POLYSACCHARIDE-OLIGOAMINE CONJUGATES AS ANTI- AMYLOID AND ANTI-VIRAL AGENTS

FIELD OF THE INVENTION The present invention generally concerns the utilization of polysaccharide- oligoamine conjugates as anti-amyloid and anti-viral agents.

BACKGROUND OF THE INVENTION

There is an urgent need to develop prophylactic and therapeutic approaches for one of the most devastating age-related diseases— Alzheimer's disease (AD) and for the transmissible spongiform encephalopathies (TSE) or prion diseases.

TSE, which comprise infectious, familial, and sporadic neurodegenerations such as Creutzfeldt- Jakob disease (CJD) and Gerstmann-Straussler-Scheinker disease (GSS) of humans, scrapie of sheep, and bovine spongiform encephalopathy, are caused by prions. Prions are thought to propagate in the host cell by the self-perpetuating refolding a normal cell surface glycoprotein, the cellular prion protein PrP ^c , into an abnormal β- sheet rich conformation. The resulting pathological conformer, PrP ^Sc , is in turn the only known component of the infectious prion. Prions thus appear to function without the involvement of coding nucleic acids. Patients with prion disease develop progressive neurological dysfunction that results in death, usually within a year of the first clinical symptoms. Prion diseases share certain mechanistic and pathological features with Alzheimer's disease, a much more common cerebral amyloidosis.

PrP ^c is a copper-binding glycoprotein that is expressed in neurons and glial cells in the CNS, as well as in several peripheral tissues including leukocytes. Its normal function remains uncertain, but its location on the cell surface would be consistent with roles in cell adhesion and recognition, ligand uptake or transmembrane signaling.

Although there is still no therapy for prion diseases, many compounds with disparate chemical structures have been identified that stop the formation of PrP ^Sc in chronically infected ScN2a-M cells. In some cases, these chemicals also increased the

incubation time of experimental scrapie in rodents. Anti-prion compounds include the amyloid-binding dye Congo red, polyene antibiotics, anthracycline, dextran sulfate, pentosan polysulfate and other polyanions, tricyclic derivatives, tetrapyrroles, cysteine proteases inhibitors and certain PrP antibodies. Most of these anti-prion compounds appear to act primarily by decreasing the formation of PrP ^Sc through a variety of mechanisms. For example, blocking antibodies such as the Fabs D18 and R72 and the mAb 6H4 recognize a specific region within PrP and may prevent the productive interaction between the two PrP isoforms. Substituted tricyclic derivatives such as the antimalarial quinacrine seem to block PrP ^Sc formation by binding to specific cellular targets that play an essential role in prion replication. Likewise, the potent anti-prion tetrapyrrole, N-methyl pyridine (with an IC ₅₀ of 0.5μM in ScN2a cells), may block PrP ^Sc formation by binding to a cellular accessory factor. Finally, several polyanions may act by preventing the interaction of PrP with cellular heparan sulfates.

An additional mode of action was recently determined by Suppattapone et al [Supattapone S, et al. Branched polyamines cure prion-infected neuroblastoma cells. J

Virol.; 75(7), 3453-61 (2001)] for a group of potent anti-prion polycations, the branched oligoamines such as polyethyleneimine (PEI), polypropyleneimine (PPI) and oligoamine dendrimers. Rather than just inhibiting the de novo formation of PrP ^Sc , these compounds appear to clear preexisting PrP ^So from prion-infected cells, perhaps by destabilizating PrP ^Sc in the acidic environment of lysosomes. In ScN2a, the most potent dendrimers had an IC ₅₀ of 80 ng/ml (WO/0072851).

Alzheimer's disease (AD) is characterized by accumulation of a peptide termed the β -amyloid protein or Aβ, in a fibrillar form, existing as extracellular amyloid plaques and as amyloid within the walls of cerebral blood vessels. Fibrillar Aβ amyloid deposition in Alzheimer's disease is believed to be detrimental to the patient and eventually leads to toxicity and neuronal cell death. Evidence implicates the formation, deposition and accumulation of Aβ fibrils as the causative factor in the pathogenesis of Alzheimer's disease.

An increasing number of sporadic, hereditary, and even infectious "conformational diseases" are characterized by the deposition of aggregates of misfolded host proteins or peptides. The structural features of the aggregates further

define two types of confoπnational diseases. In the Type I disorders, the culprit proteins form amorphous aggregates. In contrast, the so-called amyloidoses (or Type II conformational diseases, Table 1) are typified by the deposition of fibrillar aggregates with amyloidic properties. Depending on the anatomical deposition of the fibrils, amyloidoses are further defined as localized or systemic. Many amyloidoses impact gravely on human health, especially of the older population. Alzheimer's disease (AD) is perhaps the gravest human amyloidosis in terms of human suffering and economic impact. On the other hand, the transmissible spongiform encephalopathies (TSE), or prion diseases, present a grave public health danger illustrated by the transmission of bovine spongiform enephalopathy to humans.

Table 1: Different types of human amyloides

In addition of their pathogenic significance, amyloids fibers are emerging as an unexpected structure that can accommodate a variety of proteins with no common primary or tertiary structure. Amyloids are unbranched fibers ranging from 6θA to

13θA in width, in which proteins are arranged in a cross β-sheet structure. Amyloid fibrils are characterized by (i) their resistance to proteolysis, hence their resistance to tissue and cell clearance, (ii) their insolubility in detergents, and (iii) their tinctorial properties. Their so-called Congophilia refers to their green-gold birefringence when stained with Congo red and examined by polarizing optics.

Many amyloids can be grown in vitro from a pure solution of their constituent protein or peptide, exhibiting a biphasic growth pattern: (i) nucleation and (ii)

elongation. The nucleation is usually time-limiting, but it can be considerably hastened by the addition of pre-formed amyloids, or seeds, to the solution. Seeding also accelerates amyloid deposition in some amyloidoses in vivo. The best example is the vast acceleration of AA amyloidosis in mice with high SAA levels are inoculated with preformed AA fibrils. In vivo, the formation and deposition of amyloids is much more complex as it often includes (i) the formation of amyloidogenic fragments from the precursor protein, such as the complex processing of APP into Aβ40-42 by the various secretases in AD, (ii) both precursor proteins and the amyloid product are subject to vast intracellular and extracellular trafficking and metabolic constraints (iii) ultimately, whether amyloids will eventually accumulate and deposit is decided by the balance between their formation and their clearance, and finally (iv) additional molecular partners have been identified, in some cases, as important players in the deposition of amyloids.

Large amyloidic aggregates such as those in plaques have long been suspected to cause toxicity, and a large body of results show indeed that amyloids exert a wide spectrum of cytotoxic effects. However, it has recently become evident that amyloidogenic peptides often form non-Congophilic, non-fibrilar pre-amyloids may be more toxic than the fibrils. Likewise, it has now been demonstrated that small PrP ^Sc aggregates carry more prion infectivity than larger ones. Several molecular species appear recurrently in a variety of amyloid plaques, including apolipoprotein E, amyloid binding factor, hsp70, and more. Among these amyloid associates, GAGs stand out because they may play a metabolic role in amylogenesis. GAGs are long, unbranched sulfated polysaccharides consisting of repeated disaccharide units, usually occurring as side chains of the proteoglycan family of glycoproteins. Of the 4 chemical varieties of GAGs, the heparan sulfate (HS) family of GAGs is mostly associated with amyloid deposits. HS may intervene directly in the metabolism and stability of amyloids. For instance, (i) HS accelerates Aβ fibril formation in vitro, (ii) removing cell surface HS reduce the pathological PrP ^Sc in prion- infected cells and prevents the endocytosis of exogenous prions and (iii) transgenic mice that overexpress heparanase are resist systemic AA amyloidosis and have a prolonged scrapie incubation time. This may be the reason that many sulfated glycans

reduce the deposition of amyloids in vitro and in cultured cells, and in some case prolong the incubation time of scrapie in mice.

Although most amyloidoses remain incurable, extensive efforts have thus far been invested. Many therapeutic strategies are under investigation, including (i) stabilizing or reducing the level of the precursor protein, (ii) slowing the processing of precursor proteins to their amyloidogenic forms; the search of beta and gamma secretase inhibitors fall in this category; (iii) Reducing the levels of facilitating factors such as heparan sulfate, and (iv) increasing the clearance of the existing amyloids, for instance using passive or active immunization in AD models, and polycations in prion diseases.

