WO1991008747A1

WO1991008747A1 - Synthetic receptor molecules recognizable by a rotavirus

Info

Publication number: WO1991008747A1
Application number: PCT/US1990/007120
Authority: WO
Inventors: Falguni Dasgupta; Cheryl A. Srnka; Roger A. Laine
Original assignee: Glycomed Incorporated
Priority date: 1989-12-13
Filing date: 1990-12-05
Publication date: 1991-06-27
Also published as: AU6902691A

Abstract

Molecules which are active as rotavirus receptor sites are disclosed. These molecules are synthetically produced sugar derivatives which have a minimum size recognized by the antireceptor of a rotavirus. The molecules are useful in studying the interaction between the rotavirus antireceptor and most receptor sites so as to develop drugs useful in blocking the connection between such viruses and the host cells. Further, the molecules can be modified in order to increase the degree to which they attract the antireceptor sites of the virus and/or modified by binding to them antiviral compounds which are effective in destroying the virus once the receptor molecule binds to the virus. Synthetic receptor molecules recognizable by a rotavirus are obtainable by modifying the hydroxyl sites of N-acetyl-D-galactosamine (GalNAc) in the immediate vicinity of the glycosidic linkage of GalNAc. Similar modifications of other sugar residues are taught in order to obtain similar synthetic receptor molecules which are recognizable by a rotavirus as a receptor.

Description

AIDS sufferers and have been implicated as a significant cause of traveler's diarrhea. Currently, there is no effective prophylactic or therapeutic drug available to combat rotaviral disorders and attempts to develop vaccines have been problematic.

In order to infect cells and replicate, viruses bind specific receptors on the target cell surface. After attachment, the virus fuses with the cell membrane and is internalized where it uses the target cell's own metabolism to replicate. The initial attachment process is therefore essential to successful infection. For several viruses, a glycoprotein on the viral surface interacts with a specific target cell carbohydrate.

Like all viruses, a rotavirus has a virion which attaches to the surface of a host cell. The attachment is brought about by the specific binding of the virion protein (the antireceptor) to a constituent of the cell surface (the receptor). A classic example of an antireceptor is the hemagglutinin of influenza virus (orthomyxovirus). The antireceptors are distributed throughout the surface of viruses which infect human cells. Complex viruses, such as vaccinia (a pox virus) and herpes symplex virus (a herpes virus), may have more than one species of antireceptor molecules. Furthermore, antireceptor molecules may have several domains, each of which may react with a different receptor. Accordingly, complex structures and interactions are clearly involved with respect to receptors and

antireceptors.

The details of the initial interaction between rotaviruses and the host cell surface have not been

completely elucidated. However, sialic acid appears to be an important component of the rotavirus receptor, Yolken et al., J. Clin. Invest. 79: 148-154 (1987), and asialo GM1 binds rotavirus and inhibits viral replication in plaque reduction assays, Willoughby et al., abstract from Proceedings of U.S.Japan International Rotavirus Meeting, Annapolis, MD, August

SYNTHETIC RECEPTOR MOLECULES RECOGNIZABLE BY A ROTAVIRUS

Field of the Invention

This invention relates generally to the field of rotavirus receptors. More particularly, this invention relates to synthetically produced compounds such as carbohydrate epitopes and analogs thereof, which compounds are of a minimum size recognized by the antireceptor of a rotavirus.

Background of the Invention

Rotaviruses are double stranded RNA viruses of the family Reoviridae. These viruses replicate in the intestinal epithelial cells of a wide range of animal species including most mammalian and avian species and are the major etiological agents of several gastrointestinal disorders in humans and other animals. For example, rotaviruses are responsible for infantile diarrhea and enteritis, causing infant morbidity and mortality. Rotaviruses also cause diarrheal illnesses in calves and piglets, as well as other mammals.

