WO1999052922A1 - ANTIMICROBIAL βGalNAc(1→4)βGal DERIVATIVES AND METHODS OF USE - Google Patents

Abstract

Compositions and methods for treating microbial infections, such as Candida infection, are disclosed. Specifically, derivatives of βGalNAc(1→4)βGal have been found to be superior to the parent compound in inhibiting adhesion and infection by Candida and related microbes.

Description

Antimicrobial βGalNAc (1-4) βGal Derivatives and Methods of Use

Field of the Invention

The present invention relates to derivatives of β-D-GalNAc(l→4)β-D-Gal, and methods of using such derivatives in treating microbial infections such as Pseudomonas aeruginosa and Candida albicans.

References

Baker, N.R., etal, Infect. Immun. 59:2859-2863 (1991). Barczai-Martos,M. and Korosy, F., Nature 165:369 (1950).

De Bruyne, C.K., and van Der Groen, G., Carbohydrate Research 25:59-65. (1972). Dubois, M., et al, Anal Chem. 28:350 (1956). Evans, M.B., et al. Chromatographia 13:5- 10 (1980). Lemieux, et al, U.S. Patent No. 4,137,401 (1979). McEachran, D. W., and Irvin, R., Can. J. Microbiol 31(6): 563-569 (1985).

Mishell, B.B., and Shiigi, S.M. (Eds.), in SELECTED METHODS IN CELLULAR IMMUNOLOGY, W.H. Freeman and Co., San Francisco (1980).

Paranchych, W., et al, Can. J. Microbiol. 25:1175-1 181 (1979). Smith, P.K., et al, Anal. Biochem.150:76 (1985). Wong, S . S . , in CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING CRC Press, Boca

Raton, Florida (1991).

Background of the Invention

Resistance of microbes to antibiotics is an increasing problem in the United States, as well as in the rest of the world. As the current armamentarium of antibiotics becomes obsolete, it is importantto find new means of preventing and eradicating infections.

An approach which has been used in recent years is to attempt to prevent adhesion of the microbe to the cells of its host by interfering with attachment of microbial appendages known as pili or fϊmbriae to their cellular receptors. Such pili are found in a number of gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Moraxella bovis, Neisseria gonorrhea, as well as certain fungal microbes, such as Candida albicans.

By way of example, Candida albicans is a dimorphic, imperfect yeast that can be isolated from the oral cavity of approximately 40% of healthy, asymptomatic individuals. It also causes a number of superficial and invasive diseases, particularly in immunocompromised or immunosuppressed individuals.

Cutaneous candidiasis is usually treated with antifungal agents, such as nystatin, ciclopirox,

1 and imidazole creams. For oral and vaginal candidiasis, the drug may be administered in the form of a topical cream, suspension or suppository. In all forms of skin and mucosal candidiasis, relapse after successful treatment is common.

For disseminated candidiasis, such as esophageal or bladder candidiasis, intravenous administration of an antifungal agent, such as amphotericin B, is required. Side effects of currently used anti-fungal agents are often severe, and the drugs have limited use because they cannot be administered over an extended time period. Since disseminated candidiasis is a common type of infection in the terminal stages of HIV (AIDS) infection, the inability to treat the candidiasis successfully has become a widespread disease-managementproblem. As another example, Pseudomonas aeruginosa is a gram-negative bacterial pathogen that causes between 10% and 20% of all infections in hospitals. Pseudomonas infection is especially prevalent in patients with burn wounds, cystic fibrosis, acute leukemia, organ transplants, and intravenous-drug addiction. The most serious infections include malignant-external otitis, endophthalmitis, endoconditis, meningitis, pneumonia, and septicemia. The likelihood of recovery from Pseudomonas infection is related to the severity of the patient's underlying disease process. The reported mortality associated with . aeruginosa pneumonia is as high as 50-80%

U.S. Patent 5,641,760 discloses the compound 2-acetamido-2-deoxy-β-D-galactopyranosyl-

(1_^4)β-D-galactopyranoside(βGal-NAc-βGal) in the form of a conjugate composed of a carrier and multiple BGal-NAc-βGal moieties attached to the carrier. The conjugate is used for treating an infection by Candida albicans.

The present invention is based on the discovery that derivatives of βGal-NAc-βGal are significantly more effective than the previously disclosed compounds in preventing adhesion of certain microbes, notably Candida and Pseudomonas.

