progressive .,u~ldlluclear palsy, multiple sclerosis and others, are char~cteri7e~1 by the presence of one or more fused fibrils of l~lilive amyloid beta proteins or other similar amyloid residues such as paired helical filaments, neurofibrillarytangles, neuritic plaques, amyloid plaques and cerebrovascular amyloidosis.
B.H. Anderson, D. Breinberg and M.J. Downes, N'at~re, 298, p. ~86 (1982). These paired helical fil~ment~ are inr1i~ uishable immlln~logically and chemic~lly from normal neurofil~ment~ and share many of the same ~.lolt;inaceous epitopes. B.H. Anderson, D. B~c~ll)el~ and M.J. Downes, Nature, 2~8, p. 84-86 (1982); B.H. Toh, L.J. Gibbs, D.C. Gajdusek, J.
Goudsmit and D. Dahl, Proc. Natl. Acad. Sci. USA; K. Iqbal, I. Grundke-Iqbal, H.M. Wi.~ni~ki and R.D. Terry, Brain Res., 142, p. 321-332 (1975).
It has been suggested that they ~ r~r~ with axonal t~ansport. P.N. Hoffman and R.J. Lasek, J. Cell. Biol., 66, p. 351-366 (1975); J.W. Griffin, P.N.
Hoffman, A.W. Clark, P.T. Carroll and D.L. Price, Science, ~, p. 633-66 (1978).
Using a cDNA clone of the gene encoding amyloid beta protein as a genetic probe, it was shown that the gene is located on chromosome twenty-one and is ~ ed in many tissues of the body. D. Goldjaber, M.I.
Lerman, O.W. McBridge, U. Suffiotti and D.C. Gaidusak, Science, 235, p.

W O 98/05350 PCT~US97/14005 77-780 (1987); R.E. Tanzi, J.F. Gusella, P.C. Watkins, G.A.P. Bruns, P.
St.George, M.L. Vankeuren, D. Patterson, S. Pagan, D.M. Kurnit and R.L.
Neve, Science, 235, p. 880-884 (1987). Q~ iQn of amyloid beta protein e,~ ssion, as seen by its mRNA levels using the cDNA probe, has revealed S that its level of expression in brain tissue of Al7hPimer's patients was not above that seen for other tissues outside the central nervous system. Such a finding was of interest to lc;se~ c~ when noting that amyloid plaque formation only occurs in the brain. R.E. Tanzi, J.F. Gusella, P.C. Watkins, G.A.P. Bruns, P. St.George, M.L. Vankeuren, D. Patterson, S. Pagan, D.M.
Kurnit and R.L. Neve, Science, 235, p. 880-884 ~1987).
Amyloid beta protein is obtained through conventional means known in the art and has been charactelized in various reports. A.S. Cohen and E. Callcins, Nature, 183, p. 1202 (1959); A.S. Cohen and E. Cs~lkin~, J.
Cell Biology, 21, p. 481 (1964); A.S. Cohen, E. CaLkins and C. Levens, Am.
J. Pathol., 35, p. 979 (1959). More recent work is Illanife~l~;d by D. Caspi, M.C. Baltz and M.K. Pepys, Mol. Biol. Med., 3, pp. 387-407 (1986); and D.
Caspi, M.C. Baltz and M.K. Pepys, Mol. Biol. Med., 3, pp. 409-424 (1986).
Amyloid beta protein exists in various structural forms. The amyloid beta protein that has been w~l~e~ Pnt~lly used and as referred to herein in terms 20 of any specific embo~limentc constitutes a mixture of such forms. It is to beunderstood that within the scope of the present invention, it is col~le,ll~latedthat any of the various forms of amyloid beta protein may be used.
Amyloid beta protein from the brain has been cDNA cloned and shown to contain a unique twenty amino acid N~2-terminal s~ue-nce.
25 Glenner, G.G. and Wong, W., Biochem. Biophys. Res. Comm., 122, No. 3, pp. 1131-35 (1984); D. Caspi, M.C. Baltz and M.K. Pepys, Mol. Biol. Med., 3, pp. 409-424 (1986); Goldgaber, D., Lerman, M.I., McBridge, O.W., Saffiotti, U. and (T~ s~k, D.C., Science, 235, pp. 777-80 (1987).
It has been observed that a buildup of abnormalty olE~ni~
30 amyloid beta protein in brain tissue is manifested in ~l~hP~imer~s Disease. See W098/053~0 PCTrUS97/14005 Dennis J. SeL~coe and Carmela R. Abraham, "Isolation of Paired Helical fil~ment~ and Arnyloid Fibers from Human Brain," 134, Methods in Immunology, 388-404 (1986). The fact that there is an accl-m~ tion of beta amyloid protein in the brain in ~l7heimer patients has been demonct~tP~I by 5 post mortem analysis of brain tissue that manifest a concentration of amyloid beta protein as part of an accllm~ tion of parallel filaments or neural fibrillatory tangles in the brain that appear ch~r~cteristic of A17hP.imer victims, along with neuritic plaque and cerebral v~c.ul~tory amyloidosis.
The presence of amyloid beta protein in fibrils and plaques in 10 Alzheimer's Disease, as well as other CNS disorders, has been s~lggest~ to - be a result of a clegr~ tiQn product of the normal neurofil~mPntc, D.
&oldjaber, M.I. T Prm~n, O.W McBridge, U. Suf~lotti and D.C. ~ k, Science, ~, p. 77-780 (1987); R.E. Tanzi, J.~. Gusella, P.C. Watkins, G.A.P. Bruns, P. St.George, M.L. Vankeuren, D. Patterson, S. Pagan, D.M.
15 Kurnit and R.L. Neve, Science, 235, p. 880-884 (1987); M. Baudry, B.R.
Dubrm, L. Beasley, M. Leon and G. Lynch, Neurobiol. Aging, 7, p. 255-260 (1986); G.&. Glenner, Arch. Pa~h. Lab. Med., 107, p. 218-282 (1983); or possibly due to improper metabolism of byproducts. Further breakdown products of amyloid beta proteins from neurofil~mPnt~ have also been 20 observed in amyloid plaques along meningeal vascular walls and i,lLlacullicalblood vessels. S. R~hm~nyar, E.J. Williams, F.B. Johnson, S. Young and D.C. (~ s:lk~ J. Comp. Path, 95, p. 1-5 (1985); M.E. Bruce and H.
Fraser, Neuropathol. Appl. Neurobiol, 1, p. 189-207 (1981); M.E. Bruce and H. Fraser, Neurophathol. Appl N'eurobiol., 7, p. 289-298 (1981); G.G.
25 Glenner and W. Wong, J. Q-laldnla and G.G. Glenner, Proc. Natl. Acad.
Sci., 82, p. 8729 (1985); D.J. SeL~coe, C.R. Abraham, M.B. Podlisky and L.K. Duffy, J. Neurochem., 46, p. 1820 (1986).
During the mid-1960's, Solomon & Moos spec~ tP~ that there was a close integration between immllnr~logical function, the central nervous 30 system, ~ycho~hysiological factors (emotions), and disease, both physical and W 098/05350 PCTrUS97/1400S

