Title:  Methods for the prevention or treatment of alzheimer's disease

United States Patent:  6,471,960

Issued:  October 29, 2002

Inventors:  Anderson; Stephen (Princeton, NJ)

Assignee:  Rutgers, the State University (New Brunswick, NJ)

Appl. No.:  660954

Filed:  September 13, 2000

Methods for preventing or treating vascular hemorrhaging such as that incident to thrombolytic therapy, or characteristic of Alzheimer's and related diseases are provided. Such methods provide improved thrombolytic therapy to individuals who receive such therapy, and permit the diagnosis and treatment of diseases, such as Alzheimer's Disease, that are characterized by the deposition of amyloid deposits.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Overview of the Invention

Tissue-type plasminogen activator ("t-PA") is the major human plasminogen activator involved in vascular fibrinolysis. As stated above, despite the thrombolytic benefits of t-PA administration in the treatment of acute cardiovascular disease, such administration has resulted in a high incidence of intracranial hemorrhage. Indeed, initial reports of the TIMI-94 and GUSTO-IIA trials of the efficacy of t-PA therapy have demonstrated intracranial bleeding rates equal to or greater than 0.5% (Sobel, B. E., Circulation 90:2147-2152 (1994)). The cause of such bleeding has not been previously recognized. However, as discussed by Sobel, B. E., factors such as hypertension, age, female gender, impaired liver function, vitamin K levels, aspirin usage, .beta.-blocker usage, or nitrate usage have been proposed as factors (Sobel, B. E., Circulation 90:2147-2152 (1994)). Bleeding has been attributed to the susceptability of the cerebral vasculature in specific patients, including those with occult amyloid deposition in vessel walls, to injury by proteolytic agents such as plasmin (Sobel, B. E., Circulation 90-2147-2152 (1994)). Although a relationship between intracranial hemorrhaging and amyloid deposit presence has been suggested, the causal relationship between amyloid deposits and hemorrhaging, and a means for circumventing this relationship has not previously been identified (Pendlebury, W. W. et al., Annl. Neourol. 29:210-213 (1991); Ishii, N, et al., J. Neurol. Neurosurg. Psychiat. 47:1203-1210 (1984); Kase, C. S. et al., Ann. Intern. Med. 112:17-21 (1990); Wijdicks, E. F. M. et al., Stroke 24-554-557 (1993)).

The present invention derives, in part, from the recognition that the .beta.-amyloid peptide is the cause of the reported intracranial bleeding, and mediates such vascular and cellular damage by interacting with, and stimulating, tissue plasminogen activator or streptokinase to produce plasmin. The presence of plasmin catalyzes proteolysis and rupture of vessel walls at the site of the amyloid deposit.

Thus, agents which suppress this interaction may be used therapeutically to attenuate or prevent the vascular damage caused by amyloid deposits. One aspect of the present invention thus concerns agents and methods for preventing hemorrhaging, especially hemorrhaging that may occur due to the administration of thrombolytic agents such as t-PA or streptokinase.

A mechanism for the involvement of amyloid peptide in the progression of Alzheimer's Disease is discussed by Selkoe, D. J. (Trends Neurol. Sci. 16:403-409 (1993), herein incorporated by reference). Additionally, thrombin deposits have been found to accumulate in the brains of patients with Alzheimer's Disease (Akiyama, H. et al., Neurosci. Lett. 146:152-154 (1992); Akiyama, H. et al., Neurobiol. Aging 15:S124 (1994)). Within the brain, thrombin appears to function as a nerve growth inhibitor, causing neurons to retract their neurites (Cunningham, D. D., Annl. N.Y. Acad. Sci. 674:228-236 (1992); Gurwitz, D. et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:344-3444 (1988)). Recently, a number of low-density lipoprotein receptor ("LRP")-binding proteins, including t-PA, have been found in amyloid plaque deposits (Rebeck, G. W. et al., Neurobiol. Aging 15:S117 (1994)).

One aspect of the present invention concerns the recognition that thrombin-mediated nerve growth inhibition occurs through the accumulation of thrombin at the site of amyloid deposits. Thus, thrombin antagonists, such as anti-thrombin or anti-amyloid antibodies or .beta.-amyloid peptidomimetic agents, which can prevent or inhibit thrombin-amyloid peptide association, can be used to prevent or treat neurological disorders.

The capacity of .beta./A4amyloid peptide to activate t-PA also provides a highly sensitive method for diagnosing the presence of the .beta./A4amyloid peptide. Similarly, agents that stimulate t-PA synthesis (for example, in the brain) can be used to induce the plasmin-mediated dissolution of amyloid deposits.