Summary of the Invention

The present invention is directed to antimicrobial compositions and methods of preventing microbial infection.

According to one embodiment, the invention includes a βGalNAc( 4)βGal derivative having the general formula shown in FIG. 2 herein. Such compounds are further characterized as having activity in inhibiting binding of microbial fimbriae to epithelial cell targets is significantly greater than that exhibited by βGalNAc(l-.4)βGal. According to this embodiment of the invention, the R- group substitutions may be made as follows: R¹ is an alkyl group containing 1 to 4 carbons, R² is independently H or a lower alkyl group containing 1 to 4 carbons, R² is an alkyl group containing between about 5 and about 12 carbons, R⁴ is independently H or a lower alkyl group containing 1 to 4 carbons.

In a preferred embodiment, R¹ is COCH₃, R³ is a heptyl, octyl, nonyl or decyl straight chain hydrocarbon group, and R² and R⁴ are independently H or lower alkyl groups selected from the group consisting of CH₃, CH₂CH₃, C₃H₇, and C₄H₉. In another preferred embodiment, R¹ is COCH₃, R² is independently H or CH₃, R³ is (CH₂)₇CH₃, and R⁴ is independently H or (CH₂)₂CH₃.

In yet still other preferred embodiments, the composition of the present invention is selected from octyl 0-(2-acetamido-2-deoxy-4 -methyl-β-D-galactopyranosyl)-(L4)-β-D- galactopyranoside (compound 3), octyl 0-(2-acetamido-2-deoxy-β-D-galactopyranosyl)-(L4)-2-0- propyl-β-D-galactopyranoside (compound 7), or octyl t -(2-deoxy-2-propionamido-β-D- galactopyranosyl)-( l-.4)-β-D-galactopyranoside(compound 8).

According to a related aspect, the invention is directed to a method of treating a microbial infection in an individual. Accordingto this aspect of the invention, a compound is selected from the compounds described above, and is further tested for efficacy in inhibiting binding of microbial fimbriae to test epithelial cells. Compounds having sufficient potency in this regard and which are further found to have appropriately low levels of toxicity in human or veterinary subjects are then considered candidates for antimicrobial therapeutics. In accord with this embodiment of the invention, therapeutic βGalNAc(l-.4)βGal derivatives are administered to an infected site in a pharmaceuticallyeffective dose.

In a preferred embodiment, the infection is a Candida albicans infection. In a further preferred embodiment, the infection is an oral or vaginal infection, and the compound is administered by topical application to the infected site. In yet another preferred embodiment, the infection is a Pseudomonas infection. Generally, the routes of administration for treatment of Pseudomonas infection will depend on the site of infection. One common site, the lungs, may be treated by nasal insufflation.

In still further aspects, the invention includes new methods of preparing selected βGalNAc( l-.4)βGal derivatives described herein.

Brief Description of the Figures

FIG. l(A-C) shows a diagram of the structures of some of the βGalNAc(l-.4)βGal derivatives disclosed in conjunction with the present invention;

FIG. 2 shows a generalized scheme for βGalNAc( 4)βGal derivative compositions in accordance with the invention;

FIG. 3(A-N) shows structures of βGaINAc( 4)βGal derivative compounds and other compounds identified as numbers 1-14 herein;

FIG. 4 (A-B) shows a schematic diagram of the synthetic steps involved in synthesizing 2- propyl-octyl-βGalNAc( 4)βGal (Compound 7); FIG. 5 shows a schematic diagram of synthetic steps involved in synthesizing octyl- βGalNAc( 1 →4)βGal (Compound 1 );

FIG. 6 shows a saturation binding curve of binding of C. albicans fimbriae to human buccal epithelial cells (BECs);

FIG. 7 shows inhibition by varying concentrations of compoundsof the invention of binding of Candida fimbriae to asialo GM, ;

FIG. 8 shows inhibition of binding of Pseudomonas pili to asialo-GMl using varying concentrations of compounds of the invention;

FIG. 9 shows the effect of various treatment regimens on fungal burden in a rat model of oral Candidiasis from microcurette samples taken from the buccal cavities; FIG. 10 shows the effect of various treatment regimens on fungal burden in a rat model of oral

Candidiasis from tongue homogenate samples;

FIG. 11 shows the effect of various doses of Fimbrigal-P after varying time intervals on fungal burden in rat buccal cavities in a rat model of oral candidiasis; and

FIG. 12 shows Fimbrigal-P dose dependent decrease in fungal burden in rat buccal cavities (tongue homogenate).