These viruses are responsible for debilitating diarrhea in immune-compromised patients such as transplant recipients and 1989 J. Virology (1990) in press. Furthermore, bovine submaxillary mucin and chicken ovoinhibitor have been shown to prevent rotavirus gastroenteritis in mice. Yolken et al., supra. Additionally, it has been shown that rotavirus strains isolated from one species cross-react with hosts of another species (see e.g. Leece et al., Infect. Immun. 14: 816-825 (1976); Mebus et al., Infect. Immun. 14: 471-474 (1976); Wyatt et al., Science 207: 189-191 (1980)), suggesting conservation of rotaviral receptors between species.

Viral binding molecules to which a rotavirus binds could be bound to antiviral drugs and rotaviral binding molecules by themselves could be used to hinder or prevent the subsequent infection of host cells. A distinct advantage of such an approach over traditional methods of preventing viral infections, e.g. vaccines, is that the portion of the viral protein normally binding to the specific cell surface carbohydrate does not mutate. Thus, antiviral agents which act by preventing viral binding to host cells are likely to remain effective in the face of mutations to other parts of the viral genome.

Summary of the Invention

The present invention is based on the discovery of a variety of synthetic compounds of minimal size (such as carbohydrate epitopes or analogs thereof) which avidly bind rotavirus. The compounds can be used to prevent interaction of the virus with a target cell and thus prevent viral infection. These compounds can be bound to antiviral drugs and advantageously employed to treat rotaviral infections. The discovery and chemical characterization of relatively small sugars able to bind rotavirus allows for the efficient production of compositions useful in the prevention and treatment of rotaviral infections.

A primary object of the present invention is to provide a rotavirus receptor compound. Another object of the subject invention is directed to a composition for preventing or treating rotaviral infection comprising a therapeutically effective amount of

synthetic rotavirus receptor compound in combination with a pharmaceutically acceptable carrier.

Yet another object of the subject invention is directed to methods of producing molecules able to bind rotavirus.

Still other objects of the subject invention include methods for preventing or treating rotaviral induced disorders by administering to a subject a therapeutically effective amount of a synthetic rotavirus receptors.

A feature of the present invention is that the molecules are of the smallest possible size which can still be recognized by the antireceptor of a rotavirus.

An advantage of the present invention is that the small sized receptor molecules can be linked to antiviral drugs to provide effective antiviral compositions.

Another advantage of the present invention is that the compounds can be liked to probes such as radioactive probes to form conjugates which can be used to test for the presence of rotavirus and/or the binding effectiveness of compound with respect to a rotavirus.

These and other objects, features and advantages of the present invention will become apparent to those persons skilled in the art upon reading the details of the structure, synthesis and usage as more fully set forth below, reference being made to the accompanying general structural formulae forming a part hereof.

Detailed Description of Preferred Embodiments

Before the present synthetic receptor molecules and processes for making and using such are described, it is to be understood that this invention is not limited to the particular sugar residues described. For example, the specific description relates to GalNAc and Gall-->3GalNAC whereas all deoxy-halo-, deoxy amino-, deoxy acetamido, OSO₃- , etc., derivatives of such mono- and di-saccharides can be used. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims.

It must be noted that as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus for example, reference to "a synthetic receptor molecule" includes mixtures of such molecules, reference to "an antireceptor" includes mixtures of antireceptors of the type described and reference to "the process for synthesizing" includes similar processes and so forth.

A. Definitions

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

By "isolated rotavirus receptor glycolipid" is meant any sugar containing lipid that is capable of binding rotavirus. The glycolipid can be isolated from intestinal cells as described in U.S. patent application Serial No.

07/450,026, filed December 13, 1989, which is incorporated herein by reference in its entirety. The term also refers to those glycolipids which can be synthetically produced by applying the present disclosure and by using methods well known in the art, which are able to bind rotavirus.

By "rotavirus receptor compounds" is meant any of the synthetic compounds of the invention such as the carbohydrate epitopes or analogs therof which are capable of binding rotavirus as determined by standard viral binding assays including but not limited to overlay assays with thin layer chromatography plates and by probing receptor compounds adsorbed to PVC wells with labeled virus. A receptor compound of the invention will bind a rotavirus more avidly than asialo G_M1, as determined by orcinol staining in comparison with known amounts of asialo G_M1.