Thus, in one embodiment, the present invention provides an improved therapy for thrombolytic intervention in the case of cardiovascular disease, as well as a diagnostic and therapeutic approach to the management of Alzheimer's Disease.

II. The Interrelationship Between Amyloid Deposits and t-PA

The main component of the amyloid deposit is a 39-43 amino acid peptide with a molecular weight of approximately 4,200 D that is derived from the much larger membrane-bound .beta.-amyloid precursor protein (APP) (sang, J. et al., Nature 325:733-736 (1987); Robakis, N. K. et al, Proc. Natl. Acad. Sci (U.S.A.) 84:4190-4194 (1987)). hi HCHWA-D, the .beta.-amyloid peptide has a glutamic acid to glutamine substitution at position 22 (Levy, E. et al., Science 248:1124-1126 (1990)). Although Alzheimer's Disease and HCHWA-D are both characterized by .beta.-amyloid deposition, the two diseases differ in the major sites at which .beta.-amyloid deposition occurs. In Alzheimer's Disease .beta.-amyloid deposits are found predominantly in cerebral cortex, but in most cases there is also some degree of amyloid deposition in the walls of cerebral vessels (Tomlinson, B. E., In: Greenfield's Neuropathology, 5th edition (eds. Adams, J. H. & Duchen, L. W.), 1284-1410 (Edward Arnold, London. 1992)). Clinically, Alzheimer's Disease is characterized by progressive dementia.

In HCHWA-D .beta.-amyloid is predominantly found in the walls of small and medium sized vessels of the leptomeninges and the cerebral cortex and in parenchymal deposits that resemble the early preamyloid deposits of Alzheimer's Disease (van Duinen, S. G. et al., Proc. Natl. Acad. Sci (U.S.A.) 84:5991-5994 (1987); Giaccone, G. et al., Neurosci. Lett. 97: 232-235 (1989). Patients with HCHWA-D develop recurrent hemorrhages which are ultimately fatal (Wattendorf, A. R. et al., J. Neurol. Sci. 55:121-135 (1982); Luyendijk, W. et al., J. Neurol. Sci. 85:267-280 (1988)). In both of these diseases and in other conditions giving rise to cerebral amyloid angiopathy, the .beta.-amyloid found in blood vessel walls appears to accumulate within the tunica adventitia and the tunica media of the muscle layer (Tomlinson, B. E., In: Greenfield's Neuropathology, 5th edition (eds. Adams, J. H. & Duchen, L. W.), 1284-1410 (Edward Arnold, London. 1992); Vinters, H. V., Stroke 18:311-324 (1987)). However, fibrils may first be formed closer to the vessel lumen, within the abluminal vascular basement membrane (Yamaguchi, H. et al., Amer. J. Pathol. 141:249-259 (1992)).

The origin of .beta.-amyloid in both Alzheimer's Disease and HCHWA-D has not been dearly established but it has been proposed that the vascular system is one source of APP (Tagliavini, F. et al., Lab. Investigation 62:761-767 (1990); Selkoe, D. J., Neurobiol. Aging 10:387-395 (1989)). The amyloid precursor may first pass through the endothelium to be deposited within the vessel musculature. This mechanism is supported by the frequent finding of many serum proteins in vessel walls affected by CAA, suggesting that the microvasculature exhibits a relatively non-specific leakiness to some macromolecules (Powers, J. M. et al., J. Neuropathol. Exper. Neurol. 40:592-612(1981); Goust, J. M. et al., J. Neuropathol. Exper. Neurol. 43:481-488 (1984)).

Human tissue-type plasminogen activator (t-PA) is a major extrinsic thrombolytic agent, originating from the vascular endothelium. Plasminogen activation by t-PA is stimulated by fibrinogen, and more dramatically by fibrin and fibrin analogs (Holyaerts, M. et al., J. Biol. Chem. 259:2912-2919 (1982)).

The discovery that .beta.-amyloid peptides function as fibrin or fibrinogen mimics that stimulate t-PA is consistent with observations that anti-.beta.-amyloid peptide antibodies cross-react with conformational epitopes on human fibrinogen and that anti-fibrinogen antibodies cross-react with .beta.-amyloid peptide (Stern, R. A. et al., FEBS Letters 264:43-47 (1990)). The t-PA -.beta.-amyloid interaction causes amyloid peptide fibrils in blood vessel walls to promote high local concentrations of t-PA and consequently high local concentrations of plasmin which result in proteolysis, rupture, and hemorrhaging of vessel walls.

III. The Prevention or Treatment of Vascular Hemorrhaging Incident to Thrombolytic Therapy

A central aspect of the present invention concerns the recognition that the undesired hemorrhaging which is observed in some individuals receiving thrombolytic therapy (especially t-PA) is caused by the presence of .beta.-amyloid deposits which stimulate the ability of the administered t-PA to produce plasmin at the site of the deposit.