mental. G.F. Solomon and R.H. Moos, Arch. Gen. Psychiatry, 11, p. 657-674(1964). The i~e~ ion of those systems was initially suggested through ~ observation of the presence of abnormal imml~nnglobulins in sch~;ophrenic patients. G.F. Solomon and R.H. Moos, Arch. Gen. Psychiatry, 11, p. 657-674 (1964); J.G. Knight, Loncet, 82, p. 1073 1076 (1982); W.J. Fessel and M. Hirata-Hibi, Arch. Gen. Psychiatry, 9, p. 601-613. These immllnP
aberrations (termed ~l-to~ntihodies), which seemed to target certain body cellular structures, G.F. Solomon, Psychoneuroimmunology, p. 259-278 (1985); G.F. Solomon and R.H. Moos, Psychosom. Mod., 27, p. 135-149 (1981), ~uppo~led the concept that there is a close co,l-.l.u.~ication between the - CNS and the immune system. For in~t~n~e, met-enkephalin is a nt;ulo~ cmittPr in the CNS and is a product of activated T-helper cells. G.
Zurawaki, M. RenP,flik, D.J. Kamb, J.S. Abrams, S.M. Zurawaki and F.O.
Lee, Science, 232, p. 772-775 (1986).
The app~allce of 71n~o~ntihodies specific to the CNS
neurofl~men~c in patients with ~1~hPimer's and other CNS disorders suggests that the body's i.. -"lP- system may play a role in the disease process. S.
R~hm~nyar, R.K.H. Liem, J.W. Griffin and D.C. Gajdusek, J. Neuropathol.
E~p. Neurol., 53,p.85-90 (1984);S. R~ y~ M.C. Moreau-Dubois, P.
Brown, F. Catala and D.C. Gajdusek, J. Neuroimmunol., 5, p. 191-196 (1983); T.S. li7an, J. Casals and M.D. Yahr, J. Neurol. Sci., 59, p. 341-347(1983). The ~ ;bodies against normal CNS neurofi1~mPnt~ react with the paired helical filaments in n~ul.,r,l,lillary tangles ch~r~çt~ristic of Al,l..-i...e.'s Disease. D. Dahl and A. Bignami, EXp. Neurol., 58, p. 74-80 25 (1978); M.E. Bruce, J. Neuropa~ol. Exp. Neurol., 37, p. 595, abstract (1978) Animal models for these CNS disorders, which are int7uced with al~ .. chloride or B,B'-iminodipropionitrite (IDPN) to form paired helical fil~mt-ntc in neurofibrillary tangles, also react with antibodies directed CA 02233987 l998-04-06 W O 98/053S0 PCT~US97/14005 against CNS neurofil~ment~. J.W. Griffen, P.N. Hoffman, A.W. Clark, P.T.
Carroll and D.~. Price, Science, 202, p. 633-665 (1978).
Control of such au~o;...~ e reactions may lead to the alleviation of ~y~ lollls manifested by such reactions. Over the past two 5 ~e~ ~-lPs, a body of clinical lileldlul~ has accum~ tPfl relating to the treatment of autoi~ luuc disease (or, more a~p,~ iately, rli~P~es reflecting immnnP
dysfunction) using a technique called provocative-neutralization therapy.
Miller, Ann~ls of Allergy, ~8, p. 185-191 (1977); Miller, Irans. A~n. Soc.
Opth. & Otol~r. Allergy, 14, p. 159-168 (1974); Miller, Clinical Medicine, 81, p. 16-19 (1974). In short, this method, which is commonly employed for allergy therapy, involves sub~ P~us or sublingual introduction of an antigen known, or sn~recte~l, to provoke symptoms reflective of immnnP
dysregulation. l~y serial titration of the provoking material, a concentration of that agent may be determined which will nP,utrAli7P those symptoms in~ ce l 15 by the sarne s~lbst~n~e at a dirrer~ concentr~ti(-n. That is a prime example of a dose--ippen-l~nt phenomenon in which one dose induces a positive reaction while another dose of the same agent induces a negative l~*,onse.
Although it is thought that nP~It~li7~ti~)n occurs as a con~e~nen~e of reestablishing homeostatic functional levels of T8 :iu~ ;;SSul 20 cells, it is quite possible that the same antigen used at a nP~Itr~li7ing conl ent~tion to reverse immlme dysregulation could also, or in~te~l, trigger endocrine and/or nt~ ~nal control m~-~h~ni.~m~ to reverse ~ym~ lls. Re~ se of the i.~ association between the three control systems (endocrine, imml-n~, nervous) and proven co.. ~ tiQn pathways between and among 25 the cells comprising these respective systems, a single active molecule, suchas amyloid beta protein in the ~l~hPimer~s victirn, and related CNS disorders, may reverse symptoms via any or all of these routes.
Plaque formation is a common component in the etiology of numerous other disease as well. Print~.ip~l among those are :~r~prins~lerotic 3~ diseases. Like ~17h~imer's and related ~ e~es, arteriosclerotic ~ e~es~

W O 98/053~ PCTrUS97/14005 such as atherosclerosis, are plaquing diseases. Such ~ e~es are ch~r~7rt~rized by arterial plaque forrnation. These plaques commonly occur in large and medium-sized arteries and generally comprise cells, connective tissue (usually elastin, collagen, and glycosaminoglycans), and lipid deposits. The mixture 5 of those components is usually complex, forming lesions which may be calcified in advanced stages of the disease. Plaque mass slowly increases throughout life, as blood vessels undergo progressive concentric r~ u~.." sc~ r thi~kP~.i..g. In atherosclerotic patients, rblo...lls~-u1~r thiç~Pning of the intima of blood vessel walls proceeds rapidly and contributes, along with lipid 10 deposition, to restricted blood flow. In non-atherosclerotic patients, the - normal thickPning of the walls of blood vessels does not contribute to increases in blood ~ ,ssult; and does not co-llplull.ise blood flow. In fact, plaquing ~ e~ces often occur together and p~tiPntc with neural plaques also have vascula,- plaques.
15It is upon the matrix of rll"u~ c~ r thir~ning~ that atherosclerotic plaques develop. Such plaques generally become more prevalent in the third decade of life, with loc~li7~ti~-n being most common in the corullal y arteries. Atherosclerotic lesions are generally thought to develop from fatty deposits which transiently occur in all h~lm~nc in the developed 20 m11~c~ r lining of blood vessels. The mechanism of transformation from fatty deposits or "streaks" to a~l,en~sclerotic lesions appears to be unknown.
However, at least one report suggests that a virus may cause ~ lsru..~ n of the normal lipid streaks to atherosclerotic plaques. MPinir~, et al., JAMA, 263: 2204-207 (l990~; Wht;ll;;i'l it was reported that an avian herpesvirus 25 stim~ tP~ atherosclerotic lesions in chickens. The above-cited authors also correlated the presence of cytomegalovirus in h11m~nc with atherosclerotic lesions in hl1m~nc A finding of herpesvirus and cytomega1Ovirus antigens, as well as nucleic acids encoding those viruses, in arterial smooth muscle suggests that viral infection of arterial cells may be COi-lri~1ellt with the development of atherosclerosis. However, a causative relationship between any virus and atherosclerosis has yet to be conclusively de~....i.~A
Of interest to the present invention is the chronic fatigue syndrome. Chronic fatigue syndrome is a disorder of which the major 5 symptom is chronic, debilitating fatigue that is not resolved with bed rest, and which is severe enough to reduce daily activity below 50% for at least six months. In order to confirm diagnosis, eight of the following symptoms must have also begun at the onset of fatigue and have persisted or recurred over a period of at least six months. These ~y~ s include, mild fever, sore 10 throat, painful Iymph nodes, muscle we~kn~ss, muscle aches, fatigue after - exercise, h~d~ches, painful joints, neuropsychiatric complaints, sleep disturbances, and sudden onset in a healthy person. A diagnosis of chronic fatigue syndrome further involves eliminz-tinn of a va~ety of other illn~es ch~ t~.rized by fatigue th~ough personal history, physical eX;~ ()n and 15 laboratory fin-ling.~
Chronic fatigue syndrome is a disorder that may have sevelal causes. Much of the early lileldtUlt; on chronic fatigue syndrome focuses on the Epstein-Barr virus as a causative agent. The Epstein-Barr virus is a helpes-l~e virus that is the major cause of acute infectious mononucleosis, a 20 common syndrome ch~ teri7~d by fever, sore throat, extreme fatigue, and swollen lymph glands.
It was later reported in the art that the rubella virus may have a possible role in the etiology of chronic fatigue syndrome. Studies contl~lct~lon patients having chronic fatigue syndrome have shown that many of those 25 p~ti~nt~ have abnormally high levels of antibody to the rubella virus.
The use of the inflllen7~ virus vaccine and the rubell~ virus vaccine both sep.. ~l~ly and together have been reported in the art for the treatment of herpes (Epstein-Barr vims) virus infections. Liebe~
Clinical Ecology, 7(3~:51 (1990) ~ o.~ed the use of patients ~urL~ g from 30 Epstein-Barr virus with infl~len7zl virus vaccine given together with hi~t~min~