The glycolipid Ggose₄Cer (also known as G_A1) will run on thin layer chromatography (TLC) silica plates using chloroform:methanol:water (60:40:10) as solvent to provide a standard. The oligosaccharide moiety of the glycolipid G_A1 is shown below as structural formula I and is Ggose₄ (also known as asialo GM1 OS). (Note that Ac is an acetyl moiety throughout.)

(I)

The oligosaccharide Ggose₃ is the oligosaccharide moiety of the glycolipid Ggose₃Cer. The compound of structural formula II below is Ggose₃ (also known as asialo GM2 OS).

By "oligosaccharide able to bind rotavirus" is meant a sugar containing at least two, and preferably three or more saccharide units that has the ability to bind

rotavirus as described above. These oligosaccharides are equivalent to those produced by enzymatic cleavage of sugar moieties from isolated natural cell surface glycolipids able to bind rotavirus using arrays as defined above. The

oligosaccharides can either be synthesized by direct cleavage of the isolated glycolipids or can be chemically synthesized using the present disclosure in combination with methods of chemical synthesis, cleavage and separation well known in the art.

The term "therapeutically effective amount" refers to the amount of a rotavirus receptor compound or conjugate of such a compound with an antiviral drug sufficient to prevent, (i.e., treat prophylactically), or treat, (i.e., reduce or eliminate the symptoms of rotavirus) induced disorders in a recipient subject when administered.

B. Receptor Molecules for Rotavirus

Information regarding receptor sites for the antireceptor of a rotavirus are of particular interest to those involved in the research of the rotavirus and its infectious process. Since a rotavirus will readily attach to a receptor site, the production of receptor sites other than those present on cell surfaces would provide false hosts to the viruses and aid in preventing infection of the host cells to be protected. Further, by providing such receptors, it is possible to link other compounds to the receptors, such as antiviral drugs which can act to destroy the rotavirus after the rotavirus antireceptor has attached. A probe could be attached to or incorporated as part of the receptor so that the "tagged" receptor could be used to detect the presence of rotavirus. This invention relates to (1) naturally cleaved and isolated and to (2) synthetically produced compounds such as carbohydrate epitopes or analogs thereof which are of a minimum size recognized by the antireceptor of a rotavirus. The rotavirus receptor molecules of the invention are

structurally small, but functionally similar to the naturally occurring glycolipid receptor sites which are described further below.

Glycolipids can be isolated from cells receptive to rotavirus using any of several techniques for purifying glycolipids, well known in the art. A particularly useful technique is a modified Folch extraction as developed by Svennerholm and Fredman, Biochim. Biophys. Acta 617: 97-109 (1980), the disclosure of which is incorporated by reference herein in its entirety to disclose such extraction

techniques. Isolated glycolipids known to act as receptors can be used for comparison purposes with the synthetic receptors of the present invention.

Carbohydrates and related compounds can also be conjugated or attached to support base surfaces using

standard biochemical techniques, well known in the art. For example, the methods described by Tang et al., Biochem.

Biophys. Res. Comm. 132: 474-480 (1985); Schnaar et al., J. Biol. Chem. 253:7940-7941 (1978); and Laine et al., J. Biol. Chem. 249:4460-4466 (1974), all incorporated herein by reference in their entirety to disclose techniques which can be used in combination with the present disclosure to attach the compounds of the invention to a support surface. Once the receptor compound is attached to a support, such as thin layer chromatography plates or glass beads, the beads can be contacted with a sample to be assayed for the presence of rotavirus.

Once purified or synthesized, the receptor compounds such as the carbohydrates or glycolipids can be tested for their ability to bind labeled rotavirus using thin layer chromatography plates, developed in a suitable solvent or by adsorbing the glycolipids to PVC wells and subsequently probing the immobilized receptor compounds with labeled virus. The efficacy of the binding receptor compounds can be tested in vitro in standard plaque reduction assays. It is pointed out that saccharides per se cannot be directly tested in some of the assays. When such direct testing is not possible, the saccharide can be converted to its neoglycolipid form as explained further below.