Such undesired stimulation of t-PA can be attenuated or prevented by providing the patient with an effective amount of an agent that can specifically bind to the .beta.-amyloid peptide or its fibrils. Most preferably, the agent will be selected such that it is incapable or substantially incapable of binding fibrin.

The term "specific binding," as used herein refers to the capacity of two or more molecules to bind together due to structural attributes of each molecule. A molecule is said to be capable of "specific binding" to another molecule if such binding is dependent upon the respective structures of the molecules. The term is intended to distinguish such binding from non-specific binding that occurs without regard to the particular structures of the molecules involved (e.g., the binding of proteins to nitrocellulose is an example of non-specific binding). Examples of specific binding include the binding of an antibody to its antigen, the binding of a hormone to its receptor, etc. A molecule is substantially incapable of binding to another molecule where the extent, if any, of such binding fails to cause a physiologically relevant change in the concentration or activity of the un-bound agents. Most preferably, the molecules of the present invention will exhibit "highly specific binding," such that they will be incapable or substantially incapable of binding to closely related molecules.

As used herein, vascular hemorrhaging is said to be prevented by the administration of an agent when such administration decreases the probability of hemorrhage in that patient relative to the probability of hemorrhage in patients who have not received that agent. Such administration may be either "prophylactic" or "therapeutic." A prophylactic treatment is one that is provided in advance of any symptom of hemorrhage in order to prevent or attenuate any subsequent hemorrhage. A therapeutic treatment is one that is provided in response to the detection of hemorrhage, and serves to attenuate the degree, extent, or severity of such hemorrhaging. An amount of a therapeutic agent is said to be an "effective amount" if it is sufficient to mediate a clinically significant change in the severity of a symptom, or a clinically significant delay in the onset of a symptom.

In some embodiments, the molecules of the present invention may be used in a "purified" form. As used herein, a molecule is said to be in a "purified" form if it is present in a preparation that lacks a molecule that is normally associated with that molecule in its natural state.

The preferred binding agents of the present invention are antibodies or antibody fragments. Such antibodies may be intact immunoglobulins, or may be antibody fragments (F(ab'), F(ab')₂, single chain antibodies, etc.), recombinant antbodies, antibody fusion proteins, chimeric antibodies, etc. Such molecules may be obtained by screening among antibodies elicited in response to immunization with either a .beta.-amyloid peptide or a peptide or peptidomimetic molecule that is a "functional analog" of a .beta.-amyloid peptide

As used herein, the term "functional analog" includes both "classical analogs" and "mimetic analogs." A classical analog of a molecule is one that has a similar biological activity, and is chemically related to the molecule. By way of illustration, a non-naturally occurring mutant of t-PA would comprise a classical analog of t-PA. Similarly, a mutated .beta./A4-amyloid peptide would comprise an example of a classical analog of the .beta./A4-amyloid peptide. Likewise, an molecule isolated from a non-human mammalian species (such as a mouse, monkey, etc.) would comprise an example of a classical analog of that molecule. In contrast, a "mimetic analog" of a molecule retains the biological activity of the molecule, but is unrelated chemically. A peptidomimetic molecule whose structure mimics a binding site of t-PA or of .beta./A4-amyloid peptide would comprise a "mimetic analog" of such peptides.

The amino acid sequence of the .beta./A4-amyloid peptide is shown as SEQ ID NO:1. Preferred biologically active fragments of the .beta./A4-amyloid peptide lack amino acid residues 29-42 of SEQ ID NO:1. The fragments may be composed of only those amino acid residues present in SEQ ID NO:1, or may contain deletions, insertions, additions or substitutions of one, two or more amino acids from either terminus, or from an internal site. Examples of such fragments include a peptide comprising SEQ ID NO:1 residues 1-28, and a peptide comprising SEQ ID NO:1 residues 1-28, wherein the amino acid at residue 22 (Glu) is replaced with Gln.

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala SEQ ID NO:1

Additional t-PA or antibody binding fragments may be readily identified. Such molecules may be fractionated from the .beta./A4-amyloid peptide by proteases, cyanogen bromide, etc. and the resultant fragments assessed for their capacity to specifically bind and/or activate t-PA. More preferably, however, such fragments may be identified through the use of Epitope Scanning.TM. strategy (Cambridge Research Biochemicals, Inc.). Thus, the linear sequence of amino acids of the .beta./A4-amyloid peptide is evaluated to define a set of fragments of predefined length which overlap other members of the set by a preselected number of residues. The predefined peptide length may be any number. However, it is preferred that the length be great enough to confer some amount of secondary structure to the peptide, and to be small enough that the entire library of peptides can be synthesized. Thus, lengths of from about 6 to about 25 amino acids are preferred. In selecting the predefined length, a general consideration is that 90% of linear epitopes recognized by antibodies are six amino acids or less in length (Geysen, H. M. et al., Proc. Natl. acad. Sci. (U.S.A.) 81:3998-4002 (1984)). The preselected extent of overlap will generally exceed 50%, and will preferably be selected such that the overlap will be from about (n-1) to (n-3), where "n" is the predefined peptide length. An overlap of (n-1) is particularly preferred.