Development of successful drug candidates for treating brain disorders requires the design and synthesis of compounds that not only exhibit potent and specific pharmacological activity but also the ability to reach the drug's target site. Typically, this requires partitioning through membrane barriers such as the blood-brain barrier (BBB). In addition, drugs must be resistant to enzymatic degradation in the gastrointestinal tract, blood, and target tissues, and must avoid extensive binding to circulating blood-borne proteins. In addition to overcoming the cellular membrane barrier, a compound that is supposed to exert its action in the brain must also be able to cross the BBB. For many potential pharmaceuticals, such as neurotrophins, the capillary endothelial wall of the BBB is not entirely permeable and brain uptake that has been measured could, in part, be due to experimental artifacts. Alternatively, the BBB can be reversibly disrupted by osmotic shock to allow treatment with pharmacological agents. This treatment, however, bears the high risk of neuronal damage. Another approach, the mechanical delivery of proteins into the brain, has been hampered by serious drawbacks in many cases. Polybasic proteins like poly-ornithine and poly-lysine and histones highly enhance protein uptake in the brain. However, the knowledge and application of the uptake-enhancing properties by smaller basic peptides have developed more recently. Nanoparticles can cross the BBB if they are in the proper size and surface properties. The bioactive polycations tested in this project have a good chance to cross the BBB after iv administration similar to cationic peptides and proteins that cross the BBB. Alternatively, fatty chains or PEG chains attached to the poly cation to provide a hydrophobic site that enhances crossing the BBB.

Viruses are among the smallest (20-300 nm in diameter) infectious agents known today. They contain a genome, consisting of RNA or DNA, and can grow only in live cells. They cause the normal metabolic processes of the host cell to be diverted into synthesizing viral nucleic acids and proteins and producing the progeny viruses. Each virus has a very limited host range and usually can reproduce only in a small group of closely related species.

Human viral diseases, varying in severity from AIDS and rabies to the common cold, have no doubt plagued mankind since the dawn of history. One third of the world's population is affected by recurrent herpes simplex virus (HSV) infections, with a large reservoir of virus, or viral DNA, causing this disease. Herpes simplex labialis/facialis, also known as cold sore, is mainly a skin disease. However, illnesses caused by HSV type 1 (HSV-I) and type 2 (HSV-2) include genital herpes, gingivostomatitis, herpetic keratoconjunctivitis, herpetic whitlow, herpetic encephalitis and eczema herpeticatum. Although herpes simplex labialis/facialis has usually a self- limited course, the disease can affect quality of life, mainly due to pain disfigurement and the psychological impact of recurrent herpes episodes.

The current treatment of choice for herpes simplex infections is acyclovir (ACV). It is a nucleoside analogue of guanosine that has to be activated by three steps of phosphorylations. The first phosphorylation is performed by the herpes virus encoded thymidine-kinase, allowing ACV to become active only in virus infected cells. The second and third phosphorylations are achieved by cellular thymidylate-kinases. ACV triphosphate acts as a competitive inhibitor of the viral DNA polymerase and it is a DNA chain terminator.

The requirement for additional anti herpes simplex drugs, acting by a different mechanism of antiviral activity to that used by ACV, is of very high priority in antiviral drug research today. Finding new drugs, acting on other steps during the virus growth cycle, can be an important contribution for the use of combination therapy, in order to lower the risk of the emergence of drug resistant mutants and in treatment of those patients who already carry, as a latent infection, an acyclovir resistant mutant of HSV. Some sulfated polysaccharides from red algae show inhibitory activities against infectious agents causing human diseases. For example, galactan sulfate acts against

Aghardhiella tenera and Nothogenia fastigiata, and xylomannan sulfate against human immunodeficiency virus (HIV), HSV-I, HSV-2 and respiratory syncytial virus (RSV). Xylomannan sulfate is active during an early stage of the virus growth cycle, such as virus adsorption, taking place on the surface of the mammalian cell.

SUMMARY OF THE INVENTION

It has now been surprisingly determined that conjugates formed by reacting at least one polysaccharide with at least one oligoamine, herein referred to as a "polysaccharide-oligoatnine conjugates" and which had previously been used as carriers or vectors having no therapeutic activity by themselves, indeed posses such therapeutic activity. These conjugates have now, for the first time, demonstrated their therapeutic activity as being effective in the prophylaxis and/or treatment of various viral diseases and diseases and disorders associated with accumulation of amyloid fibrils (amyloidoses). Thus, this invention provides the use of polysaccharide-oligoamine conjugates in the preparation of pharmaceutical compositions in which the polysaccharide- oligoamine conjugates are the active ingredient. These pharmaceutical compositions may be used for the treatment of various diseases and disorders, as will be disclosed herein, both in humans and animals. By a first aspect of the invention, there is provided a method of inhibiting a viral infection in a biological specimen, comprising contacting the biological specimen with an effective amount of at least one polysaccharide-oligoamine conjugate.

The term "biological specimen" refers to isolated cells (eukaryotic, prokaryotic, of animal or plant origin), cell cultures, tissue cultures, organs, cells isolated from a cell bank, animal, or blood bank, or secondary cells cultured from one of these sources or long-lived artificially maintained in vitro cultures which are widely available. The biological specimen may also be fluids obtained from biological samples such as blood, plasma and serum. The virus may be inside the cells of the biological specimen (in the genome, in the cytoplasm, or budding from the membrane) or may be present outside the cells in the form of a virion or viral particle. Preferably the specimen is of an animal

origin; more preferably it is part of said animal, and most preferably said animal is a human. The animal may also be a non-human subject.

The term "viral infection" refers to a condition caused by infestation by a virus. In one embodiment, the viral infection is caused by double stranded DNA virus. The DNA virus may be selected from human and animal pathogens such as adenoviruses, hepatitis B virus, herpesviruses (e.g., HSV, CMV, EBV), papilloma virus (e.g., HPV), flaviviruses such as dengue fever and yellow fever, pestiviruses (a genus of the Flaviviridae family) such as BVDV (bovine viral diarrhea virus), hepatitis C viruses (also a genus of the Flaviviridae family), filoviruses such as ebola virus, influenza viruses, parainfluenza viruses, including respiratory syncytial virus, measles, mumps, papovaviruses, the picornaviruses, including the echoviruses, the coxsackieviruses, the polioviruses, the togaviruses, including encephalitis, coronoviruses, rubella, bunyaviruses, reoviruses, including rotaviruses, rhabdoviruses, and arenaviruses such as lymphocytic choriomeningitis. Preferably, the DNA virus is selected from herpesviruses. More preferably, the DNA viruses are HSV-I and HSV-2.

As used herein, the term "inhibition" refers to the complete or partial reduction in viral load (e.g. number of active viruses, viral particles or virions), complete or partial reduction in the activity of the virus, or in decrease of the pathological effect caused by the activity of the virus on living tissue. The viral infection does not need to be completely inhibited for the conjugate or the method utilized to be effective. For example, the use of the conjugate can decrease viral infection by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or in the case of complete inhibition even at least 100%, as compared to an amount of viral infection in the absence of the conjugate. This decrease or inhibition can result in halting or slowing the progression of, or inducing a regression of a pathological condition caused by the viral infection, or which is capable of relieving signs or symptoms caused by the condition. The inhibition of the virus by the conjugate should typically result in a decrease in measured virulency or in colony size. The present invention further concerns a method for the treatment and/or prevention of a viral induced disease or disorder in a subject in need of such treatment

comprising administering to said subject a therapeutically effective amount at least one polysaccharide-oligoamine conjugate. The subject may be a human or a non-human animal.

The present invention further concerns a method for disinfecting a desired region, suspected of having a virus load comprising: applying onto said region at least one polysaccharide-oligoamine conjugate. The region may be a surface, a vessel, a part of a medicinal equipment or apparatus, or agricultural equipment suspected of having a viral load. The application may be by any manner known in disinfection procedures such as by spraying, dipping, brushing on, and coating with a substance comprising at least one polysaccharide-oligoamine conjugate, etc.

In connection with this aspect, in one preferred embodiment, said conjugate is a conjugate wherein said polysaccharide is dextran. hi another preferred embodiment, said conjugate is a conjugate wherein said oligoamine is spermine. More preferably, the conjugate is a dextran-spermine conjugate. By a second aspect of the invention, there is provided a method of preventing and/or treating a disease or disorder associated with amyloidosis, comprising administering to a subject in need thereof an effective amount of at least one polysaccharide-oligoamine conjugate. In one preferred embodiment, said amyloidoses is Alzheimer's disease. In a second preferred embodiment, said amyloidosis is selected from human or non-human prion diseases.