Once receptor compounds such as natural glycolipids with the ability to bind rotavirus have been isolated, the sugar moieties can be cleaved therefrom using enzymes, including but not limited to neuraminidase, beta- glucuronidase, alpha-galactosidase, beta-galactosidase, endogalactosidase, beta-hexosaminidase, ceramide glycanase, alpha-fucosidase, and the coenzymes sulfatase and

phosphatase. The ability of these cleaved carbohydrates to bind to a rotavirus antireceptor can be tested and compared with the binding of synthetic compounds of the invention to the same viral antireceptor.

The cleavage products can be tested for their ability to bind rotavirus as described above and compounds (such as the various carbohydrates of the invention) with binding capability can be further tested in standard rotavirus plaque reduction assays, such as described below. Receptor

compounds with inhibitory activity can be used directly, or equivalent receptor compounds can be synthesized by applying methods known in the art in combination with the present disclosure. For example, receptor oligosaccharides can be synthesized by the sequential addition of appropriate single sugar units to a growing chain covalently linked to an insoluble solid support or by "block synthesis" where di- or trisaccharide blocks are synthesized which are then complexed to provide larger size oligosaccharides. The minimum binding and inhibitory carbohydrate epitope can be determined and used. Further, these sugars can be structurally modified to create more avidly binding analogs. In order to determine the minimum size of a receptor molecule recognized by a rotavirus certain characteristics of such receptors were taken into consideration.

First, it was noted that the conversion of asialo G_M1 into asialo G_M2 did not cause a difference in the ability of a rotavirus to bind to these molecules as measured by the TLC assay. The microtiter well assay showed some decrease in binding, although G_A2 still bound some virus, in comparison with other tested glycolipids which did not. G_A2 does support viral binding but greater concentrations are needed when compared with G_A1 in the PVC adsorption assay. This is in contrast to the TLC viral binding experiments in which G_A1 and G_A2 appear to be more equivalent. Further, it was noted that GalNAc by itself is of insufficient size to bind a rotavirus. Finally, it was noted that the removal of the terminal Gal from Ggose₄Cer (G_A1, ) generates Ggose-Cer (G_A2), a glycolipid which retains viral binding activity although at a reduced potency.

Based on the above discoveries, it was deduced that at least some of the hydroxyl groups in the immediate vicinity of the glycosidic linkage of GalNAc must be necessary for the overall binding of the rotavirus antireceptor to the molecule. These hydroxyl groups are encompassed by blocks in the structural formula put forth below:

In the above structural formula where R is H, the compound is asialo GM2 OS also known as the asialo GM2 trisaccharide which is the saccharide portion of the entire glycolipid asialo GM2. When R is beta-D-galactopyranosyl the compound is asialo GM1 OS also known as the asialo GM1 trisaccharide which is the saccharide portion of the entire glycolipid asialo GM1. In the structure relevant hydroxyls for viral recognition are shown in boxes.

In order to determine the minimum size for the receptor molecules, a synthesis was carried out in order to produce tri-(asialo GM2) and tetra-(asialo GM1) saccharides. The synthesis of each was carried out in such a manner that the lactose portion was blocked with benzyl groups whereas acyl functions protected the remainder of the molecule.

Hydrogenation was carried out in order to remove the benzyl groups followed by periodate treatment and finally reduction in order to obtain mono and disaccharide compounds having a polyhydric aglycone which retained one of the optical centers (C5 of glucose). This can be seen in Reaction Scheme (I) below. It should be noted that conventional abbreviations are used throughout, e.g., 0 is oxygen; H is hydrogen; Bn is benzyl; Ac or Acyl is an acyl moiety; F.A. is a fatty acid generally less than 30 carbons and conventional sugar abbreviations are used. Unless indicated otherwise, each "R" and "R" used with a super - or subscript is independently H, an alkyl which can be a simple straight or branched chain hydrocarbon or a hydrocarbyl, i.e., a hydrocarbon containing one or more heteroatoms most typically -OAc or a sugar. The superscripts on the "R"s are generally used to show that each "R" is independently defined. Typically, each "R" is independently H, -OAc, a hydrocarbon containing 1 to 6 carbons, or a sugar, e.g., beta-D-galactopyranosyl. A wavy line in a structural formula indicates that the attached atom or moiety may be in either the alpha or beta position.