Once the sequences of the entire library of peptide fragments have been ascertainted, the peptides are synthesized, preferably using automated synthesizers such as a multipin peptide synthesis system. Suitable systems or peptide synthesis services are available from Cambridge Research Biochemicals, Inc.; ICI Biological Products, Inc.; Chiron Mimitopes, Inc.; Lab Products International, Ltd.

In order to identify peptides having desired determinants, each peptide is introduced into a well of a microtiter plate, and evaluated for its capacity to bind and/or activate t-PA. Such determinations may be made in any of a variety of ways. Preferably, the effects of such peptides on the conversion of plasminogen to plasmin by t-PA are determined using a spectrophotometric method that permits the determination of the apparent first-order rate constant of plasminogen activation.

In one embodiment, the peptide is immobilized to the well surface, and the assay is conducted by determining the extent of antibody that becomes immobilized to the support. More preferably, a competitive ELISA is conducted in which the ability of the peptide to compete with .beta.-amyloid peptide antigen binding to antisera is determined. The extent of antibody bind by each peptide is determined and used to map the antigenic determinants of the molecule. Where no determinants are observed, the method is repeated using peptides of greater predefined length. When all observed determinants are present in only a single fragment, the method may be repeated using peptides of lesser predefined length. In such manner, the library of peptides is evaluated and members containing the antigenic determinants are identified.

Once a particular peptide has been found to have an immunological determinant, the peptide can be used to elicit antibody production in naive animals (i.e., animals that have not been previously exposed to human .beta.-amyloid peptide). Where desired, the peptides can be modified to increase their immunogenicity. Thus, they may be modified to contain an amino-terminal and/or a carboxyl-terminal cysteine or lysine residue with or without spacer arms. The peptides may be conjugated to carriers such as bovine serum albumin, ovalbumin, human serum albumin, KLH (keyhole limpet hemocyanin), or tetanus toxoid. The use of human serum albumin is preferred over ovalbumin or bovine serum albumin, since it causes lower background levels in ELISAs and dot blots than do the albumins of other species.

Since the administration of t-PA to dissolve dots of individuals suffering or recovering from acute cardiovascular disease occurs over a brief and discrete time period (generally ranging from a few hours to a few days), non-human origin antibodies may be used. Thus, polyclonal antibodies of non-human animals that bind to .beta.-amyloid peptides may be administered in accordance with the methods of the present invention, irrespective of any anti-idiotypic or anti-heterologous immune reaction that may occur. Suitable polyclonal antibodies may be prepared, for example, by immunizing female rabbits or castrated male sheep with 50 to 500 .mu.g of a .beta.-amyloid peptide preparation. The immunogen is preferably suspended in water and emulsified with Freund's Complete Adjuvant prior to injection. Animals may be injected in multiple intradermal sites (preferably subcapsularly) and are preferably boosted after 4 weeks with .beta.-amyloid peptide (in Freund's Incomplete Adjuvant) at one half the amount of peptide used for the initial immunization. If desired, additional boosts at monthly intervals using Freund's Complete Adjuvant may be given to obtain even higher antibody, titers.

In this embodiment of the invention, adverse immune responses are not relevant to the treatment provided by the antibodies since the duration of treatment is of the same or lower order of magnitude than the time needed for the patient to clear the foreign origin antibodies. To the extent that such undesired immune reaction occurs, the number of doses or the amount of each dose is increased to compensate.

Although polyclonal antibodies can be used, murine monoclonal antibodies are particularly preferred (Koprowski, H. et al., U.S. Pat. Nos. 4,172,124 and 4,196,265). BALB/c mice are preferred for this purpose, however, equivalent strains may also be used. The animals are preferably immunized with approximately 25 .mu.g of affinity purified .beta.-amyloid peptide (or an equivalent thereof) that has been emulsified with a suitable adjuvant (such as TiterMax adjuvant (Vaxcel, Norcross, Ga.)). Immunization is preferably conducted at two intramuscular sites, one intraperitoneal site, and one subcutaneous site at the base of the tail An additional i.v. injection of approximately 25 .mu.g of antigen is preferably given in normal saline three weeks later. After approximately 11 days following the second injection, the mice may be bled and the blood screened for the presence of anti-.beta.-amyloid antibodies. Preferably, a direct binding ELISA is employed for this purpose.