The "amyloid disease" or "amyloidosis" refers to a disease associated with the formation, deposition, accumulation of an amyloid protein selected from Aβ amyloid, AA amyloid, AL amyloid, LAPP amyloid, PrP amyloid, α2-microglobulin amyloid, transthyretin, prealbumin and procalcitonin. Such diseases may be selected from the group of Alzheimer's disease, Down's syndrome, dementia pugilistica, multiple system atrophy, inclusion body myositosis, hereditary cerebral hemorrhage with amyloidosis of the Dutch type, cerebral β-amyloid angiopathy, the amyloidosis of type 2 diabetes, the amyloidosis of chronic inflammation, the amyloidosis of human or non-human prion diseases, Creutzfeldt- Jakob disease, Gerstniann-Straussler syndrome, kuru, scarpie, the

amyloidosis associated with carpal tunnel syndrome, senile cardiac amyloidosis and amyloidosis associated with endocrine tumors.

Aβ amyloid deposition in patients suffering from Alzheimer's disease occurs in the brain. Deposition of amyloid with other diseases may occur also for example in the liver, heart, GI tract, kidney, skin and/or lungs. Some amyloid diseases affect a myriad of organs or tissues and others may affect a single organ or tissue such as observed with patients suffering from the so-called human prion diseases such as Creutzfeldt- Jakob disease, Gerstmann-Straussler syndrome and kuru.

Prions are infectious agents which do not have a nucleic acid genome. Prion diseases are often referred to as "transmissible spongiform encephalopathies" because of the post mortem appearance of the brain with large vacuoles in the cortex and cerebellum. The term "prion disease" as used herein, refers to transmissible spongiform encephalopathies. Non-limiting examples of non-human prion diseases are Bovine spongiform encephalitis (BSE) of cattle and cows, new variant of the Creutzfeld- Jakob disease (vCJD) caused by BSE or scarpie, transmissible mink encephalopathy and chronic wasting disease of animals. Examples of the human prion diseases are kuru,

Alpers syndrome, sporadic Creutzfeld- Jakob disease, familial CJD ₅ iatrogenic CJD,

Gerstmann-Straussler-Scheinker (GSS) disease, fatal familial insomnia and the vCJD.

In one embodiment of the invention, the human prion disease is sporadic Creutzfeld- Jakob disease.

The present invention further concerns a method of treating, decreasing or preventing the formation, deposition, or accumulation of amyloid fibrils within cells, in the extracellular fluid or matrix or in tissue comprising contacting the said cells, fluid or tissue with an effective amount of at least one polysaccharide-oligoamine conjugate. In one embodiment the amyloid fibrils are Aβ amyloid fibrils. Preferably, said cells, fluid or tissue is in a living subject, most preferably a human subject.

Thus, the present invention also concerns a method of treating, decreasing or preventing the formation, deposition, or accumulation of amyloid fibrils comprising administering to a subject in need thereof an effective amount of at least one polysaccharide-oligoamine conjugate.

The invention further provides a method of preventing and/or treating

Alzheimer's disease, comprising administering to a subject in need thereof an effective amount of at least one polysaccharide-oligoamine conjugate. In a preferred embodiment, said conjugate is a dextran-sperniine conjugate. In a another preferred embodiment, the conjugate is dextrane-prapane 1,3 diamine conjugate.

The invention further provides a method of preventing and/or treating animals suffering from a prion disease or disorder associated therewith, comprising administering to said animal in need thereof an effective amount of at least one polysaccharide-oligoamine conjugate. Said animals may be selected from cows, sheeps, goats, minks, deers, elks and others. In a preferred embodiment, said conjugate is a dextran-spermine conjugate. In a most preferred embodiment, the conjugate is dextrane- prapane 1,3 diamine conjugate.

The invention still further provides a method for the eradication of PrP ^Sc , comprising contacting a cell with a polysaccharide-oligoamine conjugate. In one embodiment, said cell is a collection of cells such as those of cell cultures. In another embodiment, said cell resides outside of an animal body, as is for example the case with a cell culture. These cell-culture cells may for example be cells isolated from a cell bank, animal, or blood bank, or secondary cells cultured from one of these sources or long-lived artificially maintained in vitro cultures which are widely available. In yet another embodiment said cell is part of an animal body.

The invention further concerns a method for disinfecting a desired region, suspected of having a prion particles load comprising: applying onto said region at least one polysaccharide-oligoamine conjugate. The region may be a surface, a vessel, a part of medicinal equipment or apparatus, or agricultural equipment suspected of having a prion particles. The vessel may be one which is used with farm animals or slaughter houses, such as slaughter machine and knives.

Alternatively, said vessel may be used with non-farm animals, e.g. during hunting of wild animals. The region may be any food product derived from farm or wild animals, said food product being for human or non-human consumption, e.g. meat of various sources, bones, muscles, or any other animal product which may not traditionally be used for consumption but rather for other purposes, e.g. animal skin.

The anti-prion disinfecting composition comprising said conjugate may be applied to said region by any method known such as spraying, dipping, brushing on, or coating.

As used herein in reference to all aspects of the invention, the term "preventing and/or treating" or any lingual variation thereof concerns the administering of an effective amount of any of the compositions of the present invention which is effective to ameliorate undesired symptoms associated with a disease, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disease, slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease form occurring or a combination of two or more of the above.

For example, the anti-prion composition is capable of reducing at least one side effect of the prion associated disease, preventing an infected subject from developing clinical signs of the disease, decreasing mortality and decreasing infectiousness of a subject.

The anti-viral composition is capable of reducing at least one side effect of the viral infection, shortening the acute phase of the infection, decreasing the number severity of lesion caused by the viral infection, preventing an infected subject from developing clinical sign of the disease, and decreasing the infectiousness of a subject.

The anti-Alzheimer's disease composition is capable of reducing at least one side effect of Alzheimer's disease, delaying or preventing the onset of a condition associated with the Alzheimer's disease, slowing down or stopping the progression, aggravation, or deterioration of the symptoms, bringing about ameliorations of the symptoms of the condition, or curing the specific condition or the disease itself. Prevention of Alzheimer's disease includes, for example, delaying the onset of the disease or stopping progression of the disease beyond the early stage in a treated population compared to untreated population; prevention of symptoms of neurodegenerative diseases such as motor impairment or vision loss includes, for example, slowing the progression of loss of function or delaying the appearance of such loss of function in a population of

- In patients receiving the prophylactic treatment relative to an untreated control population; prevention of symptoms of neurodegenerative diseases such as memory impairment or deficiency in cognitive functions, includes, for example, reducing the number of episodes of failed recollection or cognitive impairment in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of memory deficiency in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.

The "effective amount" for purposes herein is determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect as described above, depending, inter alia, on the type and severity of the disease to be treated and the treatment regime. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the affinity of the ligand to the receptor, its distribution profile within the body, a variety of pharmacological parameters such as half life in the body, on undesired side effects, if any, on factors such as age and gender, etc.

By a third aspect of the invention, there are provided pharmaceutical compositions which comprise at least one polysaccharide-oligoamine conjugate as an active ingredient.

Thus, the present invention provides pharmaceutical compositions comprising at least one polysaccharide-oligoamine conjugate and a pharmaceutically acceptable carrier, diluent or excipient. These compositions may be used to prevent or treat viral infections, diseases and disorders associated with amyloidoses, and may also be used as veterinary compositions for the treatment of animals.

As used herein, the term "polysaccharide" refers to polymers of more than about ten monosaccharide residues linked glycosidically to each other in branched or unbranched chains. This term includes both naturally occurring polysaccharides as well as synthetic polysaccharides. The monosaccharides in the polymer may be of the same

or different type. For example, dextran is made of D-glucose units connected by a 1,6- glucoside bond, forming a linear chain of various lengths.

In one preferred embodiment of the present invention the polysaccharides are selected from dextran, pullulan and arbinogalactan. The term "oligoamines" refers to chemical entities having at least two amino groups; preferably, the oligoamines have 2, 3 or 4 amino groups and are linear (unbranched). The amino groups may be substituted (thus forming secondary, tertiary or quaternary amine or ammonium groups) with, for example, aliphatic groups of various chain lengths, functional groups, for example, for increasing solubility, chemical or physical affinity, and other groups as may be necessary. Examples of oligoamines are spermine, propane- 1,3-diamine, spermidine, N,N-bis(2-aminoethyl)-l,3- propanediamine, and N,N-bis(2-aminopropyl)-l,2-ethylenediamine. Preferably, the oligoamine is spermine or propane- 1,3 -diamine. In one specific embodiment, the conjugate is dextran-spermine. The term "conjugated" or any lingual variation thereof refer to a product of chemical reaction between at least one polysaccharide and at least one oligoamine. In the resulting conjugate the oligoamines are preferably covalently linked to the polysaccharide. The covalent bond is preferably a biodegradable bond, e.g. by enzymes naturally occurring in mammals, such as peptidases. The covalent conjugation may occur via any synthetic method known to a person skilled in the art, for example, reductive amination, imine bond formation, amidation, amine conjugation, carbamate (urethane) and urea conjugations. In one specific embodiment, the synthetic approach is reductive amination which involves the conversion of a carbonyl group to an amine. The carbonyl group, which is preferably a ketone or an aldehyde, is reacted with the oligoamine, as shown in Scheme 1, to form the imine intermediate. This intermediate may then be isolated and reduced with a suitable reducing agent (e.g. NaBH4). It is also possible to carry out the same reaction all in one pot, with the imine formation and reduction occurring concurrently.