Detailed Description of the Invention

I. Definitions

The term "fimbrial subunit" or "pilus subunit" as used herein, refers to a protein that is the predominant component of fimbriae or pili, respectively, present on microbes, as described herein.

For example, the fimbrial subunit that is the predominant component of fimbriae isolated from

Candida albicans is a 66 kD glycoprotein, as described herein. This protein is referred to as

"Candida fimbrial adhesin protein." The pilus subunit that is the predominant component of pili isolated from Pseudomonas aeruginosa is referred to as "pilin" and has been well characterized. The term "adhesin" refers generally to molecules present on a microbial cell or appendage, such as a fimbria. that mediate adherence of the microbe to a cell that is susceptible to infection by the microbe.

The term "βGalNAc( 4)βGal" or "βGal-N Ac-Gal" refers to octyl-0-(2-acetamido-2-deoxy- β-D-galactopyranosyl)-( l→4)β-D-galactopyranoside(Compound 1 herein). The term "parent compound" or "parent βGalNAc( 4)βGal" or "parent βGal-NAc-Gal" refers to Compound 1. of FIG. 1.

The term "βGal-NAc-Gal derivative" refers to βGalNAc( 4)βGal where one or more hydroxyl groups are substituted by O-alkyl groups, such as O-methyl or O-propyl groups, as described herein. The term "potential therapeutic compositions" refers to compounds which possess requisite biological activity to be considered as therapeutic agents, but which have not necessarily undergone the stringent toxicological testing to qualify as therapeutic agents for veterinary or human medicine.

Unless otherwise specified herein, all other terms should be interpreted using standard meanings as commonly used in the art.

II. BGalNAc( 4)βGal Derivatives

It is the discovery of the present invention that certain derivatives of βGalNAc(l~,4)βGal are significantly more efficacious than the parent compound in inhibiting adhesion of microbes to host epithelial cells. As will be shown, the derivatives of βGalNAc(l→4)βGal of the invention are significantly more potent than the parent compound βGalNAc(l-.4)βGal in inhibiting fimbrial adhesion. This section describes the structures and synthesis of such compounds.

A. Nomenclature and Structures of βGalNAc( 4)βGal Derivatives With initial reference to both FIG. 1 and FIG. 2, βGalNAc( 4)βGal derivatives that have been synthesized and tested in conjunction with the present invention are shown. Compound 1 of FIG. 1A shows the parent compound βGalNAc( 4)βGal (octyl-(9-(2-acetarnido-2-deoxy-β-D- galactopyranosyl)-(l→4)β-D-galactopyranoside). The compound consists of two galactosyl moieties, designated as rings A and B herein (see FIG. 2), where 2-acetamido-2-deoxy •β-D-galactopyranoside is designated as Ring A and β-D-galactopyranoside is designated as Ring B. According to an important feature of the present invention, it has now been discovered that substitution of one or more of the hydroxyl groups by a lower O-alkyl substituent can provide even greater activity and/or potency in inhibiting adhesion by microbes such as Candida or Pseudomonas than does βGalNAc( 1 - 4)βGal. Methods for assessing such binding will be described in Section IV below. The structures of eight βGalNAc(l-4)βGal derivatives, Compounds 2-8, synthesized in support of the present invention are also shown in FIG. 1 A, which will now be discussed.

1. N-Substitution Derivatives. Compounds 2-9 have substitutions on the A-ring subunit of the parent compound. As discussed above, the A-ring is 2-deoxy-D-galactosaminewith an N-acetyl substitution at the amine (2-acetamido-2-deoxy-β-D-galactopyranoside). A substitution at this position, designated as R¹ in FIG. 2, can be selected from lower alkyl groups, C1-C4. Compounds 2-7 and 9 have a two-carbon substitution at R¹, while compound 8 is exemplary of a three-carbon substitution at R¹. 2. 2-deoxy-D-galactosamine(Ring A) Hydroxyl Substitutions The A-ring subunit-of βGalNAc(l→4)βGal includes hydroxyl groups at ring positions 3, 4 and 6, which are designated in FIG. 2 as R². Compounds 2-4 as illustrated in FIG. 1 have substitutions on the A-ring subunit at ring positions 6, 4 and 3, respectively. R² is selected from H, or a lower alkyl substitution, having between one and four carbons.