Most preferably, the mouse having the highest antibody titer is given a third iv. injection of approximately 25 .mu.g of .beta.-amyloid peptide or fragment. The splenic leukocytes from this animal may be recovered 3 days later, and are then permitted to fuse, most preferably, using polyethylene glycol, with cells of a suitable myeloma cell line (such as, for example, the P3X63Ag8.653 myeloma cell line). Hybridoma cells are selected by culturing the cells under "HAT" (hypoxanthine-aminopterin-thymine) selection for about one week. The resulting hybridoma clones may then be screened for their capacity to produce monoclonal antibodies ("mAbs) to .beta.-amyloid peptide, preferably by direct ELISA.

Thus, in another embodiment, this invention contemplates a novel continuous hybridoma cell line which expresses monoclonal anti-.beta.-amyloid peptide antibody, as well as the use of such cell line to produce such antibody. The present invention also contemplates a novel continuous hybridoma cell line which expresses anti-.beta.-amyloid peptide antibody obtained by immunizing an animal with .beta.-amyloid peptide. Antibody may be obtained through the in vitro culturing of the cells, or by injecting the cells into a histocompatable animal where they can proliferate and produce high levels of anti-.beta.-amyloid peptide antibody. Such antibody can be recovered from the animal's ascites fluid, lymph, blood, etc.

In a highly preferred embodiment, populations of polyclonal .beta.-amyloid peptide antibodies, or species of monoclonal .beta.-amyloid peptide antibodies, are further screened to remove those antibodies that are additionally capable of specifically binding to fibrin. In the case of polyclonal sera, such removal can readily be accomplished by passing the sera through a column containing immobilized fibrin. In the case of monoclonal antibodies, such removal can be accomplished by evaluating the capacity of the molecule to bind fibrin, and then discarding those hybridomas that produce antibodies that specifically bind both .beta.-amyloid peptide and fibrin. The elimination of antibodies that bind fibrin serves to ensure that the antibodies will not disrupt the desired ability of the administered t-PA to dissolve fibrin clots.

Where chronic or prolonged administration is desired, the use of non-immunogenic antibodies is preferred. Such molecules can be pseudo-homologous (i.e., produced by a non-human species, but altered to a form that is immunologically indistinct from human antibodies). Examples of such pseudo-homologous molecules include humanized" (i.e., non-immunogenic in a human) antibodies prepared by recombinant or other technology. Such antibodies are the equivalents of the monoclonal and polyclonal antibodies, but are less immunogenic, and are better tolerated by the patient.

Humanized antibodies may be produced, for example, by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e., chimeric antibodies) (Robinson, R. R. et al., International Patent Publication PCT/US86/02269; Akira, K. et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison, S. L. et al., European Patent Application 173,494; Neuberger, M. S. et al., PCT Application WO 86/01533; Cabilly, S. et al., European Patent Application. 125,023; Better, M. et al., Science 240:1041-1043 (1988); Liu, A. Y. et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, A. Y. et al., J. Immunol. 139:3521-3526 (1987); Sun, L. K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, Y. et al., Canc. Res. 47:999-1005 (1987); Wood, C. R. et al., Nature 314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst. 80:1553-1559 (1988); all of which references are incorporated herein by reference). General reviews of "humanized" chimeric antibodies are provided by Morrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi, V. T. et al., BioTechniques 4:214 (1986); which references are incorporated herein by reference).

Suitable "humanized" antibodies can alternatively be produced by CDR or CEA substitution (Jones, P. T. et al., Nature 321:552-525 (1986); Verhoeyan et al., Science 239:1534 (1988); Beidler, C. B. et al., J. Immunol. 141:4053-4060 (1988); all of which references are incorporated herein by reference).

In an alternate embodiment of the invention, a mutant derivative of t-PA may be administered in order to achieve a more desirable thrombolytic therapy. Preferably, such a t-PA derivative will retain an ability to bind to fibrin, but will be substantially or completely incapable of binding to amyloid peptides of amyloid deposits. Such t-PA mutants may be obtained by, for example, mutating a nucleic acid molecule that encodes t-PA, expressing such molecules in a mammalian host cell line (e.g., Chinese hamster ovary cells), and determining whether such mutagenesis has resulted in a t-PA variant which can bind to fibrin, but which has a decreased ability to bind to amyloid peptide.