The conjugates formed may be charged or neutral. Typically, charging of the conjugates may occur in physiological medium at physiological pH or by treating the

conjugates with, for example, an aqueous solution at a specific pH, typically an acidic pH, thus obtaining a polycation.

In a conjugate comprising a polysaccharide and an oligoamine, the charged entities or groups are typically the amine groups which may be charged in the presence of free protons or proton-contributing agents. The polycation may not be homogenous or complete, namely, only a certain number of the amine groups may be charged, enough to induce the conjugate with an overall positive charge.

In case of pharmaceutical compositions which comprise the conjugates, the charging of the conjugate may be necessary dependent on the mode of administration. In case of oral administration, for example, the conjugate may be presented in its neutral form and charging may occur in situ, e.g. in the stomach. In other cases, charging may not be necessary. Alternatively, charging of the conjugate may take place during the process of preparing a composition containing thereof, prior to administering the composition, and, for example, in order to increase solubility of said conjugate. The charged conjugates, i.e. the salts of the conjugate may be prepared by any method known to a person skilled in the art.

The compositions of the invention may be homogenous, namely comprising a single type of conjugate, e.g. dextran-spermine or heterogeneous in nature, namely comprise two or more types of conjugates, e.g dextran-spermine conjugate and dextran- propane- 1 ,3 -diamine, at varying ratios.

The covalently bonded oligoamines may all be identical or may be of various structures. The ratio between the polysaccharide and the oligoamine may vary. Typically, the conjugates are made of 1 oligoamine for any 1 to 20 sacchride units making the polysaccharide. For example, in a polysaccharide X having 20 saccharide units, the conjugate with an oligoamine Y may be in a ratio of IX to 2OY, IX to 1OY, IX to 5 Y, IX to 2Y ₅ IX to IY, etc.

The conjugates may be further derivatized in order to improve at least one of the following: decrease their degradation, increase circulation time, increase their penetration either through the skin (especially for the anti-vial treatment) or through the blood brain barriers (BBB, especially for anti-prion, and anti-Alzheimer's disease

treatments), decrease their immunogenicity, decrease their clearance by the liver and the renal system and the like. Specific derivatives are, for example, those prepared by the addition of PEG and in particular methoxy poly(ethylene glycol) [MPEG] in order to provide sterically stabilized compounds and increase BBB penetration. Additional derivatization is the addition of hydrophobic groups, especially unsaturated fatty acids and cholesterol derivatives in order to produce a hydrophobic shield and promote colloidal dispersion.

The compositions of the invention are adapted for the prophylactic and/or treatment of the viral or amyloid diseases (prion and Alzheimer's disease) and may be formulated as pharmaceutical compositions for human use or as veterinary compositions for animal use. These compositions all aspects of may be administered to the subject in need thereof (being a human or a non-human animal) by any method known to a person versed in the art, for example by intraocular administration, oral ingestion, enteric administration, inhalation, cutaneous, subcutaneous, intramuscular, intraperitoneal, intrathecal, intracerbral, intracheal, by intravenous injection, or by topical administration.

The administration of the compositions of all aspects of the invention by the various modes of administrations exemplified may require the presence of pharmaceutically acceptable carriers, such as vehicles, adjuvants, excipients, or diluents. Such carriers are well-known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the conjugate or conjugate mixture being administered and one which has no detrimental side effects or toxicity under the conditions of use. The choice of carrier will be determined in part by the particular conjugate, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following compositions for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are merely exemplary and are in no way limiting. Compositions suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of the conjugate dissolved in diluents, such as water, saline,

or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the conjugate, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.

The conjugates of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

The conjugates may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

Oils, which may be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils.

Suitable preservatives and buffers can be used in formulations comprising the conjugates. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile- lipophile balance (HLB) of from about 12 to about 17. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

As stated above, the conjugates used with the present invention may be made into injectable formulations. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4 ^th ed., pages 622-630 (1986).

Preferably the compositions of the invention should be in a form which readily crosses the blood brain barrier and may be further conjugated, embedded, encapsulated etc, to, in or by any moiety which facilities such transfer through the blood brain barrier.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

MATERIALS AND METHODS Dextran of an average molecular weight of 40 kDa was obtained from Sigma

Chemical Co. (St. Louis, MO). Arabinogalactan with an average molecular weight of 19 kDa was obtained from Larex International (St. Louis, MO). Pullulan of 40 kDa was received from Sigma Chemical Co. (St. Louis, MO). Potassium periodate (KIO ₄ ), sodium borohydride (NaBH ₄ ), polyethylene glycol monomethyl ether (MPEG ₂ ooo), p- nitrophenyl chloroformate, oleate-NHS, spermine, spermidine, Propane- 1,3 -diamine, N,N-bis(2-aminopropyl)- 1 ,2-ethylenediamine and N,N-bis(2-aminoethyl)- 1 ,3 -propane diamine were all purchased from Aldrich (Milwaukee, WI). AU solvents and reagents were of analytical grade and were used as indicated.

Water-free methoxy-PEG2ooo was obtained by azeotropic distillation from toluene and vacuum-dried over P ₂ O ₅ . A sage-metering pump model-365 (Orion, NJ) was used for slow and reproducible addition of reactants.

Average molecular weights of the conjugates were estimated by GPC-Spectra Physics instrument (Darmstadt, Germany) contaim ^' ng a pump, column (Shodex KB- 803) and refractive index (RI) detector. Average molecular weights were estimated

according to pullulan standards (PSS, Mainz, Germany) with molecular weights between 5,800 and 212,000. Eluents used were 0.05M NaNO ₃ for the uncharged polymers and 5% (w/v) Na ₂ HPO ₄ in 3% (v/v) acetonirile (pH 4) for the cationic conjugates. The degree of conjugation was estimated by elemental microanalysis of nitrogen

(%N) using a Perkin-Elmer 2400/11 CHN analyzer.

¹ H-NMR spectra (D ₂ O) were obtained on a Varian 300-MHz spectrometer in 5 mm o.d. tubes. D ₂ O containing tetramethylsilane served as solvent and shift reference. FT-IR spectra were recorded on a Perkin Elmer, 2000 FTIR. Cell culture reagents were from Biological Industries (Beit Haemek, Israel).

Opti-MEM was from Gibco. Tissue culture dishes were from Miniplast (Ein Shemer, Israel). Multiwell plates were from Nunc (Roskilde, Denmark). Secondary antibodies were from Jackson Immunoresearch (West Grove, PA).

SYNTHESIS OF THE CONJUGATES

Example 1: General Synthesis of cationic polysaccharides

The desired polysaccharide such as dextran, pullulan, arabinogalactan, galactomannan, cellulose, amylase and other polysaccharides, (62.5 mmol of glucose units) was dissolved in 200 ml of double deionized water (DDW). Potassium periodate was added at either 1:1, 1:2, or 5:1 mole ratio (glucose/IO ₄ ^' ) and the mixture was stirred in the dark at room temperature for 6-8 hours. The resulting polyaldehyde derivatives labeled as compounds E, F and G were purified from iodate (1O ₃ ^" ) and unreacted periodate (1O ₄ ^' ) by Dowex-1 (acetate form) anion exchange chromatography, followed by extensive dialysis against DDW (3500 cutoff cellulose tubing) at 4 ⁰ C for 3 days. Purified polyaldehyde derivatives were freeze-dried to obtain white powder in 70% average yield. FT-IR (KBr) =1724cm ^'1 (C=O). The aldehyde content was determined by the known hydroxylamine hydrochloride method.

Example 2: Conjugation with oligoamine

Oxidized polysaccharide (Ig, 0.75-6.56 mmol of aldehyde groups) was dissolved in 100 ml of DDW. The dialdehyde solution was slowly added over several hours to a basic solution containing 1.5 equimolar amount of oligoamine dissolved in 50 ml of borate buffer (0.1 M, pH 11). The mixture was stirred at room temperature for 24 hours. NaBH ₄ (1 g, 4 equimolar) was added to reduce the imine bonds to amines and stirring was continued for 48 hours under the same conditions. The reduction was repeated with additional portion OfNaBH ₄ (1 g, 4 equimolar) at the same conditions for

24 hours. The resulting light-yellow solution was poured into a dialysis membrane (3500 cutoff cellulose tubing) and dialyzed against DDW at 4 ⁰ C for 3 days. The dialysate was lyophilized to dryness and stored under nitrogen atmosphere.