3. Galactopyranoside (Ring B) Hydroxyl Substitutions. The B-ring subunit βGalNAc( 4)βGal includes at the Cl position of the ring, designated as R³ in FIG. 2, an O-alkyl group containing between about 5-12 carbons. With respect to the parent compound, it is standard practice to include an O-octyl group, -0(CH₂)₇CH₃, at this position to stabilize the interaction between the βGalNAc(l-,4)βGal and a receptor {c.f Compound 1 of FIG. 1A). The derivatives of the present invention also include the O-octyl group at R³, except Compound 9 which has an 8- (methoxycarbonyl)octyl ether substitution at R³. It will be appreciated that other moieties are suitable for the R³ position and still preserving activity of the compound. Other substitutions include other straight chain hydrocarbons such heptyl, nonyl or decyl or hyderocarbon rings or branched groups. Typically, uncharged alkyl groups are preferred at position R³.

Hydroxyl substitutions at the remaining positions of the B-ring subunit (C6, C3 and C2) are designated "R⁴" in FIG. 2. Substitutions at these groups may be lower alkyl groups. Compounds 5, 6 and 7 have three-carbon substitutions (propionate groups) at each of positions C6, C3 and C2 of Ring B, respectively.

FIG. 3(A-N) show the structures for the parent Compound 1 (FIG.3A) and for the derivative Compounds 2-9 (FIGS. 3B-3I) described above, and other derivatives in accordance with the invention (FIGS.3 J-3N).

B. Synthesis of βGalNAc(l→4)βGal and BGalNAc(l-,4)BGal Derivatives

Detailed methods for synthesizing the parent Compound 1 and derivative Compound 7 are presented in Example 1. Briefly, with reference to FIG. 4, 2-0-propyl-octyl-βGalNAc( 4)βGal (Compound 7) is synthesized using as starting material tetra-O-acetyl-β-D-galactopyranosyl bromide (compound I, FIG.4). Details of the synthetic steps are provided in Example IB. Octyl (2-acetoamido-2-deoxy-β-D-galactopyranosyl-( l→4)-β-D-galactopyranoside

(Compound 1, FIG. 1 and Compound XV, FIG. 5) is synthesized using octyl-β-D-galactopyranoside (Compound II, FIG. 4) as starting material, as described in Example 1 herein. With the guidance found herein, with reference to Compounds 1 and 7, other octyl-βGalNAc( 4)βGal derivatives can be synthesized, using appropriate substitutions, according to methods known in the art. III. Assays for Determining Anti-adhesive Activity

As mentioned above, Candida albicans and other microbes possess fimbriae or pili, long filimentous structures that protrude from the microbial cell surface. These structures can be isolated and used in binding assays that provide quantitative measurements of the anti-adhesive properties of test compounds. Methods for isolating Candida fimbriae are detailed in Example 2. Fimbriae from other suitable organisms can be isolated according to methods similar to those described herein or by other methods known in the art. For example, Pseudomonas pili can be isolated according to methods known in the art. (Paranchych, et al, 1979). The reagents and assays described in this section are representative of reagents and assays for testing the anti-adhesive properties of derivative compounds in accordance with the present invention.

A. Isolation of Candida Fimbriae

Fimbriae are conveniently purified from the yeast phase of C. albicans according to the methods described in Example 2. Briefly, C. albicans cells are cultured and harvested according to standard methods, then subjected to homogenization to shear off fimbriae. The fimbriae are then separated from the cellular material by centrifugation. The resulting supernatant is then processed to yield a crude fimbriae (CF) preparation.

The CF preparation may be fractionated by size-exclusion high performance liquid chromatography according to methods now known in the art {c.f, U.S. Patent 5,641,760, incorporated herein by reference) and detailed in Example 2. The resulting fraction is termed "enriched fimbriae" (EF).

B. Purification of Candida Fimbrial Adhesin Protein

Fimbrial fractions prepared as generally described above, are used to prepare a purified fimbrial proteins as detailed for Candida and Pseudomonas in Example 2 herein. Fractions are analyzed for purity as detailed in Example 3.