and the immune enh~ncer Staphage 1ysate. Patients were also s13cGessfully treated with the same composition further in combination with rubella virus vaccine and with rubella virus vaccine alone.
Also of interest to the present invention is the disclosure of McMichael U.S. 4,521,405 that patients experiencing recurrent herpes simplex virus type II infection have reported relief of lesion pain and lesion enl~ell.cnt upon tr~tmPnt with compositions including hi~t~mine, mç~1P~s inactivated, attenuated virus and inFlllP.n7~ vaccine (killed) virus. McMichael, U.S.
4,880,626 taught a composition for alleviating the symptoms of AIDS
comprising human chorionic gonadotropin, Staphage lysate, an influen_a virus - vaccine, such as Fluogen~ and fractionated inactivated H[V virus.
Also of interest to the present invention is hypertension. The increases in vascular permeability generally observed in hy~ ension may increase influx of lipopl~Leil- into cells, thus increasing the likelihood of atheroma formation. Eyyellension may also COllllilJUlt; to atherosclerosis in blood vessels surrounding the brain. A reduction in hy~ Lt;llsion has been shown to significantly reduce the incidence of myocardial infarction associated with atherosclerosis. Other factors in the development and ~ ,glGs~ion of atherosclerosis include f~i~betes mellitus, which may reduce lipid efflux from 20 cells in the arterial wall. In addition, cigarette smoking dr~m~ti- ~lly increases the risk of developing atherosclerosis and assochted hy~ellension, including their sequelae, such as infarction of the myoc~ll.liulll and brain. Obesity is another factor which may contribute, especially in an individual who smokes.
Overall, hylJ~lk;llsion is the single g~l~lesl risk factor in COl~ P~ÇS as 25 well as cerebrovascular stroke.
The presence of hyp~llensioll is a ~li,nal~y in~lic~tor of an arteriosclerotic condition and is often used by physicians as the sole diagnostic measure of .li~ P.s such as atherosclerosis. Moreover, a reduction of blood ~JlC;S~ul~;iS thought to have an effect in reducinp~ the severity of alhe~ a 30 plaques. The mech~ni~m for such reduction rnay be a reduction in the W 098/053SO PCT~JSg7/14005 transport of lipids and proteins into blood vessels which is coincident with a reduction in blood P1~S~7U1G. The diastolic component of blood PI~S:,UI'~ iS
generally thought to be the primary in-lic~t-~r of hy~ellen~.ion. While the systolic component may vary greatly llepen-iing upon nervousness, anxiety and 5 the ILke, diastolic blood IJlt;SbUl~ generally remains Coll~kull and is more reflective of a patient's general vascu}ar state. A diastolic reading of over 90is considered mild hypertension in an adult and a ~ ctoli~ reading of over 100 is considered hy~t;lLellsi~re and an m-lir~t- r of arteriosclerotic disease.