IV. The Diagnosis of Alzheimer's Disease and Related Conditions

A second aspect of the present invention is derived, in part, from the recognition that the amyloid peptide of Alzheimer's Disease and HCHWA-D stimulate t-PA to cleave plasminogen into fibrin. Thus, the present invention provides a means for diagnosing the presence of amyloid peptide associated with brain (i.e., "CNS" or central nervous system) cells of a patient. As used herein, a "CNS cell" is a neuron, or a cell in contact with nerve cells, such as a glial cell.

In one embodiment, such diagnosis is conducted in vivo, by imaging the location and extent of amyloid peptide deposit. In accordance with this embodiment of the invention, an analog of t-PA is provided to an individual, and its binding to amyloid deposits is monitored. Most preferably, such t-PA analogs will lack the capacity to bind to fibrin, or to activate plasminogen, but will retain t-PA's capacity to bind amyloid peptide.

Such derivatives can be readily isolated by mutating (or synthesizing) t-PA molecules that are substantially incapable of activating plasminogen (e.g., the t-PA variant in which the serine residue at position 478 has been replaced with an alanine residue), and then screening such molecules for those capable of binding to .beta.-amyloid peptides. The use of such molecules in vivo has the advantage that their administration will not comprise an undesired thrombolytic therapy.

Alternatively, the above-described antibodies to .beta.-amyloid peptides may be used for such diagnosis. Most preferably, such antibodies will be capable of binding to .beta.-amyloid peptides but be substantially incapable of binding to fibrin.

Most preferably, for such in vivo use, such molecules will be detectably labeled, as with radioisotopes, paramagnetic labels, etc. so as to facilitate the imaging of the location of any amyloid deposit.

In yet another embodiment, material (such as blood, sera, urine, cerebrospinal fluid, tissue biopsies, etc.) may be withdrawn from a patient and evaluated for the presence of amyloid peptide using the above-described anti-.beta.-amyloid peptide antibodies or t-PA analogs. The detection of these molecules may be done by any of a variety of methods. In one embodiment, antibodies are employed that are capable of binding to the .beta.-amyloid peptides, and the presence of such molecules is determined via an immunoassay. A large number of suitable immunoassay formats have been described (Yolken, R. H., Rev. Infect. Dis. 4:35 (1982); Collins, W. P., In: Alternative Immunoassays, John Wiley & Sons, NY (1985); Ngo, T. T. et al., In: Enzyme Mediated Immunoassay, Plenum Press, NY (1985); incorporated by reference herein.

The simplest immunoassay involves merely incubating an anti-.beta.-amyloid peptide antibody with a sample suspected to contain the target .beta.-amyloid peptide molecule. The presence of the target molecule is determined by the presence, and proportional to the concentration, of any antibody bound to the target molecule. In order to facilitate the separation of target-bound antibody from the unbound antibody initially present, a solid phase is typically employed. Thus, for example the sample can be passively bound to a solid support, and, after incubation with the antibody, the support can be washed to remove any unbound antibody.

In more sophisticated immunoassays, the concentration of the target molecule is determined by binding the antibody to a support, and then permitting the support to be in contact with a sample suspected to contain the target molecule. Target molecules that have become bound to the immobilized antibody can be detected in any of a variety of ways. For example, the support can be incubated in the presence of a labeled, second antibody that is capable of binding to a second epitope of the target molecule. Immobilization of the labeled antibody on the support thus requires the presence of the target, and is proportional to the concentration of the target in the sample. In an alternative assay, the target is incubated with the sample and with a known amount of labeled target The presence of any target molecules in the sample competes with the labeled target molecules for antibody binding sites. Thus, the amount of labeled target molecule that is able to bind the antibody is inversely proportional to the concentration of target molecule in the sample.

As indicated above, immunoassay formats may employ labeled antibodies to facilitate detection. Radioisotopic immunoassays ("RIAs") have the advantages of simplicity, sensitivity, and ease of use. Radioactive labels are of relatively small atomic dimension, and do not normally affect reaction kinetics. Such assays suffer, however, from the disadvantages that, due to radioisotopic decay, the reagents have a short shelf-life, require special handling and disposal, and entail the use of complex and expensive analytical equipment. RIAs are described in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S., et al., North Holland Publishing Company, NY (1978), with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein.

Enzyme-based immunoassay formats (ELISAs) have the advantage that they can be conducted using inexpensive equipment, and with a myriad of different enzymes, such that a large number of detection strategies--colorimetric, pH, gas evolution, etc. --can be used to quantitate the assay. In addition, the enzyme reagents have relatively long shelf-lives, and lack the risk of radiation contamination that attends to RIA use. ELISAs are described in ELISA and Other Solid Phase Immunoassays (Kemeny, D. M. et al., Eds.), John Wiley & Sons, NY (1988), incorporated by reference herein. For these reasons, enzyme labels are particularly preferred.