Analysis of the fimbrial fractions described above indicated that the major component of the purified fimbrial fraction from Candida is a 66 kDa protein, as visualized by SDS PAGE.

To further purify the isolated fimbrial protein, gel slices containing the 66 kDa protein are isolated and electroeluted, according to the protocols given in Example 3A, or by other equivalent procedures well known in the art. For use in experiments described herein, the eluted protein fraction was further purified by reversed-phaseHPLC. The peak fraction may be used as a source of purified fimbrial adhesin protein for further characterization. C. Properties of Purified Fimbrial Protein

This section describes certain physical and biochemical characteristics of fimbrial fractions used in assays carried out in support of the present invention. While it is understood and taught that other equivalent assay methods can be used to assess the antimicrobial efficacy of compositions, these parameters are illustrated in order to provide a benchmark for assay conditions.

The EF preparation was used to determine the protein and carbohydrate composition of the 66kDa C. albicans fimbrial subunit protein. To this end, a combination of techniques including phenol-sulfuric carbohydrate assay, BCA protein assay and amino acid analysis were used to analyze protein derived from the EF fraction, as detailed in Example 3, parts B and C, and described below. The fimbrial subunit is approximately 85% carbohydrate and approximately 15% protein.

1. Molecular Weight. The molecular weight of the fimbrial subunit protein was determined to be 66,000, as determined by SDS polyacrylamide gel electrophoresis. The molecular weight of the peptide component of the protein was determined to be 8,644, based on compositional analysis of the peptide, which was found to consist of 79 amino acids, as described below.

2. Protein/Carbohydrate Composition The amount of carbohydrate present in the fimbrial protein sample can be determined according to standard methods, one of which is the phenol-sulfuric acid carbohydrate assay detailed in Example 3C. From this assay, in conjunction with the measured protein content of the same sample, the relative percentages of carbohydrate and protein in the sample are determined. In the samples prepared from C. albicans 40 described herein, it was found that carbohydrate comprises about 80-85% of the fimbrial protein, while the peptide portion of the protein constitutes only about 15% of protein.

Further characterization of the carbohydrate portion of the protein can be carried out according to standard procedures, such as the gas chromatographic procedure detailed in Example 3C. Using this method, it was found that the main carbohydrate moiety present in the fimbrial preparation used in experiments carried out in support of the present invention is D-mannose. Several additional minor components were also observed in the samples derived from EF.

3. Amino Acid Composition The amino acid composition of the fimbrial protein subunit is also determined according to standard methods. As described in Example 3, acid hydrolysis followed by amino acid analysis by automated analyzer was used in experiments carried out in support of the present invention .

D. Binding Properties of Fimbria and Fimbrial Proteins Candida fimbriae mediate binding of the fungal cells to target human epithelial cells. FIG. 6 shows the results of a saturation binding assay in which binding of fimbriae to human buccal epithelial cells was determined. In this assay, as detailed in Example 5A, epithelial cells were added to filtration chambers equipped with polycarbonate filters, and aliquots containing varying amounts of EF were added to each chamber. Following washing of the cells to remove unbound fimbriae, mouse anti-Candida albicans fimbrial antibodies (Fml6) were added to each sample. Fimbrial binding was measured by determining binding of horseradish peroxidase goat anti-mouse antibodies to the preparation, according to standard methods set forth in Example 5. The data plotted indicate that the binding is saturable, with an approximate maximal binding (B_max) of 50 μg of protein per milliliter. Half maximal binding was observed at about 10 μg protein/ml, indicating a high affinity interaction. The ability of the purified (EF) fimbrial preparation to interfere with binding of native Candida fungal cells to buccal epithelial cells (BECs) was measured in further experiments, as described in Example 5B. In these assays, BECs were pre-incubated with fimbrial EF fraction, then incubated with Candida albicans yeast cells. At a concentration of 7.5μg/ml, EF fimbria were able to inhibit binding of the fungal cells to buccal epithelial cells by about 40% Similarly, binding of pili of Pseudomonas aeruginosa strains PAK to asialo-GM, was evaluated as previously reported (Lee, et al, 1994), using biotinylated pili binding to asialo-GM„ coated on microtitre plates. Bound pili were detected using streptavidin conjugated alkaline phosphatase and p-nitrophenylphosphateas the color developing reagent at an absorbanceof 405 nm.