Average yield: 50% (w/w). ¹ H-NMR (D ₂ O): 1.645 ppm (m, 4H, Dextran- NH(CH ₂ ) ₃ NHCH ₂ CH ₂ CH ₂ CH ₂ NH(CH ₂ ) ₃ NH ₂ ), 1.804 ppm (m, 4H ₅ Dextran- NHCH ₂ CaCH ₂ NH(CH2) ₄ NHCH ₂ CH ₂ CH ₂ NH ₂ ), 2.815 ppm (m, 12H, Dextran- NHCSC^CH^NHCaCHzC^C^NHCH^C^CSNH^, 3.30-4.45 ppm (m, glucose hydrogens), 5.01 ppm (m, IH, anomeric hydrogen). FT-IR (KBr): 1468 cm ^"1 (-CH ₂ - aliphatic), 1653 cm ^"1 (-NH ₂ , primary amine), 2935 cm ^"1 (C-C, aliphatic) and 3297 cm ^"1 (-NH, -OH groups). The primary amine content was determined by the TNBS method.

Example 3: Dextran-spermine-methoxypoly(ethylene glycol) (MPEG) conjugation

Dextran-spermine conjugate (100 mg, 123 μmol of ε-NH ₂ ), was dissolved in 2.5 ml DDW. Aqueous solution of MPEG ₂ oo ₀ -p-nitrophenyl carbonate (1%, 5% and 10% mol/mol to ε-NH ₂ ) was added to the dextran-spermine solution. The mixture was stirred at room temperature for 16 hours. The modified derivative was purified from p- nitrophenol and unbound MPEG by Sephadex G-25 column chromatography using DDW as eluent. Fractions containing the pegylated derivative were defined by ninhydrin test, collected and freeze-dried to obtain a white powder. Average yield: 80% (w/w). The degree of modification was calculated by spectrophotometric measurement of the released p-nitrophenol (UV, λ=410 nm) after conjugation which was found to be about 95%.

Example 4: Dextran -spermine oleate conjugation

Dextran-spermine conjugate (40 nig, 49.2 μmol of S-NH ₂ ), was dissolved in ImI of DDW and in 2 ml of THF. To this solution 5% or 20% mol/mol to ε-NH ₂ of oleate-

NHS dissolved in anhydrous THF (21 mg in 5 ml THF) was added. The mixture was stirred at room temperature over night and evaporated to dryness. The yellow powder was washed with diethylether and vacuum-dried over NaOH.

Average yield: 85% (w/w). The degree of substitution with oleate-NHS was determined by ¹ H-NMR and found to be 95%. ¹ H-NMR (D ₂ O): 0.696 ppm (t,3H, terminal methyl group of oleate), 1.095 ppm (m,24H, oleate aliphatic hydrogens), 1.437 ppm (m, 4H, Dextran-NH(CH ₂ ) ₃ NHCH ₂ C/iC^CH ₂ NH(CH ₂ ) ₃ NH ₂ ) ₅ 1.617 ppm (m, 4H, Dextran-NHCH ₂ CH^CH ₂ NH(CH ₂ ) ₄ NHCH ₂ CH ₂ CH ₂ NH ₂ ), 2.15-3.26 ppm (m, 12H, Dextran-NHCHjCH ₂ CH^NHCH^CH ₂ CH ₂ CHiNHCHjCH ₂ CH^NH2), 3.30-4.45 ppm (m, glucose hydrogens), 5.01 ppm (m, IH, anomeric hydrogen) and 5.14 ppm (m, 2H olefin hydrogens of oleate). Example 5: Synthesis of cellobiose-spermine

2 gr of cellobiose (5.84 mmol) was dissolved in 35 ml of DDW. The cellobiose solution was slowly added over several hours to a basic solution containing 1.5 equimolar amount of spermine dissolved in 50 ml of borate buffer (0.1 M, pH 11). The mixture was stirred at 37 ⁰ C for 96 hours. NaBH ₄ (1 g, 4 equimolar) was added to reduce the irnine bonds to amines and stirring was continued for 48 hours under the same conditions. The reduction was repeated with additional portion of NaBH ₄ (1 g, 4 equimolar) at the same conditions for 24 hours. Resulted white solid was purified by precipitation in EtOH followed by purification in Sephadex G-10.

¹ H-NMR (D ₂ O): 1.5 ppm (m, 4H, Cellobiose- NH(CH ₂ ) ₃ NHCH ₂ CZf _a CH _a CH ₂ NH(CH ₂ ) ₃ NH ₂ ), 1.7 ppm (m, 4H, Cellobiose-

NHCH ₂ CH ₁ CH ₂ NH(CH ₂ ) ₄ NHCH ₂ CH ₂ CH ₂ NH ₂ ), 2.7 ppm (m, 12H, Cellobiose-

NHCaCH ₂ CSNHCHjCH ₂ CH ₂ CaNHCHaCH ₂ CHjNH ₂ ), 3.30-4.45 ppm (m, glucose hydrogens).

Example 6: Synthesis of Dextran-Monoquaternary ammonium spermine conjugate

Oxidized polysaccharide (6.25 mmol of aldehyde groups) was dissolved in 100 ml of double deionized water (DDW) and the solution was slowly added over several hours to a solution containing 6.25 mmol monoquaternary ammonium spermine dissolved in 50 ml of borate buffer (0.1 M, pH 11). The mixture was stirred at room temperature for 24 h. NaBH ₄ (Ig ₅ 4 equimolar) was added to reduce the formed imine bonds and stirring was continued for 48 h at room temperature. The reduction was repeated with an additional portion of NaBH ₄ (1 g) and stirring was continued for additional 24 h. The resulting light-yellow solution was poured into a dialysis membrane (3500 cutoff, Membrane Filtration Products, Inc., San Antonio, TX) and dialyzed against DDW at 4 ⁰ C for 3 days. The dialysate was lyophilized to dryness.

Average yield: 50% (w/w). The crude was characterized by elemental analysis and ¹ H-NMR (D ₂ O): 1.45 (m, 4H, spermine hydrogens), 1.63 (m, 4H, spermine hydrogens), 2.64 (m, 1OH, spermine hydrogens), 3.0 (s, 9H, methyl hydrogens of monoquaternary spermine), 3.0 (m, 2H, spermine hydrogens), 3.30-4.45 (m, glucose hydrogens) and 5.01 (m, IH, anomeric hydrogen).

ANTI-VIRAL COMPOSITIONS

Anti-viral activity and Toxicity Various conjugates of polysaccharide derivatives having multiple amine functionalities of different oligoamines prepared by reductive animation were tested for their antiviral activity in BS-C-I cell line.

Cell monolayers (BS-C-I, a cell line derived from green monkey kidney cells) were infected with a suspension of virus (i.e. HSV-I, HSV-2, vaccinia and poliomyelitis) in order to give confluency of plaques. When the agar containing overlay was solidified, a disc of Whatman 3 MM paper, 5 mm in diameter, was immersed in a solution containing the examined compound at a desired concentration and layered on the agar in the center of the culture. ACV, a drug treatment currently used for the herpes simplex infections, dissolved in DMSO (4.8mg/ml), was used as a positive inhibitory

control as well as isotin beta thiosemicarbazone (IBT) that was used as a positive inhibitory control for vaccinia virus. The cultures were incubated at 37 ⁰ C for four to five days and then fixed and stained. Data analysis was evaluated by measurement of diameters: the diameter of the central area, in which the cell monolayer was destroyed by the toxicity of the compound and, as a result, lost the stain, and the diameter of the external area, in which the cells were alive but plaques were not formed. The first diameter reflected the toxicity of the tested compound, while the second, following deduction of the first, served as a semiquantitative criterion for anti-virus activity. A negative control, using just the solvent, (DMSO or DDW), is regularly added to the assay.

Results

Table 2 shows the results of the antiviral activity of the specific polysaccharide- polyamine conjugates tested. These results show that dextran-propane- 1,3 -diamine has antiviral activity against HSV-I and HSV-2. There was a strict correlation between the activity of the conjugates and their concentration and no toxicity effect on the cell viability was detected. Although the antiviral reactivity is not strong as of ACV, it is present indeed.

Although several structural parameters influence the antiviral potency of various conjugates, the most important feature appears to be the oligoamine graft onto the sugar backbone. Whereas diamine residue grafted on oxidized dextran demonstrated high antiviral potency against HSV-I and HSV-2, neither tetracaine nor triamine showed any significant antiviral activity against the three tested viruses.

Efficiency of the conjugate directly contributes to the modification of the polysaccharide backbone with appropriate oligoamine. Unconjugated dextran demonstrated no inhibitory effect on the three tested viruses, while the conjugate of dextran-propane- 1,3 -diamine was the most efficient compound.