In the above reaction scheme, it can be seen that the original orientation of the C3-OH (of galactose) has been disturbed. Rotation around the C3-C4 bond should allow the appropriate orientation of the newly formed CH₂OH moiety. The trihydroxhylic aglycone can be recognized as D-threitol. Accordingly, both asialo GM2 and asialo GM1 would end up in mono-saccharides and di-saccharides having D-threitol as the aglycone as shown in Reaction Scheme I above as well as within Reaction Scheme II below. Accordingly, the structures should provide receptor sites recognizable by a rotavirus.

Reaction II below also begins with a starting compound selected from the group consisting of D-GalNAc, D- GlcHN₂-HCl or D-GlcNHAc which compound by itself does not bind a rotavirus. The starting compound is then modified by the addition of other saccharides to provide the needed sites and structures for attachment to the rotavirus.

Reaction Schemes I and II above each show

mechanisms by which it is possible to produce receptor molecules (carbohydrate epitopes or analogs thereof)

recognizable by a rotavirus which molecules have a minimum size. These receptor molecules can be further modified in an attempt to improve the binding affinity of the rotavirus to the receptor. This can be done by incorporating a second beta-D-GalNAC (in the case of asialo GM2) and a second beta- D-Gall-->3-beta-D-GalNAc (in the case of GM1) on the free secondary hydroxyl. This type of addition is carried out by a mechanism in which suitable protected D-threitol or any other polyol with appropriately oriented hydroxyls is used as the acceptor in combination with a suitable glycosyl donor. The reaction scheme necessary in order to carry out such an addition is shown below in Reaction Scheme III.

It is pointed out that the polyolic moiety could be D-iditol. The D-iditol molecule provides different optical centers (D- and L-) at the C2 and C5. These optical centers can be O-glycosylated with GalNAC or beta-Gall-->3 GalNAc. A reaction scheme for this procedure is shown below as Reaction Scheme IV.

It should be pointed out that the iditol can be obtained commercially or can be prepared from D-mannitol as shown above within Reaction Scheme IV. By contrast, the D- galactitol would offer the same optical centers at C2 and C5 as shown below within Reaction Scheme V.

It should be noted that in the molecule produced in accordance with Reaction Scheme IV the sugar residues are oriented in opposing optical sense, i.e., one end (C2) is L-threitol and the other end (C5), has a D-sugar orientation or is D-threitol. However, in accordance with the Reaction Scheme V utilizing D-galactitol, the same carbohydrates are linked as if two D-thrietol moieties has been joined head-to- head.

Assays can be carried out on the compounds 5 and 13 shown above to determine their ability to act as receptors with respect to a rotavirus. Reactions can also be carried out on the newly formed C5-OH moieties 5 and 13 shown

respectively above in Reaction Schemes I and II. Accordingly, it is possible to introduce various linking arms for attachment to solid supports. In one modification, the C3'- OH (equivalent to the C5-OH of compounds 5 and 13) is masked with a fatty acid ether function as shown below in Reaction Scheme VI.

Compounds of the type shown within Reaction Scheme VI as well as the various rotamers of such compounds are shown below:

ROTAMERS

ROTAMERS (cont)

VARIOUS ROTAMERS POSSIBLE FOR A COMPOUND OFTYPE 24b.

Comment: R-1 Seems to be energetically most stable, having the most staggered orientation ofthe bulky groups.

interestingly,.this is also the orientation that is likely to be recognized by rotavirus. The Rotamers shown above should form micelle structures as shown below:

Micelles

Micelles (cont)

FIGURE 4: Possible Micalte rstructure of a compound like

24 b (Figure 2). USE and ADMINISTRATION

The compounds of the invention such as various carbohydrates or more specifically oligosaccharide epitopes and analogs thereof can be administered to a subject either prophylactically or after rotaviral infection. The inventive synthetic receptor molecules are administered with a

pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, oral administration, usually using a solid carrier and I.V. administration a liquid salt solution carrier. The method of choice, can be accomplished using a variety of excipients including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like. These oral compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders. Particularly useful is the administration of the subject carbohydrate receptor molecules directly in infant nutrient formulae or rehydration formulae, given to replenish electrolytes lost in severe bouts of diarrhea.