SllMMARY OF l~l~; INVENTION
The present invention provides methods for alleviating the ~y~ ls of disease states associated with plaque formation. In accordance with the invention, there is provided a method to $tim~ te the apl)r~l,ale 15 metabolic regulatory systems (immllnP, CNS or endocrine) which retard the progress of the ~7y~ Jlollls of plaquing ~ice~ce'C~ such as ~l7~hpimer7s and related ~licP.~ces and arteriosclerotic ~lice~ces. Observations by s~;ontictC have now indic~t~l that the ap~>c..~llL elevated amyloid beta protein concPnt~ti~-n in, for example, ~17hPim~r's diseases may not be due to an increase in 20 genomic e~l)r~;s~ion, but possibly to activation of a mechanism that induces the re~ p~ n of amyloid moieties from normal neurofi1~mPntc into paired helical fil~mPntc resl-ltin~ in n~ulurlblillary tangles, neuritic plaques or amyloid plaques. D. Goldjaber, M.I. Lerman, O.W. McBridge, U. Suffiotti and D.C. G~ ~k, Science, 235, p. 77-780 ~1987), R.E. Tanzi, J.F.
25 Gusella, P.C. Watkins~ G.A.P. Bn~ns, P. St.C~eorge, M.L. Vankeuren, D.
Patterson, S. Pagan, D.M. Kurnit and R.L. Neve, Science, ~, p. 880-884 (1987); M. Baudry, B.R. Dubrin, L. Beasley, M. Leon and G. Lynch, Neurobiol. Aging, 7, p. 255-260 (1986); G.G. Glenner, Arch. Palh. Lab.
Med., 107t p. 218-282 (1983). The mecl~ of the present invention may 30 result in triggcl--,g control plucesses that correct the rear~angement of W 098/05350 PCTrUS97114005 neurofil~ment~, alter abnormal amyloid protein formation including amyloid beta formation, and/or allow for clearing of axonal transport mech~ni~m~.
~ Similarly, regulatory control systems, as noted above, play a role inarteriosclerotic plaque formation, leading to arteriosclerotic tli~e~es such as 5 atherosclerosis. A ~ignifi~nt common occurrence in p~ti~nt~ having arteriosclerotic disease and/or neural plaquing disease, such as ~17hPimPr's, is hypertension. Accordingly, methods of the present invention cause a reduction in hyy~lLc;~lsion as an in-1ir~tinn of alleviation of the overall disease state. The f~i~e~es susceptible to treatment with methods according to the 10 invention have in common plaque formation. Accordingly, tr~tmPnt with - methods according to the invention provides an effective tre~tment of all such e~P.s by alleviating causative symptoms of the disease. In addition, as ~et~i1P~l below, compositions and methods of the invention are useful in the reduction of hyyt;~lellsion gPnPr~lly As one aspect of the invention, it has been discovered that a cc,ll.yonent of influenza virus vaccines that provided activity against herpes viruses as reported in Li~belll,al~, ~linical Ecology, McMich~Pl U.~.
4,521,405 and McMichael U.S. 4,880,626 was the preservative thirnerosal which was present in commercially available i ~ virus vaccines such as 20 FluogenlM (Parke Davis, Morris Plains, NJ), Fluzone~ (Conn~llght LaboldL~lics, SwirLwal~l, PA), and Flu-Tmm~lnP~ (Lederle, Wayne, NJ).
Con~eq~-Pntly, it is believed that results reported by MrMirh~Pl and Liebe~.l.dll ~IL~ uLed to the anti-herpes virus effects of in~ ,n7~ virus vaccine are due to the presence of thimerosal in the tested co,l,l~o~iLions. Further, it25 has been discovered that the anti-herpes virus activity of influenza virus vaccine is aLLlil,uLable to the presence of thimerosal in the tested vaccines and that helpes virus infections may be treated with thimerosal uncombined with or in the absence of influenza virus vaccine. Accordingly, the present invention provides mPthorls for treating subjects ~urÇe~ g from herpes virus 30 infections, comprising the step of ~lmini~tenng an effective amount of a composition comprising thimerosal free of association with inflll~n7~ virus.
More specii~lcally, the present invention provides m~tho-lc for l,~,atil-g patients having chronic fatigue syndrome comprising ~-1mini~tering an effective amount of thimerosal free of association with influenza virus to a subject ~urÇ.~ g 5 from chronic fatigue syndrome. As one aspect of this invention it has been found that the combination of thimerosal free of association with infl11en7~
virus and rubella virus vaccine is particularly effective in the tre~tm~nt of subjects suffereing from chronic fatigue syndrome. The invention further provides methods for in vitro lcilling of herpes virus by ~-lmini~t~tiQn of lO effective amounts of thimerosal. The invention further provides methods of treating plaquing ~lice~es such as atherosclerosis and hy~ellension by - combining thimerosal or Ihi~ usal cc,l.~ g compositions such as iniFluenza virus vaccines which contain thimerosal with amyloid beta protein.
In order to identify a dose of amyloid beta protein for use in the 15 invention, a wheal produced upon intradermal injection of the l~
m~t~ri~l was evaluated according to criteria set forth in Moore, Clinical Medicine, 81: 16-l9 (1974), incol~o.~led by reference herein. Upon subcutaneous injection, a wheal may be ~1elts~ ."i.1~1 to be positive ten Illil~lllr"S
after injection as a blanched, hard, raised, and discoid ~ sion from the 20 skin. A negative wheal is sufficiently absorbed at the end of ten minutes that the protrusion on the skin has grown less than an average of two mi11imçters in rli~meter from its original size.
In a ~lt;rcilled embodiment of the invention, compositions for tre~tm~ont of plaquing ~ e~C~s according to the invention comprise a dose 25 from about 10-1~ to about lO-2 mg of amyloid protein and from about O.OS ~bg to S00 ,ug thimerosal with about 0.5 ,ug to about 50 ,ug thimerosal being ~lc;rt;il~d and about S ~g thimerosal being particularly ~lc;rt;li~d. The total volume of a typical composition according to the invention for ~ ;on to a patient is about 0.05 cc, or one drop. Compositions according to the W O 98/053SO PCTrUS97/14005 invention for treatment of plaquing diseases may comprise ,B-amyloid protein or the first 28 amino acids of ,B-amyloid protein.
Preferred dosages of thimerosal for treatment of subjects suffering from herpes virus infections range from about 0.05 ,~4g to 500 ~ug thimerosal with about 0.5 ~g to about 50 ~g thimerosal being ~ d and about S ,~lg thimerosal being particularly ~ re~c;d.
Also in a preferred embodiment, compositions according to the invention are ph~ ce~-tic~l compositions for tr~tment of arteriosclerotic ~1ice~es which pharrn~re~lti~l compositions comprise amyloid protein and thimerosal in a pharrn~ce~ltic~lly-acceptable carrier.
Methods according to the present invention are useful in alleviating symptoms of arteriosclerosis generally, and atherosclerosis in particular. Such methods comprise the step of ~-imini~tering to a patient suspected or co~r~ -ed as having an arteriosclerot;c disease an effective amount of a pha~ ceutir~l compos;tion com~lisillg amyloid protein and an inflllen7~ virus vaccine. An effective amount of a co~ o~iLion according to the invention is an amount which results in a reduction in the syll-plol,ls of an arteriosclerotic disease. Most ~I~;rel~ly, an effective amount of a composition according to the invention comprises from about 10-1~ to about lO-2 mg of an amyloid protein, preferably ~-amyloid protein, and about 0.05 and about 0.05 ,ug to about 500~g thimerosal being pr~r~l~d. An amyloid protein used in methofls according to the invention may be a ,B-amyloid protein or may be the first 28 amino acids of a ~-amyloid protein. A highly pl~f~ d amount of amyloid protein used in compositions and methods according to the invention includes from about 10-5 and about 10-2 mg of amyloid protein.
Methods and compositions according to the present invention are effective in alleviating symptoms of any disease in which pl~qlling is involved and especially diseases in which aLlle,.,ma forrn~tic n is ch~ te~istic.
ColllposiLions and m.othn~s according to the invention also alleviate hylJt;llen~ion and reduce cholesterol, both of which have a direct effect in CA 02233987 l998-04-06 W 098/OS350 PCTAUS97/1400~ ~

redl-cin~ the severity of or elimin~ting symptoms associated with arteriosclerotic disease. The following f1~t~ilP~ description of the invention provides exemplification of claimed methods and compositions. However, it is understood by the skilled artisan that other uses of the invention, 5 specifically relating to the treatment of arteriosclerotic diseases, are within the scope of the present cl~ims.
Methods according to the present invention are useful in alleviating symptoms of arteriosclerosis generally, and atherosclerosis in particular. Such metho-1s comprise the step of a~lmi~ lrling to a patient 10 suspected or confirmPcl as having an arteriosclerotic disease an effective amount of a ph~rm~reutic~l composition COIIIp~ g amyloid protein and an inf~ .n7~ virus vaccine. An effective amount of a colllposiLion according to the invention is an amount which results in a reduction in the symptoms of an arteriosclerotic disease. Most preferably, an effective amount of a 15 composition according to the invention comprises from about 10-'~ to about 10-2 mg of an amyloid protein, preferably ,~-amyloid protein, and about 0.05 and about 0.05 ,ug to about 500,ug thimerosal being ~ lc;d. Preferred dosages of thimerosal for tre~tment of subjects ~urre~lg from herpes virus infections range from about 0.05 ~g to 500 ,ug thimerosal with about 0.5 ,ug 20 to about 50 ~bg Ll~imel~sal being ~It;rell~d and about 5 ,~4g thimerosal being particularly l)lc;rell-,d. An amyloid protein used in methods according to the invention may be a ~B-amyloid protein or may be the first 28 amino acids of a ,~-amyloid protein. A highly ~Ic;rel~c;d amount of amyloid protein used in compositions and methods according to the invention is from about 10-5 and 25 about 10-2 mg of amyloid protein.
Mf thor~ and compositions according to the present invention are effective in alleviating symptoms of any disease in which plaquing }s involved and especially di~e~es in which alllelullla formation is ~hz~ .t~ri.~i~.
Compositions and methods according to the invention also alleviate 30 h~el~ sion and reduce cholesterol, both of which have a direct effect in W O 98/05350 PCT~US97114005 reducing the severity of or elimin~ting symptoms associated with arteriosclerotic disease. The following ~et~ihPA description of the invention provides exemplification of claimed methods and compositions. However, it is understood by the skilled artisan that other uses of the invention, S specifically relating to the treatment of arteriosclerotic diseases, are within the scope of the present claims.
Also in a p~ lled embodiment, the invention provides a method for alleviating the symptoms of disease states associated with abnormal ?~Çcl~m~ ti~ n of and/or molecular ~3r~ 1 ion of amyloid protein or amyloid 10 placlues, which comprises ~-imini~tration to a ~ e~e(1 patient of an effective amount of amyloid protein or an effective active fragment thereof. The amyloid protein is pl~r~lably an amyloid beta protein although amyloid protein fr~gment~ such as fr~gmP-nt~ coll,plisillg the first 28 amino acid residues of the amyloid beta protein are expectP~l to be useful. The method of the invention 15 is useful against disease states associated with abnormal ~c cl-m--l~ti~ n ofand/or molecular ~,~,..~i, .~ ion of amyloid protein or amyloid plaques including disease states in which the amyloid protein or plaques are associated with the central nervous system and histopathologically related disorders. Such e~ces include, but are not limited to Alzheimer's Disease and P~hh~soll's Disease. Other disease states include those such as atherosclerosis.