No single enzyme is ideal for use as a label in every conceivable immunometric assay. Instead, one must determine which enzyme is suitable for a particular assay system. Criteria important for the choice of enzymes are turnover number of the pure enzyme (the number of substrate molecules converted to product per enzyme site per unit of time), purity of the enzyme preparation, sensitivity of detection of its product, ease and speed of detection of the enzyme reaction, absence of interfering factors or of enzyme-like activity in the test fluid, stability of the enzyme and its conjugate, availability and cost of the enzyme and its conjugate, and the like. Examples of suitable enzymes include peroxidase, acetylcholine esterase, alpha-glycerol phosphate dehydrogenase, alkaline phosphatase, asparaginase, .beta.-galactosidase, catalase, delta-5-steroid isomerase, glucose oxidase, glucose6-phosphate dehydrogenase, glucoamylase, glycoamylase, luciferase, malate dehydrogenase, peroxidase, ribonuclease, staphylococcal nuclease, triose phosphate isomerase, urease, yeast-alcohol dehydrogenase, etc. Peroxidase and urease are among the more preferred enzyme labels, particularly because of chromogenic pH indicators which make its activity readily visible to the naked eye.

In lieu of such enzyme labels, chemiluminescent, radioisotopic, or fluorescent. labels may be employed. Examples of suitable radioisotopic labels include ³ H, ¹¹¹ In, ¹²⁵ I, ¹³1 I, ³2 P, ³5 S, ¹⁴ C, ⁵¹ Cr, ⁵⁷ To, ⁵⁸ Co, ⁵⁹ Fe, ⁷⁵ Se, ¹⁵² Eu, ⁹⁰ Y, ⁶⁷ Cu, ²¹⁷ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd, etc. Examples of suitable chemiluminescent labels include luminal labels, isoluminal labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, aequorin labels, etc. Examples of suitable fluorescent labels include fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, o-phthalde-hyde labels, fluorescamine labels, etc. For purposes of magnetic resonance imaging, paramagnetic labels (³ H, ¹³ C, etc.) are preferred.

V. The Prevention or Treatment of Alzheimer's Disease and Related Conditions

Yet another aspect of the present invention concerns the prevention or treatment of Alzheimer's Disease and related conditions.

As indicated, the HCHWA-D condition reflects both the deposition of amyloid peptide on to the surfaces of blood vessels, and the proteolysis of such deposits by plasmin, in a t-PA dependent process. In accordance with the methods of the present invention, Alzheimer's Disease may be treated by a plasmin-mediated proteolysis of the .beta.-amyloid peptides of the Alzheimer's amyloid plaque deposits. Such proteolysis does not occur naturally in Alzheimer's Disease victims because the gene encoding t-PA is not highly expressed in the brain, the site of the Alzheimer's Disease deposits.

Thus, in accordance with one embodiment of the present invention, the administration of t-PA to nerve cells comprises a therapy for Alzheimer's Disease.

Alternatively, agents which induce the synthesis of t-PA may be provided to patients in order to prevent or treat Alzheimer's Disease. A preferred agent is a DNA molecule that encodes t-PA. The general principles of such gene therapy have been discussed by Oldham, R. K. (In: Principles of Biotherapy, Raven Press, NY, 1987); Boggs, S. S. (Int. J. Cell Clon. 8:80-96 (1990)); Karson, E. M. (Biol. Reprod. 42:3949 (1990)); Ledley, F. D., In: Biotechnology, A Comprehensive Treatise, volume 7B, Gene Technology, VCH Publishers, Inc. NY, pp 399-458 (1989)); all of which references are incorporated herein by reference.

In accordance with such a method, DNA molecules that encode t-PA are incorporated into a vector and delivered to brain cells or to other cells which are subsequently implanted into the brain. Recombinant adenovirus is an efficient vector for such in vivo gene transfer. The transcription of the t-PA-encoding DNA can be mediated from any suitable eucaryotic promoter. Examples of such suitable promoters include the RSV LIR, the SV40 early promoter, the cytomegalovirus (CMV) IE promoter, and the MMTV promoter.

In an especially preferred sub-embodiment, the genetic therapy will link the t-PA-encoding DNA to sequences that will direct the secretion of the t-PA into the cerebrospinal fluid. The secretion of therapeutic gene products even from a modest population of transfected cells will create a microenvironment around the amyloid deposits that will contain a high concentration of t-PA.

Although, as indicated above, such gene therapy can be provided to a recipient in order to treat an existing condition, the principles of the present invention can be used to provide a prophylactic gene therapy to individuals, including those who, due to inherited genetic mutations, or somatic cell mutation, are predisposed to Alzheimer's Disease.