The dextran-spermine conjugate was modified with MPEG (M _w =2 kDa) to obtain partially shielded molecules, with oleic acid to provide hydrophobic nature to the conjugate and with mono-quaternary ammonium spermine that imparts cationic charge to the polymer. The results show that the effectiveness of the dextran-spermine can not

be attributed to any of these modifications, because no inhibitory effect can be seen with these compounds. The nature of the polysaccharide backbone also showed minor effect on the potency of the dextran-spermine.

Table 2. Antiviral activity characterization of the conjugates. HSV-I = herpes simplex virus type 1; HSV-2 = herpes simplex virus type 2; Vaccinia = vaccinia virus, Western Reserve (WR) strain; Polio = poliomyelitis virus, Sabin type 1; T = Toxicity (diameter in mm); - = Samples not tested for antiviral activity. Explanations to results given in Table 2:

The results for the different viral activities of the conjugates tested are in the format "niT. n^\ wherein "ni" is an integer which gives the diameter (in mm) of the dead cells which in turn indicates toxicity ("T"); and "n∑" is an integer which gives the diameter (in mm) of the viral inhibition. For example, for the antiviral activity of the dextran-propane- 1,3 -diamine (4.8 mg/ml) against HSV-I, the cell toxicity can be determined according to "OT. 17", as follows: the nf=Q indicating that no toxicity effect was observed on the cell viability and the n^l7 indicating that a moderate antiviral activity was achieved (17 mm in diameter of the HSV-I inhibition). The antiviral activity can be classified based on the size of "fi ₂ ^f \ whereas for « ₂ in the range of 0 to 9 the antiviral activity is considered low; in the range of 10 to 20 the antiviral activity of the compound is considered moderate; and for the range of 21 and up the antiviral activity is considered high.

ANTI-AMYLOID COMPOSITIONS

Anti-amyloid experiments The therapeutic potential of the conjugates and their derivatives are determined both in vivo and in vitro in the following models: (i) a transgenic mouse model of Alzheimer disease (AD), (ii) prion-infected mice. Additional mechanistic and

pharmacologic information are derived from mice induced to develop AA (systemic) amyloidosis.

Conjugates of dextran, pullulan and arabinogalactan grafted with oligoamines of 2 to 4 amino groups were investigated for their ability to eliminate PrP ^Sc , the protease- resistant isoform of the prion protein, from chronically infected neuroblastoma cells, ScN2a-M. The Proteinase K (PK)-resistant PrP elimination depends on both the concentration of the reagent and the duration of exposure. The most potent compound was found to be dextran-spermine that caused depletion of PrP ^Sc to undetectable levels at concentration of 31 ng/mL after 4 days of exposure. Activity analysis revealed that grafted oligoamine identity of the poly cation plays a significant role in elimination of PK-resistant PrP from chronically infected N2a-M cells, regardless of the polysaccharide used. Dextran-spermine conjugates were modified with oleic acid and with methoxypoly(ethylene glycol) (MPEG) at various degrees of substitution for further studies and their antiprion activity was examined. Substitution of dextran- spermine with MPEG or oleic acid slightly decreases its activity as a function of MPEG/oleic acid content.

Amyloid fibril assembly using purified and synthetic proteins/peptides with wild type and mutant sequences

The three polypeptides involved in the model amyloidoses can assemble into amyloid fibrils in vitro. This process follows the standard "seeding-elongation" pattern. The influence of the conjugate molecules on the assembly and disassembly of these fibrils was measured, as follows: Recombinant PrP, Aβ, and SAA were purchased for InPro (South San Francisco) and Sigma, respectively. SAAl.1 was produced from overexpressing E. CoIi. Formation of amyloids was performed using established procedures and was monitored both by birefringent Congo red staining and by negative staining electron microscopy. The formation of β -sheets was followed by CD spectroscopy and FTIR. Fibrils were sized using biophysical methods. SD was included at various time points of the seeding and elongation processes. Both kinetics and final yields were recorded.

CeII culture methods for Alzheimer's disease and prions

Cell culture systems were used to follow the influence of conjugates on (i) the formation of Aβ, and (ii) the propagation and toxicity of prions and the metabolism of PrP. The formation of Aβ is a complex process that involves the processing of amyloid precursor protein, APP ₅ by β- and γ-secretases and decreased α-secretase activity. Several cell culture systems recapitulate these processes. The influence on the formation and precipitation of Aβ in human neuroblastoma lines, including APP overexpressors was studied. Aβ was monitored by Tricine PAGE and quantified using commercial ELISA systems. Flotation assays were performed in order to determine whether the conjugates alter the association of APP, Aβ, or the β secretase BACE with cholesterol rafts. The association of the resulting Aβ with raft lipids, and especially with ganglioside GMl, was studied following methanol extraction using cholera toxoid B as a ligand.

The influence of the conjugates of the invention on prions was studied in 2 mouse lines in culture: the neuroblastoma N2a, and the immortalized neurons GTl. These lines were infected with several strains of mouse-adapted scrapie. The GTl system was studied and was determined to include several other scrapie strains such as ME7, since the amyloids formed by various strain vastly differ in properties such as protease-resistance. (i) The formation of PrP ^Sc was monitored using established immunoassays, (ii) The accumulation of PrP ° in infected cells provoked an abnormal pattern of intracellular cholesterol deposition, a phenomenon that may explain prion toxicity, (iii) The influence of conjugate on the de novo infection of cells by prions, and especially on the binding and internalization of exogenous prions was examined. In vivo assays Animal experiments are performed in compliance with current legislation for animal welfare. The anti-amyloid activity of the lead conjugate molecules as well as various formulations thereof is assessed in vivo, using the following models:

1. Many transgenic mouse models for AD are now available. Both a fast model (such as the TGCRND 8 mouse available though the Jackson Laboratories) and one slower model are used. Mice are inoculated with the conjuagets intraperitoneally twice a week, starting either before the first neuropathological signs or at their onset. In the

short TGCRND8 model, both thioflavine S-positive Abeta deposits and behavioral changes are already evident at 3 months of age. The effect of conjugates on the clinical and the neuropethological signs and ties . of onset are recorded. Histopathology and amyloid staining are performed using established procedures. 2. The effect of conjuagtes on the development of prion diseases is assayed in

C57bl mice inoculated either intracerebrally or peripherally (intraperitoneally) with one of several strains of scrapie. The mice are kept in the P3 biocontainement animal facility at Ein Kerem. Scrapie incubation time, time of death, levels of PrP ^Sc , and the extent of neuronal vacuolation and glial activation are recorded. 3. Systemic amyloidosis is induced in C57bl mice by the AEF/ AgNO ₃ procedure. Amyloid enhancing factor (AEF) is prepared from AA amyloid fibrils and administered i.p. (200 μg per animal). Immediately thereafter, 0.5 ml of AgNO ₃ (2% solution) is injected subcutaneously. Seven day after the inoculation, mice are examined for tissue deposition of SAA amyloids using existing procedures. Animals and Amyloid Induction

The homozygous mouse strain overexpressing human heparanase (hpa-tg) and the respective control (ctr) mice (C57BL background) were generated and maintained in the animal facility. The animal experiments were performed in compliance with Swedish legislation for animal welfare (approval number C 176/2). Amyloidosis was induced as follows: amyloid enhancing factor (AEF), prepared as AA amyloid fibrils, was administered i.p. (200 μg per mouse, 200 μl, n = 6 hpa-tg and n = 6 cti" male mice, 10 weeks old). Immediately after the administration of AEF ₅ 0.5 ml of AgNO ₃ (2% solution) was injected s.c. into the loose tissue of the back, between the shoulder blades. Mice were killed by cervical dislocation 7 days after commencement of the induction protocol. Spleens, livers, and kidneys were dissected from each animal, fixed overnight in a solution containing 96% ethanol, 1% glacial acetic acid, and 3% distilled water, and stored in 70% ethanol until processed for histological analysis. Histochemical Analyses Tissues in 70% ethanol were dehydrated by using standard procedures and embedded in paraffin. Sections of 8-10 μm were stained with Congo red to detect amyloid deposition and with sulfated Alcian blue (SAB) to detect sulfated glycosaminoglycans. The percent tissue area occupied by birefringent Congo red-

positive staining in polarized light was determined as described by image analysis by using a program and apparatus from MCID M2 Imaging Research. AU comparisons were made after calibrating the apparatus against a set of standard spleen sections containing AA amyloid. Example 1: The effect of PSP on serum amyloid A (SAA) amyloidosis.