DETAILED DESCRIPTION OF TEIE INVENTION
A. Application of Materials and Methods of the Invention to the Tr~ of Alzheimer's and Related Diseases The alleviation of ~17hPimer~s Disease ~yll-y~c~l--s obseIved following ~ .a~ion of amyloid beta protein as described herein likely reflects stimulation of a~y~p-ialt; metabolic regulatory systems in the Alzheimer's Disease p~ti~nt~ such that ~rcum~ ti(~n andlor formation of the paired helical fil~ment~ in nc;ul~rlt~lillary tangle and/or amyloid plaque developments are ~ignifiç~ntly altered or slowed and ?rcllm~ ted pl~leills are W 098/05350 PCTr~S97/14005 elimin~t~ This reprogrAmmin~ to establish proper homeostasis would allow more efficient tr~n.cmiccion of nerve impulses which would result in clinical improvement of treated Alzheimer's p~tient~.
Typically, a pharrn~e~lti~l dosage unit of the present invention 5 for the delivery of amyloid beta protein in a low concentration comprises a liquid or solid carrier and an effective amount of amyloid beta protein. One suitable carrier for sublingual ~dmini~tration comprises a phenylated saline solution. ~ffective amounts of the amyloid protein range from about 10-1~ to about 10-2 mg, and preferably from about 10-5 to about 10-3 mg and most 10 preferably about 104 mg, amyloid beta protein in association with pharm~c e~lfi~lly ~ cept~hle excipients. The amyloid beta protein is ~ minict~red through standard methods, incll~tlinE sublingual, subcut~nPous and t~nsderm~l routes, and in dosage units that are either liquid or solid.
One explanation for the mode of action of this invention may 15 be that the amount of this protein ~tlmini~tp~red is sufficient to trigger a negative fee~lb~rk mP~h~ni~m to the body such that production of ~1flition~1 amyloid beta protein, possibly through breakdown of normal neurofil~mPnt~, is inhibited. Under this theory, the low level of amyloid beta protein, or a derivative thereof, gives a signal to the body to correct the abnormal 20 synthesis/degr~r1~ti~n process. The body sensors are then adjusted to normal metabolic control of amyloid beta protein prc!cçs~inE that allows the proper balance to reestablish itself, alleviating the abllo,l--al procec~ing. The immlmP
system, as well as the endocrine and CNS control systems, could play an integral regulatory role in rt;~onse to the low dose therapy, with the amyloid 25 protein functioning through mP~h~ni~m~ that not only correct the mt)lPcuhr o~ i,n~iQn of the arnyloid beta protein moieties, but clear ~he il~lt;lr~ihlg amyloid molecular constructs.
In a plc;~erled embodiment, the present invention provides ~1mini~t~ti-7n of amyloid beta protein or a derivative thereof. The amyloid 30 beta protein may be provided either as part of a liquid solution or in a solid W O 98/053~0 PCTrUS97/14005 powder matrix, and may be ~mini~tered with conventional excipients to permit ease of ~f~mini~tration and accurate dosage delivery. Patients characte~ed herein below were evaluated using a battery of objective tests ~l~sign~d to measure cognitive ability. Ihese incl~ ~1 the mini-mental State 5 Px~min~tion, the Verbal Fluency Task Ex~min~tion (word name task and category task), evaluation on the Demattis DemPnfi:- Rating scale, and the Word-Association Task ~Y~min~tion of the Wechster Memory Scale-Revised.
Not all results of all tests are provided herein, however, the results of all the tests were qualitatively the same as those below and led to the same 10 conclusions as those provided herein relative to the effect of tre~tm~nt according to the invention.

A 67 year old male with a history of Al~heimer's Disease for 15 four years prior to inil;~ g lhGl~y presented with an inability to answer questions, to place names with faces, and to complete his senten~es His wife noted a con~i~t~nt downhill progression of his condition on a monthly basis.
At the initiation of therapy, with the composition of the invention, his initialscore on the Mini-Mental State Exam was 5 of a possible 3Q. The subject was 20 treated by sublingual ~t1mini.~ration four times per day of a dosage unit COIIIp~ lg 104 mg of amyloid beta protein in a phenylated saline solution.
After five months of therapy according to the invention, the patient scored a 12 1/2 on the Mini-Mental State ~-xam, was reading road signs while travelling and was co..-...~ fing with family members. Also, the patient 25 appeared to be more relaxed and better able to respond to his wi~e's efforts to assist him.

An 81 year old male with a history of ~l7h~im~r's Disease was 30 treated according to the invention. PAor to tre~tment the subject was unable CA 02233987 l998-04-06 WO 98l05350 PCT~US97tl4005 to dress himself, had a flat affect, was poorly co.. ~ tive and scored 10 1/2 on the Mini-Mental State ~xam. The subject was treated by sublingual ~lmini~tration four times daily of the amyloid beta dosage unit of Example 1.
After three months of tre~tment, the subject scored 17 points on the Mini-S Mental State Exam, was more ~ l~i in speech, could dress himself most days and was more confident in physical actions.

In this example, the subject was a 62 year old female who was 10 originally diagnosed by the University of Pill~bu~ MPAi~l School - ~l7heimer's Disease Research Centerto be suffering from a fi.l.. i.~,.li.~g form of ~l7hpim~r~s Disease. In one year, the subject had gone from being director of nursing in a chronic care establi~hmPnt to req~irin~ constant care.
The subject was unable to communicate, did not appear to recogni7e anyone 15 and had a score of 1.5 on the Mini-Mental State Exam. The subject was treated by sublingual 7~rlmini~tration four times daily of the amyloid beta dosage unit of Eixample 1. After three months of tre~tment7 the patient's hll~k~n~ reported that warmth had returned to the patient's hands, no deterioration of any type was evident, although prior to therapy, he could note 20 weekly declines. C~ ;e~tion rPm~in~ liffl~nlt but improved for the subject, she showed increased alertness, and she was not only able to recogmze individuals con~ fently, but also was able, at times, to participate in conversations, and her test score rose to 7.75 on the Mini-Mental State Exam. Although the subiect was originally diagnosed as having ~l7hpimp~r~s 25 Disease, the results of an autopsy inrlic~tPA that she did not have ~17hPimer's Disease but suffered from a form of r~hi~son's Disease known as Striatonigral degen~tion.

W O 98/053~0 PCTAUS97/14005 In tbis example, the subject was a 79 year old male who had suffered from two transient ischemic attacks and had also been diagnosed as having Alzheimer's Disease. Prior to tre~fm~nt the subject had a score of 16 5 on the Mini-Mental State Exam. The subject was treated by sublingual jq~lmini~tration four times daily of the amylo;d beta dosage unit of ~xample 1.
While the subject became somewhat more irascible and eventuaUy died of a stroke two months after the trial period, his p~lro~ allce on various mental performance exams including the Dementi~ Rating Scale improved during the 10 initial six month evaluation period. SpecificaUy, the Mini-Mental State Exam improved during the initial six months of testing with scores of 18, 20, 21 and 25 at tbe three, four, five and six month tests, respectively. The subject's mental p~ allce at the tbree month foUow-up *x~ ion had declined ~i~nific~ntly, with the score on the Mini-Mental State Exam dropping to the 15 level of the original test with a score of 16. The subject's scores on the other mental pelr~ ce exams also showed marked decline to the original ;lrullll~ce levels. The subject died of a stroke prior to the six-month follow-up evaluation.