Alternatively, the receptor molecules of the invention can be administered orally, linked to supports such as beads, resins, or natural or synthetic polymers. Methods for binding substances to such supports are well known in the art. The receptor compounds may also be administered in small lipid particles comprising these receptor molecules, such as in vesicles, micelles, or liposomes.

A sufficient amount of receptor molecules should be administered to bind to a substantial portion of the

rotavirus expected to cause or actually causing infection so that infection can either be prevented or ameliorated.

Typically, the oral compositions of the instant invention will contain from less than 1% to about 95% of the active ingredient, preferably about 10% to about 50%. Preferably, between about 12 ug and 1.2 mg will be administered to a child and between about 200 ug and 10 mg will be administered to an adult. The frequency of administration will be

determined by the care given based on patient responsiveness. Other effective dosages can be readily determined by one of ordinary skill in the art through routine trials establishing dose response curves.

Other modes of administration will also find use with the subject invention. For instance, the rotavirus receptor molecules of the invention can be formulated in suppositories and, in some cases, aerosol and intranasal

compositions. For suppositories, the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%.

Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.

The receptor molecules of the instant invention may also be administered as injectables. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles. The receptors in the form of glycolipids and carbohydrates or more specifically

oligosaccharides can be mixed with compatible, pharmaceutically acceptable excipients.

Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or

formulation to be administered will, in any event, contain a quantity of the receptor molecules adequate to achieve the desired state in the subject being treated.

The various receptor compounds of the present invention can be used by themselves or in combination with pharmaceutically acceptable excipient materials as described above. However, it is more preferable to use the receptor compounds of the invention as conjugates wherein the

compounds of the invention are linked in some manner to an antiviral drug. By forming such conjugates, the receptor compounds of the invention act as biochemical delivery systems for the antiviral drugs . It is well known that many antiviral drugs are extremely toxic. Accordingly, it is desirable to provide such drugs to a patient in extremely small amounts. The smaller the amount of the drug delivered, the less toxic affects. However, the decrease in toxic affects must be balanced against the need to provide sufficient amounts of the drug in order to sufficiently destroy the virus. The receptor compounds of the invention aid in solving the problem by providing the biochemical delivery system. By forming conjugates of the receptor molecules of the invention with antiviral drugs, the conjugates readily attach to the viruses and destroy them. Thereby, smaller amounts of the antiviral drug need be delivered to a patient to obtain efficatious results.

The receptor molecules of the invention could also be used as laboratory probes to test for the presence of a rotavirus in a sample. Such probes are preferably labeled such as with a radioactive label. C. Experimental

The following examples are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to test the receptor molecules of the invention and are not intended to limit the scope of what the inventors regard as their invention or as being the only methods of testing such molecules. Efforts have been made to insure accuracy with respect to numbers used (e.g., amounts, temperature), but some experimental errors and deviation should be accounted for. Unless indicated otherwise, parts or parts by weight, temperature is in degrees centigrade, and pressure is at or near atmospheric. Example 1

Rotavirus Thin Layer Overlay Binding Assay

The ability of any of the compounds of the invention to bind rotavirus can be tested in the following manner.

Rotavirus SA11 for use in the following experiment can be grown in and isolated from MA104 cells, available from Whittaker Bioproducts, Walkersville MD., using previously described methods. See, e.g. Yolken et al., J. Clin. Invest. 79: 148-154 (1987) and Kabcenell et al., J. Virol. 62: 2929 (1988), the disclosures of which are incorporated herein by reference in their entirety. The virus can be iodinated using solid state iodobeads as reagent as described by

Markwell, M.A., Analyt. Biochem. 125: 427-432 (1982), incorporated herein by reference. Approximately 25 ug of gradient purified virus (by protein assay) can be incubated with 2 mCi of Na¹²⁵ -I and the virus subsequently purified by gel exclusion on a PD-10 column.