Systemic (serum amyloid A) amyloidosis is induced in C57bl mice by the AEF/ AgNO ₃ procedure. Amyloid enhancing factor (AEF) prepared from AA amyloid fibrils is administered intraperitoneally (200 μg per animal, dissolved in saline solution). Immediately thereafter, 0.5 ml of AgNO ₃ (2% solution) is injected subcutaneously. To examine the effects of PSP on serum amyloid A amyloidosis, mice are divided in four groups: Groups 1 and 2 contain animals in which amyloidosis is induced as described above on day 0. Mice in groups 3 and 4 are control age-matched mice which are inoculated on day 0 with the solvents only (saline and water). Groups 2 and 4 are administered with dextran-spermine conjugate by intraperitoneal injection either once on the day of amyloid induction (day 1), or twice (days 0 and 3), or once on day 3. On the same days, animals from parallel subgroups of groups 1 and 3 receive the equivalent amount of saline i.p.

On day 7, mice are examined for tissue deposition of SAA amyloids using histological procedures. Spleens, livers, and kidneys are dissected, fixed in 95% ethanol and 1% acetic acid and then embedded in paraffin using standard procedures. Amyloid deposits are detected in paraffin sections through their characteristic green- gold birefringence following staining with Congo red staining. The levels of circulating SAA are examined by Western immunoblots.

All the PSP administration regimes vastly reduce the amyloid deposits in all three organs. The double inoculation (days 0 and 3) in particular reduce amyloids to undetectable levels. In contrast, soluble SAA levels are not changed by these treatments, showing that it is the deposition of amyloids and not the inflammatory increase in SAA mat is targeted by these compounds.

Example 2: The effect of PSP on amyloid deposition in mice models of Alzheimer's disease (AD)

The fast TGCRND8 transgenic mouse model (Jackson Laboratories) is used for these experiments. These mice overexpress mutants human APP. Thioflavine positive

brain deposits of Aβ and behavioral changes are evident in these mice already at three months of age. Mice are divided in four groups: Groups 1 and 2 are composed of TGCRND8 mice. Groups 3 and 4 are age matched mice. Groups 2 and 4 are inoculated with dextran-sperrnine conjugate intraperitoneally twice a week, starting either before the first neuropathological signs or at their onset. Groups 1 and 3 receive the equivalent amount of saline. The effect of PSP on (i) the clinical and neuropathological signs and (ii) the time of onset are recorded. Histopathology and amyloid staining (with Congo red and thioflavine S) are performed using established procedures.

Biodegradable conjugates prepared by grafting linear (non-branched) oligoamine residues on natural polysaccharides eliminated PrP ^Sc from chronically infected ScN2a-M neuroblastoma cells in a dose- and time-dependent manner. The most effective dextran derivative was dextran-spermine with one spermine unit per 2.03 saccharide units, which completely eliminated PrP ^Sc at a conjugate concentration of 3 lng/ml. There was a strict correlation between the activity of the conjugates and between their overall oligoamine content (Table 3). The oligoamine content integrates several quantitative features of the conjugate, including (i) the M _w of the oligoamine molecule serving as a graft and (ii) the actual degree of conjugation in each particular conjugate.

Although several structural parameters influence the anti-prion potency of the various conjugates, the most important feature appears to be the identity of the oligoamine grafted onto the sugar backbone, while other parameters seem to have played a less significant role.

The nature of the polysaccharide backbone has a relatively minor effect on the efficiency of the conjugates. Dextran-spermine was the most efficient (active dose 31 ng/ml), while spermine grafted in the same manner on pullulan and arabinogalactan and chitosan (not shown) resulted in slightly less active, but still effective, conjugates (active dose=125 ng/ml). Branching of the polysaccharide backbone (in arabinogalactan) had no significant influence on the activity of the conjugate.

One important requirement for anti-prion drugs is that they cross the blood-brain barrier. Dextran-spermine was coated with either MPEG _2O oo, a hydrophilic, inert and biocompatible polymer at 1%, 5% and 10% mol/mol degrees of substitution relative to the primary amine of the spermine, or with the hydrophobic chain oleic acid at 5% and

20% mol/mol to the primary amine. Although pegylated and oleic acid substituted dextran-spermine had reduced activity as compared to the non substituted conjugates in cells (in vitro), the PEGylated and hysdrophobic sunstituents are preferred in vivo, as their bioavailability turns out to be superior to that of their non-shielded analogs. Example 3: Ex vivo biological activity

Cells: Chronically PrP ^Sc infected mouse neuroblastoma cells ScN2a-M were stably transfected with a vector that expresses the MHM2-PrP chimeric gene that reacts with mAb 3F4. Cells were grown at 37 ⁰ C in DMEM- 16 (Ig glucose/liter) supplemented with 10% fetal calf serum (FCS). Treatments with the conjugates were performed on cells grown in 12-well dishes in DMEM- 16/Opti-MEM (1:1) supplemented with 5% FCS.

Antibodies: MAb 3F4 binds to residues Met ¹⁰⁸ and Met ¹¹¹ in chimeric MHM2- PrP but does not recognize the wild-type mouse PrP endogenous to N2a cells. This antibody was used at a dilution of 1 :5000 for Western blot. PrP Analysis: PrP ^Sc was defined as the PrP fraction resistant to proteolysis catalyzed by proteinase K (20 μg/ml, 37 ⁰ C, 30 min). The protease activity was stopped by adding phenylmethylsulfonyl fluoride at 2mM. SDS-PAGE. Sample preparation and western immunoblotting of the PrP isoforms were carried as follows: cells were lysed in ice-cold "standard" lysis buffer (0.5% Triton X-100, 0.23% sodium deoxycholate, 150 mM NaCl, 10 mM Tris-Cl, pH 7.5, 1OmM EDTA), and the lysates were immediately centrifuged for 40 sec at 14,000 rpm in microcentrifuge. All of the biochemical analyses were performed on this postnuclear fraction. Protein concentration in cell lysates were measured using the Bradford assay (Bio-Rad) where needed. Lysates were resolved in 12% polyacrylamide gels and electrotransferred to PolyScreen polyvinylidene difluoride membranes (PVDF) in a Tris/glycine buffer containing Sarkosyl (48mM Tris base, 39mM glycine, 20%methanol, 0.001% Sarkosyl). The membranes were blocked for 30 min with low fat milk prior to incubation with mAb 3F4. HRP-conjugated secondary antibodies were used at 1:10,000 dilution and the blots were developed by chemoluminescence. As Table 3 shows, that complete eradication of PrP ^Sc was successfully achieved with all conjugates tested. However, the conjugate of dextran and spermine was most efficient in eradicating PrP ^Sc as only 31 ng/ml of the conjugate were needed in

comparison to 125 ng/ml for spermidine and 500 ng/ml for N,N-bis(2-aminopropyl)- 1 ,2-ethylenediamine.

% Oligoamine Primary

Code Oligoamine Activity %N binding content amine

A Propane- 1 ,3 -diamine >500 10.18 75 26 2.09

B Spermidine 125 10.05 59 35 1.48

N,N-bis(2-aminoethyl)- 1,3-

125 11.7 51 34 1.06 propanediamine

N,N-bis(2-aminopropyl)- 1,2-

D 500 9.67 40 30 1.28 ethylenediamine

E Spermine 31 10.49 48 38 1.23

Table 3: Chemical characterization and activity of dextran-oligoamine conjugates.

5 Appropriate oligoamine was reacted with oxidized dextran (-50% dialdehyde) at 1:1.5 aldehyde/oligoamine mole ratio under the same conditions as described above. The activity relates to the approximate concentration of polymer (ng/ml) required for complete eradication of PrP ^Sc from ScN2a-M after exposure for 4 days. %N- is the nitrogen content determined by elemental analysis. The %binding refers to the percent 0 of substituted saccharide units of a particular conjugate. The oligoamine content is measured in 100 g of corresponding conjugate. The amount of primary amine is measured in mmol/g in conjugates, as determined by the TNBS method. The molecular weights, Mw, of the conjugate was determined by GPC and was found to be 6,000 Da, except of spermine conjugate that was 11,000 Da. 5 Example 4: Activity of various dextran-oligoamine conjugates against PrP^ ⁰ ex vivo

Cationic polysaccharides were synthesized by conjugation of oligoamine to oxidized polysaccharides for example by reductive animation as shown in Scheme 1.

The polycations were characterized for their structure ( ¹ H-NMR), nitrogen content (%N) and primary amine content (TNBS) as shown in Table 4. The polycations had similar % amine content ranging from 9.67 and 11.7.