EXAM~E S
In this example, a 76 year old male who was diagnosed as having Alzheimer's Disease, was treated by sublingual 7~ l;on four times daily of the amyloid beta dosage unit of Example 1. The subject's ~e.~ll,~al~ce on both the Mini-Mental State Exam and the DemPnti~ Rating Scale improved ~ignifi-~ntly over five months of testing. Test scores on the Mini-Mental State Exam on the first, third, fourth and fifth months were: 20, 20, 20 and 25; while scores on the DPmenti~ Rating Scale were: 115, 117, 122 and 126. The subject showed improvement primarily in areas of attention and conceptl~li7~tion and in imme~i~t~ short term memory, with some 30 improvement in verbal fluency. In contrast, his ~elro,~ ce did not improve W O 98/05350 PCTrUS97/14005 in the area of delayed memory which rPm~inçcl severely co~ llised. In addition, there was only slight improvement demonstrated on tasks re~nirin~
new learning abilities which was also severely colllpl~l-lised. The subject was unavailable for follow-up study.

In this example, the subject was a 74 year old woman who was diagnosed as having Alzheimer's Disease so advanced that she was unable to identify a comb or a key. The subject was disoriented, incontinent and 10 severely h~ ellsi~e and required intensive around the clock care from her sister who was a nurse. The sub3ect was treated by sublingual ilclmini~trAtion four times daily of the amyloid beta dosage unit of Example 1. The subject's initial score on the Mini-Mental State l~xam was 2, and the subject demonstrated slight improvements (subsequent scores were 2, 3, 4, 8 and 7) 15 although some, but not all, of the improvement may have resulted from modification of the test procedure to accommodate the marked t;,.~ e language deficits exhibited by the subject. Significantly, the subject's blood IJlc;S:iul~ returned to normal upon tre~tm~Pnt with the amyloid protein composition. This inAi~t~s that the amyloid protein composition exhibits 20 utility in trr~tment of the symptoms of atherosclerosis which can be associated with amyloid plaques.
B. Application of Mzlt~ri~l~ and Methods to the Trç~hn~nt of Arteriosclerotic Diseases ~17hPimPr~s patients treated with amyloid protein as described 25 above show a significant decrease in blood l~lc;Si,ul~, as a result of such treatment which is conco,~ with a reduction in sy~ ollls of ~iementi~.
As noted above, Melnick, et al. report a role for a herpesvirus (Cytomegalovirus, CMV) in atherogellc~is. In co-owned, United States Patent No. 4,880,626, it is noted that, while alI Ull~ted AIlDS patients studied had 30 CMV, none of the patients treated with a inflllen7~ vaccine based (Fluogen'U) CA 02233987 l998-04-06 W O 98/05350 PCTrUS97/14005 composition had CMV. The present application teaches compositions and methods comprising the anti-plaquing amyloid protein and the anti-viral thimerosal in order to effect trç~tmPnt of patients pl~S~ g with arteriosclerotic conditions. The following examples provide exemplification S of the invention through representative embo-lim~nt~ comprising the use of claimed compositions and methods on human subjects. For each example below, original (i. e., pleL~ nf) blood pressure was taken about three times during each reading to ensure accuracy. Subsequent measures were repeated about 5 times. Unless otherwise noted, patients undergoing tre~tm~nt 10 according to the invention received 1 drop (sublingual) four times per day.
One drop is approximately 0.05 mL of a composition according to the - invention.

EXAMP~ F~ 7 A 54-year-old male patient ~ ,senled with atherosclerosis, inr1~1-1ing blood yres~ul~ of 140/90. The patient was treated with sublingual drops of a composition co~ ising 10-9 mg amyloid protein in a 1:25 ~lilntinn of 0.05 mL thimerosal cc~ influenza vaccine (Fluogen~) in saline. The patient was not treated with any other merli~tion during the period in which he was treated with the above composition. In addition, the patient reported that he rem~in~l on a high fat diet and reported no exercise during the tr~tment period. After daily sublingual trP~fm~nt (1 drop 4 times per day) for 90 days, the patient's blood pressure had decreased to 117/72. After two years of taking the above composition, the patient's blood l~lcs~urt; has stabilized at about 115/70. The patient's cholesterol also ~ignifi-~nt1y decreased after snst~in~ tr~-~tme-nt A 44-year-old, moderately obese male presented with blood plC;:~:iUl~, of 140/110. The patient was treated with daily sublingual doses (4 WO 9810~350 PCT~US97114005 times daily) of a composition according to the invention, as recited above in Example 1. The patient received no other m~Aic~tion and did not otherwise alter his lifestyle during the treatment period with the result that his blood pressure had decreased to 120/90. After continued tr~Atm~nt as described 5 above and with no change in lifestyle or diet during the tr~tment period, the patient had a blood plG:~UlG of 123/78. Blood plGSsulG was taken up to five times during each mea~ulGIl~ent to ensure accuracy.

10A 55-year old female with initial (pl~ -ent) blood pl'GS~UlG
- of about 138/90 began treatment according to methods of the invention and with compositions according to the invention. The patient showed a steady improvement in the diastolic component of blood pl~S;,ul~G through three months of trP~tm.~,nt At that point, the patient disco~ ued tre~tmt~nt and 15 three months later showed increases in diastolic blood plGS~ult. Upon .c.~.. ;.-~ tr~ tment~ diastolic blood plc;S~ul'e again decreased. During trP~tment7 the patient was ~lmini~tP~red compositions as described above in Examples 1 and 2. The following table provides a partial t~lring of the patient's blood ~l~;S~ulG during trP~tment and non-tr~tmP,nt periods.

W098/05350 PCTrUS97/14005 T~LE 1 TrP~tmf nt Blood Pressure pre-treatment 138/90 yes 150/88 yes 144/82 no 136/82 no 118/74 no 122/80 no 142/86 yes 140/80 yes 130/78 A S0-year-old male patient presented with blood pl~;S~u~; of 150/104 and began tre~tmPnt as described above. The results of that trs~tm~nt are ~res~ Led in Table 2.

W 098/05350 PCT~US97/14005 TABLE: 2 Treatment Blood I~GS~U
yes 146/95 yes 155/94 yes 138/90 yes 138/78 yes* 150198 yes* 140/96 yes* 156/108 no 160/102 no 160/100 yes 130/78 *dose of 1-2 drops/day The results l~rGse~ ;d in Examples 3 and 4 demonstrate not only the effect of compositions according to the invention in reducing symptoms of arteriosclerosis, but also demonstrate that such ~y~ L~llls return upon cessation of L~ L according to the invention.

E~NIPLE 11 A 76-year-old female with blood ~JlGS~iUlG of 210/110 began trP~tment according to the invention and as described above, using 4 drops per day. After one month of tr~?tm~nt~ the patient's blood plG;i:iUlG was reduced to 200/90. After 90 days of tre3tmPnt her blood pleS;!iUl'e was reduced to 160/90.