Silica 60 high performance thin layer

chromatography plates (Merck) can be spotted with the

isolated glycolipids and from 50 to 500 pmoles of asialo GM1 as standard. The plates can be run in

chloroform:methanol:water 60:40:10 (v:v:v). The plates can be probed with 10⁷ dpm of ¹²⁵I-labeled rotavirus per 100 sq.cm. using a modification of the technique of Magnani et al., Anal. Biochem. 109: 399-402 (1980).

Autoradiography can be used to determine if the molecules bind rotavirus, and whether they bind to a higher degree than asialo GM1 which normally binds virus if present in 50 pmoles in a 2 mm streak. The location of glycolipids on a plate can be detected by treatment with iodine before probing, or by spraying with orcinal after probing.

The above binding assay can be used for testing cleaved or synthesized sugar moieties and derivatives thereof after their attachment to lipids by means well known in the art of neoglycolipid synthesis. For example, the Ten Feizi method can be used whereby oligosaccharides are attached to phosphatidylethanolamine by reductive amination to make neoglycolipids.

Example 2

Rotavirus Binding to Adsorbed Glycolipids

The ability of (1) isolated glycolipid receptors;

(2) receptor molecules obtained therefrom; or (3) synthetic molecules which are believed to be receptors to bind

rotavirus can be tested by synthesizing the neoglycolipid form of a saccharide and using the following technique.

Compounds (i.e., glycolipids or saccharides in their

neoglycolipid form) to be tested are first adsorbed to PVC wells by evaporating the compound be tested out of

chloroform:methanol:water (4:8:3, v:v:v) and resuspending by sonication in an appropriate volume of 2.6 uM phosphatidyl choline/2.6 uM cholesterol in absolute ethanol. The

resuspended compound being tested should then be diluted with an equal volume of water to give stock solutions between 0 and 20 uM of test lipid. Prior to use, the PVC 96 well microtiter plates should be washed by immersion and agitation in n-butanol for 30 minutes, rinsed in absolute ethanol and air dried. 50 ul of test solution should be added to the microtiter wells to yield between 0 and 1 nmole of test lipid with 130 pmoles each of phosphatidyl choline and cholesterol. The PVC plates should be allowed to stand at room temperature for 80 minutes or just before the meniscus of the solution contacts the well bottom. Microtiter plates should then be washed three times with water and stored with water in each well until probed with labeled virus.

The putative receptor compounds (glycolipid or neoglycolipid) can be probed as follows: ¹²⁵I-Rotavirus is to be diluted in complete PBS supplemented with 10 mg/ml BSA (Fraction V), pH 7.4, to give approximately 20,000 cpm/100ul. This volume should be added to each microtiter well and the plates incubated at 4°C. for 3 hours with gentle agitation. Following incubation, plates are to be washed three times with ice cold PBS to remove unbound virus. The bottom half of each well should then be clipped off and placed in vials for measurement of bound radioactivity. This procedure will indicate if the tested compound demonstrates the ability to bind rotavirus.

Soluble rotavirus binding substances can also be tested (for their ability to inhibit binding to an immobilized glycolipid) with this technique by first adding the putative rotavirus binders to microtiter wells in 50 ul volumes of PBS-BSA at twice the desired final concentration. Labeled rotavirus should then be added to the wells at approximately 20,000 cpm/50 ul and the radioactivity in the wells determined as above.

Example 3

Rotavirus Plaque Reduction Assay

The ability of the above-described synthesized sugar moieties to inhibit rotavirus infection can be tested in vitro using the following plaque reduction assay. SA11 rotavirus (at 100 pfu) is to be incubated for 1 hr. with the compounds to be tested. The monitor is to be added to 6 well plates containing confluent MA104 cells. The cells are to be incubated for one hour at 37°C, the inoculum removed and the cells washed once with EBSS medium. The cells are then to be overlaid with 3 mis of medium containing 0.7% agarose and the compounds to be tested. Known inhibitors, such as ovalbumin and bovine submaxillary mucin and substances known not to inhibit rotavirus, such as globoside and trihexosyl ceramide, can be added as positive and negative controls, respectively. After the overlay gels, the plates are to be incubated for 48 hours and the number of plaques counted.