Primary Oligoamine %

% dialdehyde P Mw %N Activity Polymer amine content binding

12 1.4 11.600 0.45 9.6 8 2.65 >2000 F

10.4

48 1.2 11.000 1.23 38 48 31 E 9

13.2

105 2.5 91.500 1.23 47.8 72 500 G 5

5 Table 4: Chemical characterization of dextran-spermine conjugates at different degrees of substitution. Oxidized dextran was reacted with spermine at 1:1.5 aldehyde/oligoamine mole ratio under the same conditions as described above. The activity relates to the approximate concentration of polymer (ng/ml) required for complete eradication of PrP ^Sc from ScN2a-M after exposure for 4 days. %N- is the 0 nitrogen content determined by elemental analysis. The % binding refers to the percent of substituted saccharide units of a particular conjugate. The oligoamine content is measured in 100 g of corresponding conjugate. The amount of primary amine is measured in mmol/g in conjugates, as determined by the TNBS method. Average molecular weight (Mw) and polydispersity (P=Mw/Mn) were determined by GPC. 5 Molecular weight of the FI-70 differs from other conjugates due to slight crosslinking between the polyaldehyde and spermine during conjugation. The % dialdehyde refers to the content of the oxidized dextran prior to spermine conjugation as determined by hydroxylarnine hydrochloride titration method.

Scrapie-infected mouse neuroblastoma ScN2a-M cells were exposed to various 0 concentrations (0-2000ng/ml) of the polycations for four days (because PrP ° is very stable in cells, several days of treatment are required to eliminate preexisting PrP ^Sc ). Elimination of scrapie was demonstrated by the disappearance of the proteinase K (PK)- resistant core of PrP ^Sc , as monitored in Western blots (WB) developed with the PrP Ab

3F4. Most effective was dextran-spermine, which eliminated PrP ^Sc at 31ng/ml, whereas dextran-propane-l,3-diamine reduced PrP ^Sc only slightly even at 500 ng/ml. The triamines and tetramines: dextran-spermidine, dextran- N,N-bis(2-aminoethyi)-l,3- propanediamine and dextran-N,N-bis(2-aminopropyl)-l,2~ethylene diamine were less effective (125, 125 and 500 ng/ml, respectively) than dextran-spermine, but better than the propane- 1,3 -diamine derivative (not shown). Based on these results dextran- spermine was selected for further studies.

Example 5: Activity of dextran-spermine with increasing spermine content In order to determine whether the nitrogen content directly contributes to the

10 potency of the compounds, we tested dextran-spermine conjugates with various degrees of spermine substitution (prepared by conjugation of increasing amounts of spermine onto oxidized dextran). The compounds also differed in their M _w (see legend to Table 4). The polycations were characterized for their average molecular weight (GPC), structure ( ¹ H-NMR), nitrogen content (%N) and primary amine content (TNBS) as

15 shown in Table 4. Interestingly, most efficient was the moderately substituted conjugate E, whereas both smaller and higher degrees of substitution reduced the anti-prion efficiency of the conjugates. The reasons for the reduced potency of G remain to be determined.

Oligoamin Primary

Polymer Polysaccharide Activ. %N %binding Mw P content amine

H Dextran 31 10.49 48 38 1.23 11.00 1.2

I Pullulan 125 8.86 37 31 1.35 17.00 1.4

J Arabinogalactan 125 8.86 37 31 1 17.00 1.5

Table 5: Chemical characterization and activity of polysaccharide-spermine conjugates. 0 Appropriate oxidized polysaccharide (~50% dialdehyde) was reacted with spermine at 1:1.5 aldehyde/oligoamine mole ratio under the same conditions as described above. The activity relates to the approximate concentration of polymer (ng/ml) required for complete eradication of PrP ^Sc from ScN2a-M after exposure for 4 days. %N~ is the nitrogen content determined by elemental analysis. The %binding refers to the percent

of substituted saccharide units of a particular conjugate. The oligoamine content is measured in 100 g of corresponding conjugate. The amount of primary amine is measured in mmol/g in conjugates, as determined by the TNBS method. Average molecular weight (Mw) and polydispersity (P=Mw/Mn) were determined by GPC. Molecular weight of the FI-70 differs from other conjugates due to slight crosslinking between the polyaldehyde and spermine during conjugation. The % dialdehyde refers to the content of the oxidized dextran prior to spermine conjugation as determined by hydroxylamine hydrochloride titration method.

Example 6: Effect of the polysaccharide backbone To examine the effect of the polysaccharide structure on the activity of the agents, spermine grafted on pullulan, arabinogalactan and dextran were examined. Dextran and pullulan are linear chains with glucose units connected by 1,4 and 1,6- glycoside bonds, respectively. Arabinogalactan is a branched polymer and its units are connected by 1,3-glycoside bonds. The polycations were characterized for their average molecular weight (GPC), structure ( ¹ H-NMR), nitrogen content (%N) and primary amine content (TNBS) as shown in Table 5. According to WB analysis (data not shown) all three cationic polysaccharides inhibited PK-resistant PrP from ScN2a-M cells at a very low concentration ranging from 31 to 125 ng/ml (Table 5).

Example 7: Activity of methoxypoly(ethylene glycol) PMPEG] conjugated dextranspermine

The purpose of this part of the study was to evaluate the shielding effect of MPEG and its influence on PrP ^Sc by applying pegylated dextran-spermine conjugates, with the aim of finding effective compounds that can be delivered to the brain. Highly charged polycations can form aggregates with serum components and thus might not cross the blood-brain barrier. Polycations that are shielded with an inert and biocompatible polymer such as PEG are defined as "sterically stabilized" compounds. For this reason dextran-spermine was modified with increasing amounts of MPEG to obtain partially shielded molecules. MPEG (M _w =2 kDa) was activated with p- nitrophenyl chloroformate to obtain p-nitrophenyl carbonate derivative. Substitution of dextran-spermine was carried out at room temperature in aqueous solution. The amount of MPEG was fixed at 1%, 5% and 10% (mol/mol) to the primary amines of spermine.

The pegylated derivatives were characterized for their average molecular weight (GPC) and structure ( ¹ H-NMR) as shown in Table 6.

The WB analysis indicated that the MPEG derivatives were active in the elimination of PrP ^Sc at concentrations ranging from 80 and 500 ng/ml. Increase in MPEG content in the conjugate decreased the anti-prion effectiveness of the compound.

% Substitution to ε- Activity"

Polymer No. P NH ₂ (ng/ml)

E 0% 11.000 1.2 31

K 1%MPEG2OOO 10300 3 250

L 5% MPEG2000 11000 3.1 500

M 10% MPEG2000 7400 2.9 >500

N 5%Oleate 22.000 1.6 125-250

O 20%Oleate 6.500 2.3 250

Table 6: Chemical characterization and anti-prion activity of pegylated and oleate dextran-spermine derivatives. %N of dextran-spermine conjugate (#E) was 10.49% and the amount of primary amines was 1.23 mmol/g as determined by TNBS method. ^a Average molecular weight (Mw) and polydispersity (P=Mw/Mn) were determined by GPC. ^b See notes in Table 2.

Table 6 demonstrates the effect of modification by PEGilation or fatty chain conjugation on the anti-prion activity using dextran spermine, the most effective anti- prion agent in this study (31mg/ml) as starting compound. As can be seen, PEGilation reduces the activity as a function of the amount of PEGilation applied. Same is true for the fatty chain modification.

Example 8: Activity of oleate dextran-spermine derivative as PrP ⁸⁰ elimination agent

The activity of oleate conjugated dextran-spermine was determined. Oleic acid is an unsaturated fatty acid that imparts hydrophobic nature to the conjugate. Addition of oleic acid to the polycation provides a hydrophobic shield to the polymer and

condensates into a colloidal dispersion if enough oleate groups are introduced. Dextran- spermine was hydrophobized with increasing amounts of oleic acid by adding oleic acid N-hydroxy succinimide (NHS) ester to concentrated solution of dextran-spermine in a mixture of DDW/THF. The hydrophobized conjugates were characterized for their average molecular weights (GPC) and structure ( ¹ H-NMR) as shown in Table 4.

Oleate derivatives showed moderate potency in scrapie inhibition (250 ng/ml) relative to unmodified dextran-spermine. No distinct difference in anti-prion activity between the hydrophobized conjugates of increasing oleate content was observed (data not shown). Example 9: Kinetics of PrF^ ⁰ elimination by dextran-spermine as compared to pentosan polysulfate

The following results demonstrate the potency of dextran-spermine to eliminate PK resistant PrP from ScN2a-M cells after short treatments. ScN2a-M cells were treated with either dextran-spermine (2 μg/ml) or pentosan polysulfate (PPS) (10 μg/ml) for the indicated times, and the elimination of PrP ^Sc was assessed by WB developed with mAb 3F4. Although the two compounds caused a substantial reduction in PrP ^Sc levels after 100 hours, 2 μg/ml of dextran-spermine eliminated PrP ^Sc much more quickly (24 hours, as compared to 100 hours for PPS).

Scheme 1

(Dialdehyde derivative)

Dextran-Spermine Dextran-Spermine (Imine conjugate) (Amine conjugate)