W 098/~50 PCTAUS97/14005 EXAMPI~l~ 12 According to this example v~;le~ y clinical trials were carried out involving the ~Amini~tration of amyloid beta protein and a thimerosal cont~ining fraction of inflllen7~ virus vaccine for the tre~tm~nt of S atherosclerosis in a rabbit atherosclerosis model. Specifiica11y, a filter centrifugation technique was used to isolate a 30kD fraction of commercially available influenza virus vaccine (FluvironlU) co.-~;.i..i..~ t~imerosal as a preservative at a con~Pnt~tion of 0.01% wherein the vaccine was loaded onto an Ultrafree low-binding spin-filter unit with a 301000 nominal molecular 10 weight limit and centrifuged in a microfuge until all of the fluid had passed- through the filter. The filtrate was further filtered through a S,000 nominal molecular weight spin filter to yield a thimerosal cont~ining SkD filtrate fraction.
According to the trial, four groups of six atherosclerosis-prone 15 Watanabe rabbits apiece were treated with A) saline as a control; B) a composition acco~ g to the invention comprising 10 4 mg of amyloid beta protein and the 51cD thimerosal co..~ g influenza virus vaccine fraction; C) a composition according to the invention cc,lllplis..lg 10-3 mg amyloid beta protein and the 5kD Ll~ihlle~ co..l;.i.)il-g influenza virus vaccine fraction;
20 and D) a composition according to the invention con~ g 10 4 mg of amyloid beta protein and the 5kD thimerosal co.~ -g inflllen7~ virus vaccine fraction. The ~nim~l.c were treated for six months at which time the surviving ~nim~l~ were tested for weight gain and two ~nim~l~ for each group were sacrificed and vessel patency was determined by a gross pathological 25 e~min~tion of plaque formation in the lumen of the aorta. The ~nim~l~
~ treated with the compositions of the invention had clear birur~dlions and exhibited fewer and smaller plaques than did the control ~nim~lc which were aged and sex matched and which exhibited plaque accum~ ti- n and occlusion of vessels exiting the aorta. The average weight gain over six months for the 30 control :~nim~l~ was 690 grams as co---pa-~d to the three test groups which W 098105350 PCTrUS97/1400S

showed, respectively, average weight gains of 450 grams (Group B); 450 grams (Group C); and 525 grams (Group D).

SAccording to this example, a U.S. FDA Phase 1/2 controlled double blind human clinical trial was conducted using Ll-i~llclvsal co.~ g compositions for the treatment of chronic fatigue syndrome. Thirty-six (36) patients suffering from documented chronic fatigue syndrome were studied of whom thirty-three (33) completed the study. Of the subjects who completed 10the study, 17 were treated with placebo and 16 were treated by sublingual mini~t~ti-)n six times daily of 1 drop (0.05 mL) of a composition comprising 0.0020 mL (2 ,uL) infl~lçn7~ vaccine cont~ininf~ 0.01% (0.2 ~bg) thimerosal, 0.0004 mL (0.4 ,bL) rubella virus vaccine and 0.0576 mL saline.
After ten weeks of tr~tmPnt, the subjects were evaluated and were taken off 15of either the drug or placebo and were further evaluated after an additional four weeks of no tre~tmçnt ~The subjects were evaluated by means of two principal art recognized efflcacy p~r~m~te~rs: (1) a visual analogue scale for subjective evaluation of fatigue, and (2) a fatigue impact scale Co~ isil~g 36 ~uestions 20related to cognitive, psychologic and social disorders. Analysis of the results using the visual analogue scale showed no st~ti~ti~ y $ignifi~nt dirrer~ilce at the 95 % confi-len~e level belweell the therapy and the control. Analysis of the results using the fatigue impact scale also failed to demonstrate a ~t~ti~ti~lly ~ir..ir;.~ dirr~ ce at the 95% confidence level bt;lw~n the 25 therapy and placebo groups but did in~ te a trend in favor of the IIW14JY
over the placebo indicative of a therapeutic effect.

W098/05350 PCTrUS97/14005 C. Application of M~tf~,ri~lc and Methods of the Invention to the Tr~7,tmçnt of Herpes Infections The following examples relate to work establishing the in vitro and in vivo anti-herpes viral activity of thimerosal as the anti-herpes active 5 fraction of inflll.o,n7~ virus vaccines.
Typically, a pharrn~- entical dosage unit of the present invention for the delivery of thimerosal comprises a liquid or solid carrier and an effective amount of thimerosal. One suitable carrier for sublingual ~Amini~tration comprises a phenylated saline solution. Effective amounts of 10 thimerosal range from about 0.Q5 ~g to 500 ~g thimerosal with about 0.5 ,ug - to about 50 ~g thimerosal being ~ fe~l~,d and about 5 ,ug ll~illlen~sal being particularly preferred. The thimerosal is preferably ~f1mini~t~,red in association with pharm~ce~1tically acceptable excipients. The thimerosal is ~lmini~t~red through standard methods, including sublingual, subcutaneous 15 and transdermal routes, and in dosage units that are either liquid or solid.

EXAMPI,E 14 According to this exalnple, a filter centrifugation technique was used to isolate a 301cD fraction of commercially available influen_a virus 20 vaccines (Fluviron~ and Fluzone~) wherein the vaccine was loaded onto an Ultrafree low-binding spin-filter unit with a 30,000 nominal molecular weight limit and centrifuged in a microfuge until all of the fluid had passed through the f~ter. In vitro assays with the 30kD filtrate fractions (which contained thimerosal present at a concentration of 0.01% as a preservative in the 25 commercial vaccine) saw complete inhibition of herpes virus in cell culture assay l~tili7in~ HSV-l and HSV-2 infection of A549 (Human Lung Carcinoma) cells in vitro. The fraction obtained was also used in place of dilute influenzavirus vaccine in human subjects and was found to improve the clinical response to chronic fatigue syndrome and other herpes infections.

E~AMPLlE 15 According to this example, a further filtration was carried out isolating a 5kD fraction from the Fluvirin~ inflnen7~ vaccine. This fraction which also contained thimerosal was effective at inhibiting growth of the herpes virus in the cell culture of ~xample 14 and a~l,ea.~d to clinic~lly superior in in ~vo ~Amini~fration to 30kD fraction of ~cample 14.
In the following set of examples 16 through 21 the 30kD
thimerosal cot.l;.ini~-~ influe.n7~ vaccine isolate was ~dmini~ctered to subjects ~urre~ g from herpes virus infections.

In this example, a 38 year old female presented with acute onset of pain on the left side of the head, neck, shoulder, chest and arm which she had suffered for fve days. She also suffered from similar pain on the dorsal left foot. P.x~ l ion revealed three small papular lesions in a quarter-sized red area on the left upper chest. Herpes zoster was s-l~rect~l The 30kD
thimerosal cc,-~ i--g fraction of example 15 was ~1mini~tered sublin~l~lly with one drop (0.05 mL) four times daily for a dosage of about 5 ,ug thimerosal. The subject reported a g5% reduction in pain 30 to 60 minllt~s after ~rlmini~t~ti~n of the thimerosal co~ g flaction. The subject continued to do well after one week of QID treatment with one drop of the composition.

E~ IPT F.17 In this example a 66 year old female with a history of lG~;ull~llL
herpes zoster plCS~llLed during the inithl stages of such an outbreak.
~lmini~tr~ti~n of one drop sublingually of the composition of ~xample 16 P~ d discolllrolL. The sub3ect took a second drop of the c~ osiLion 24 hours later and did not e~pçrience further pain or discol~ -l for the next two weeks.

W O 98/0~350 PCT~US97/14005 In this example a 19 year old male pl~sellLGd with a history of having developed acute infectious mononucleosis a~pru~ill,ately five days previously with ~ym~ llls of sore throat, 4+ fatigue, lymph node enl~GIllGll~
5 ~4+ cervical), hP~ chP, dizziness, splenomegaly. The subject was treated with one subcutaneous injection of 0.2 mL of the composition of example 16 and reported 60-70% improvement within 30 mimlt~p~s~ Seven hours later, the subject reported feeling 90% better.