A further aspect of the present invention concerns the recognition that the Alzheimer's amyloid .beta. peptide is a potent functional mimic of fibrin, the main protein component of blood clots. As indicated above, tissue plasminogen activator is the body's main defense against spontaneous blood clotting, such as that which occurs in heart attacks and stroke The mechanism of t-PA action involves its recognition, targeting, and destruction of the fibrin molecules in blot clots.

It has been known for a number of years that the amyloid .beta. peptide can form deposits in the blood vessels of the brain (cerebral vasculature) as well as in the neuritic plaques associated with Alzheimer's Disease; this condition is known as cerebral amyloid angiopathy or CAA. CAA is a very significant risk factor for brain hemorrhages.

One aspect of the present invention relates to the recognition that too much t-PA can also cause brain hemorrhage. This recognition led to that aspect of the present invention that is concerned with whether the amyloid .beta. peptide, might appear to t-PA as resembling fibrin, and thereby cause t-PA to target and attack amyloid deposits in the cerebral vasculature as if they were fibrin-containing clots--a molecular example of mistaken identity. Because t-PA triggers a powerful cascade of local digestive action, collateral damage from this attack could be responsible for the bursting of the cerebral blood vessels in a brain hemorrhage. One aspect of the present invention thus concerns the recognition that the amyloid .beta. peptide indeed interacts functionally with t-PA. Any of a variety of t-PA antagonists may be employed to modulate or block this effect. Such antagonists include antibodies to t-PA (particularly humanized antibodies) or antibody fragments, t-PA ligands, t-PA analogues that bind amyloid .beta. peptide without causing proteolysis, etc.

The implications of this recognition to the treatment of Alzheimer's Disease are potentially profound. In the brain as well as in the blood t-PA appears to have a dual character. In the blood, the right amount of t-PA removes blood clots and protects the body against heart attacks and strokes; too much t-PA, however, causes hemorrhagic complications. In the brain, the right amount of t-PA allows for the synaptic plasticity that is vital for learning and memory; too much t-PA, however, can cause neurotoxicity. It is possible that amyloid .beta. peptide deposits are toxic to neurons because they attract toxic levels of t-PA to the vicinity of the nerves and trigger neuronal dystrophy. This hypothesis is consisent with what is known regarding the pathology and physiology of neural degeneration in Alzheimer's Disease.

Thus, one aspect of the present invention involves providing neuronal therapy (to, for example, treat or prevent Alzheimer's Disease) by "correcting" the effective concentration of tissue plasminogen activator in contact with the nerve cells of treated individuals. Depending upon whether the treated individual has a deficient or an excess effective concentration of tissue plasminogen activator in contact with his/her nerve cells, such "correction" may entail either increasing t-PA concentration or expression (optionally with additionally administered plasminogen) or providing an antagonist of t-PA activity or expression. It will be understood that such correction is intended to modulate the concentration of tissue plasminogen activator in effective contact with the nerve cells of treated individuals so that it is within a bernficial range encountered in individuals who do not have Alzheimer's Disease.

VI. Administration of the Molecules of the Present Invention

The above-described therapeutic agents of the present invention can be formulated according to known methods used to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in. Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed, Mack, Easton Pa. (1980)). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of such agents, together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the agents. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the agents into particles of a polymeric material such as polyesters, polyamino acids, hydro-gels, poly(lactic add) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly-(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980).

In a preferred method for treating hemorrhaging, the antibody and other amyloid peptide-binding agents of the present invention are provided concurrently with, or more preferably, prior to, the administration of a thrombolytic agent. Such antibody and, other amyloid peptide-binding agents are preferably provided by injection, most preferably by intravenous infusion.

Previously, despite the urgency of acute cardiovascular illness, the hemorrhaging associated with the administration of thrombolytic agents led health providers to avoid providing such agents until the diagnosis of cardiovascular disease had been confirmed by a cardiologist. Since the present invention attenuates a possibility of hemorrhage, it (either alone, or in conjunction with the administration of the thrombolytic agent) may be provided by acute care providers (such as paramedics, emergency room attendants, etc.). Moreover, since no adverse side-effects of anti-amyloid antibodies are known, and since a delay between the administration of the antibodies and the administration of the thrombolytic agent is desirable, the anti-amyloid antibodies of the present invention are particularly suitable for administration by emergency medical personnel in the treatment of suspected or potential acute cardiovascular disorders.

Claim 1 of 2 Claims

What is claimed is:

1. A method for increasing plasmin-mediated proteolysis of .beta.-amyloid peptides in brain cells or tissues comprising contacting brain cells or tissues with a purified tissue plasminogen activator so that .beta.-amyloid peptides in said brain cells or tissues are proteolyzed.

 

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