The instant invention is shown and described herein in what is considered to be the most practical, and preferred embodiments. It is recognized, however, that departures may be made therefrom which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art upon reading this disclosure.

Claims

1. A composition for preventing or treating rotaviral infection comprising a therapeutically effective amount of a rotavirus receptor carbohydrate epitope or analog therof of minimum structural size in combination with a pharmaceutically acceptable carrier.

2. The composition of claim 1 wherein the

rotavirus receptor carbohydrate of minimum size has the following structural formula:

wherein R is H or a beta-D-galactopyranosyl moiety and wherein the hydroxyls relevant for rotavirus recognition are shown in the boxes and the waved line indicate that -OH may be in either the alpha or beta position.

3. The composition of claim 1 wherein the rotavirus receptor carbohydrate of minimum size has the following structural formula:

( I )

β

4. A compound capable of binding to a rotavirus receptor, the compound having the following structure:

(II)

5. A composition for preventing or treating rotaviral infection as claimed in claim 2 wherein the pharmaceutically acceptable carrier is a carrier for oral delivery.

6. A composition for preventing or treating rotaviral infection as claimed in claim 5, wherein the pharmaceutically acceptable carrier is an infant nutrient formula.

7. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 5.

8. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 6.

9. A composition for preventing or treating rotaviral infection comprising a pharmaceutically acceptable carrier having dispersed therein a therapeutically effective amount of a rotavirus receptor compound having the following structural formula:

10. A composition for preventing or treating rotaviral infection as claimed in claim 9 wherein the pharmaceutically acceptable carrier is a carrier for oral delivery.

11. A composition for preventing or treating rotaviral infection as claimed in claim 10 wherein the pharmaceutically acceptable carrier is an infant nutrient formula.

12. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 10.

13. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 11.

14. A composition for preventing or treating rotaviral infection comprising a pharmaceutically acceptable carrier having dispersed therein a therapeutically effective amount of a rotavirus receptor carbohydrate having the following structural formula:

15. A composition for preventing or treating rotaviral infection as claimed in claim 14 wherein the pharmaceutically acceptable carrier is a carrier for oral delivery.

16. A composition for preventing or treating rotaviral infection as claimed in claim 15 wherein the pharmaceutically acceptable carrier is an infant nutrient formula.

17. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 15.

18. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 16.

19. A composition for preventing or treating rotaviral infection comprising a pharmaceutically acceptable carrier having dispersed therein a therapeutically effective amount of a rotavirus receptor carbohydrate having the following structural formula:

wherein each R¹¹ is indeependently H or a beta-D- galactopyranosyl moiety

20. A composition for preventing or treating rotaviral infection as claimed in claim 19 wherein the pharmaceutically acceptable carrier is a carrier for oral delivery.

21. A composition for preventing or treating rotaviral infection as claimed in claim 20 wherein the pharmaceutically acceptable carrier is an infant nutrient formula.

22. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 20.

23. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 21.

24. A composition for preventing or treating rotaviral infection comprising a pharmaceutically acceptable carrier having dispersed therein a therapeutically effective amount of a rotavirus receptor compounds selected from the group of compunds having the following structural formula:

wherein R in each of R-1, R-2 and R-3 is independently H or BETA-D-galactopyranosyl and F.A. is independently a fatty acid containing 10 to 30 carbon atoms.

25. A composition for preventing or treating rotaviral infection as claimed in claim 24 wherein the pharmaceutically acceptable carrier is a carrier for oral delivery.

26. A composition for preventing or treating rotaviral infection as claimed in claim 25 wherein the pharmaceutically acceptable carrier is an infant nutrient formula.

27. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 25.

28. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 26.

29. A composition for preventing or treating rotaviral infection comprising a pharmaceutically acceptable carrier having dispersed therein a therapeutically effective amount of a rotavirus receptor compound having the following structural formula:

wherein R is H or a beta-D-galactopryranosyl moiety R¹ is H, an alkyl moiety containing 2 to 5 carbon atoms or a fatty acid containing 10 to 30 carbon atoms and n is an integer in the range of from 8 to 28.

30. A micelle structure formed by a plurality of compounds as claimed in claim 29.