10E2~AMPLE: 19 - According to this example a 30 year old woman presented with - predictable oral herpes simplex outbreak on the 23rd day of her menstrual cycle. Associated with the herpes outbreak were plG~ ual syndrome symptoms which were sufficiently severe as to require anti-psychotic 15 metlic~tion. The patent ~(1mini~tPred 2 drops of the composition of Example 16 sublingually at the f~st sign of herpes outbreak (that being sensitivity of the lip in the area of the typical lesion expression). No cold sores developed in or on her mouth for the first time in several years and the prçm~n~trual syndrome was not Gxllibiled.

~Y~ikrPl~E 20 A 63 year old female subject with a history of lesions lasting several weeks pl~sc;llLed with a four day old HSV-l lesion on her lip. One drop of the ~;olllpo~ilion of example 16 was ~-lmini~tered sublingually and 25 within 30 Illi lult;s the subject reported a m~rkP~l improvement. The lesion resolved in two days with ~rlminict~ti(m of two drops of the composition per day.

W098/05350 PCT~US97/14005 ~X A~PLE 21 A 50 year old plus male presented with chronic fatigue syndlullle having pronounced lethargy, a history of mental fogginess and poor quality of life. Two drops of the composition of example 16 were 5 ~rlmini~tered to the subject sublinqually and the subject reported improvementin excess of 70% with an increase in energy and mental clarity for the first time in several years. After three weeks, the subJect contlnued to do well with ~lmini~t~tion of two drops of the composition daily.

~ccording to this example, a wide variety of tests were carried out to tletf~rminp the anti-herpes active component of the 51cD infh-en7~
vaccine fraction with the result that it was deL~ ed that thimerosal possesses the anti-herpes effect. In vitro assays with purified thimerosal demonstrate activity against herpes viruses. Of interest is the observation thatwhen thimerosal is mixed in a test tube with herpes virus and is subseqllently introduced into susceptible cells viral pl~Jp~;~-lion is not inhibited. This suggests that the anti-herpes viral activity of ~l~ih~ osal is not by direct action on the virus. Further study shows that thiosalicylic acid and ~lithio~lihe~oic acid which are the breakdown products of thimerosal do not provide antiviral activity in v~vo although dithiodibenzoic acid shows anti-herpes vims activity in vitro.

According to this example, a double-blind study was carried out on sixteen subjects ~urr~ g with chronic ~atigue syndrome with thr~e compositions (lecign~t~ A, B, and C ~?mini~tered over the course of one month as daily sublingual drops. Each subJect was treated by sublingual .dlion of one drop (0.05 ml) four to six times daily. Conlpo~iLion A
comprised 0.0004% weight per volume thimerosal (0.2 ,~cg per drop) and 3.2 W 098/05350 PCTrUS97/14005 x 10-' units nt;u~ e per drop in saline. Composition B co~l~prised 0.0004 % weight per volume thimerosal (0.2 ~g per drop) in combination with 0.00032 mL (0.32 ~L) mbella virus vaccine (Meruvax, Merck & Co.) in saline. Composition C comprised 0.00()4 % weight per volume thimerosal (0.2 5 ~g per drop) alone in saline. The following p~r~mPtPrs were measured by patients' self-reported scores: overall level of fatigue; overall level of pain;severity of flare-ups; muscle cramps; hP~ c hPs; mental alertness and memory; and overall or average sleep. According to evaluation of these parameters, compositions A and B exhibited ~igni~ nt improvements in the 10 severity of CFS ~y",plo,l,s. Moreover, if the results of one subject treated - with composition B are omited (because physical therapy starting and ending about the same time as the therapy may have adversely affected the results for that subject) the l~--lA;ll;l~ subjects treated with composition B comprising the combination of thimerosal and rubella virus vaccine exhibited the ~;l~le~L
15 decrease in the severity of chronic fatigue syndrome syl"~)Lo~lls.
The fo~ uihlg representative results demonstrate that application of compositions accoldillg to the invention reduce blood ~ UI~, and other ~ylll~Lollls associated with arteriosclerosis. Thelt;~l~ n~ methods and compositions according to the invention con~liLule an effective means for 20 alleviating the ~ylllplollls of arteriosclerotic ~ e~ces and for completely alleviating such rii~e~es in some cases.

Claims (25)

WHAT IS CLAIMED IS:

1. A method for alleviating the symptoms of disease states associated with amyloid plaque formation and/or formation of arterial plaques comprising the step of administering to a patient of an effective amount of amyloid protein or a therapeutically-active fragment thereof and thimerosal free of association with influenza virus.

2. The method of claim 1 wherein the amyloid protein is an amyloid beta protein.

3. The method of claim 1 wherein the amyloid protein is a fragment comprising the first 28 amino acid residues of the amyloid beta protein.

4. The method of claim 1 wherein from about 10-10 to about 10-2 mg of amyloid protein is administered per dose.

5. The method of claim 1 wherein from about 10-5 to about 10-3 mg of amyloid protein is administered per dose.

6. The method of claim 1 wherein the disease state is associated with abnormal accumulation of and/or molecular organization of amyloid protein or amyloid plaques associated with central nervous system and histopathologically related disorders.

7. The method of claim 1 wherein the disease state is Alzheimer's Disease.

8. The method of claim 1 wherein the disease state is Parkinson's Disease.

9. The method of claim 1 wherein the disease state is atherosclerosis.

10. The method of claim 1 wherein said composition comprises from about 10-10 to about 10-2 mg of amyloid protein and from about 0.05 µg to about 500 µg thimerosal.

11. The method of claim 10 wherein said composition is administered to a patient in an amount of about 0.05 cc of the composition.

12. A pharmaceutical composition for treatment of disease states associated with abnormal accumulation of and/or molecular organization of amyloid protein or amyloid plaques which comprises amyloid protein or a therapeutically active fragment thereof, thimerosal free of association with influenza virus in amounts effective to alleviate one or more symptoms of said disease state and a suitable carrier.

13. The pharmaceutical composition of claim 12 wherein the amyloid protein is amyloid beta protein.

14. The pharmaceutical composition of claim 12 wherein the amyloid protein is a fragment comprising the first 28 amino acid residues of the amyloid beta protein.

15. The pharmaceutical composition of claim 12 in the form of a single dosage unit which comprises from about 10-10 to about 10-2 mg of amyloid protein and from about 0.05 µg to about 500 µg of thimerosal.

16. A method for treating hypertension, comprising administration of an effective amount of a pharmaceutical composition comprising amyloid protein or a therapeutically active fragment thereof and from about 0.05 µg to about 500 µg of thimerosal free of association with infuenza virus.

17. A method for treating subjects suffering from herpes virus infections, comprising the step of administering an effective amount of a composition comprising thimerosal free of association with influenza virus.

18. The method of claim 17 wherein the subjects is suffering from chronic fatigue syndrome.

19. The method of claim 17 wherein from 0.05 µg to 500 µg of thimerosal is administered per dose.

20. The method of claim 17 wherein from 0.5 µg to 50 µg of thimerosal is administered per dose.

21. The method of claim 18 wherein the composition further comprises a rubella virus vaccine.

22. The method of claim 21 wherein the composition comprises from about 0.01 to about 10 TCID50 of a rubella virus.

23. The method of claim 17 wherein said composition is administered to a patient in a single dose of 0.05 cc in a pharmaceutically acceptable carrier.

24. The method of claim 17 wherein the composition is administered sublingually.

25. A method for in vitro killing of herpes virus comprising administering an effective amount of thimerosal.