Assay conditions similar to those described above are used to evaluate inhibition of binding by test compounds in accordance with the present invention.

IV. Selection of Inhibitory βGalNAc( 4)βGal Derivatives βGalNAc(l→4)βGal derivatives are selected for use as potential therapeutic compositions if they are significantly more potent or effective than βGalNAc(l-,4)βGal in inhibiting binding of microbial pili or fimbriae to target epithelial cells or to asialo-GMl, which has been shown to be predictive of epithelial cell binding. By "significantly more potent or effective than βGalNAc( l-4)βGal" is meant that the compound exhibits either (i) at least a two-fold higher potency than βGalNAc( 4)βGal as evidenced by about a corresponding lower inhibitory concentration, measured by IC₅₀ or K-, (determined according to standard methods known in the art), or (ii) at least 1.5 times the efficacy, as evidenced by a 1.5X higher killing percentage in a standard antimicrobial assay, as described herein.

This section describes exemplary screening tests of the parent compound and derivative Compounds 2-11 (see FIG. 1 and/or FIG. 3) designed and synthesized in accordance with the present invention. Based on these tests, certain of the compounds are selected as potential therapeutics. A. Inhibition of C. albicans Binding to BECs by βGalNac(l→4)βGal and βGalNacC 4)βGal-Conjugates βGalNAc(l→4)βGal derivatives were synthesized as described in Section II above. Assays for binding inhibition were carried out essentially as described in Example 5 herein. Tables 1 and 2 show results of experiments carried out in support of the present invention, in which biotinylated fimbriae prepared from Candida and biotinylated pili prepared from Pseudomonas were tested for binding to asialo-GM,, and compounds were assessed for inhibitory activity. Compound 1 served as a reference for "inhibitory power", defined herein as the percent improvement (lowering) of the IC₅₀ of the test compound with respect to Compound 1. From the results of experiments shown in Table 1, Compounds 3, 7 and 8 are clear candidates for therapeutics. Data used to generate Table 1 are shown in FIG. 7, where percent inhibition is shown as a function of concentration of selected test compounds (Compounds 1 (closed squares) and Compounds 3, 4, 7 and 9). The assay method is detailed in Example 7, except that biotinylated fimbriae were used instead of biotinylated pili.

Table 1

Inhibitory Binding of Biotinylated Fimbriae of

Candida albicans to asialo-GM, by

GalNAcGal Derivatives

Compound Inhibitory Power^* (%)

1 100

2 <100

3 230

4 110

5 150

6 <100

7 750

8 300

9 <100

10 no inhibition

11 no inhibition

'inhibitory power is calculated as the percentage improvement of the IC₅₀ of the derivive compounds as compared with that of βGalNAc(l-.4)βGal (Compound 1):

IC₅₀ of compound 1

100

IC₅₀ of derivative

Further studies in support of the present invention tested the effects of the exemplary compounds described herein on binding of Pseudomonas to target sites. Here, Compounds 2-9 as (FIG. 3) were tested in along with the monosaccharide Compound 10 (the terminal monosaccharide

10 of the natural ligand; FIG. 3J) and monosaccharide Compound 11 (an unrelated negative control having an octyl aglycon, FIG. 3K). Compound 11 was used as reference and did not show significant inhibition under the present assay conditions. Typical inhibition curves are shown in FIG. 8. The IC₅₀ values and relative inhibitory power are presented in Table 2. Most of the mono-O-alkylated compounds bind with affinities similar to that of Compound 1. A notable observation is that the aglyconic methyl ester Compound 9 decreases the binding affinity about three-fold compared to that of the parent glycoside.

Based on the data presented below and the criteria discussed above, Compounds 3 and 7 are significantly more potent than parent Compound 1 and are therefore candidates compounds for Pseudomonas therapy, subject to further toxicity and pharmaceutical testing, according to methods known in the art.

Table 2

ICsn Values of Compounds 1-11

Compound IC_So (/ VI) Inhibitory Power

1 79 + 18 100%

2 56 ₊ 13 140%

3 30 + 7 260%

4 65 + 7 120%

5 130 ₊ 36 60%

6 98 ₊ 1 1 81%

7 8 ₊ 4 990%

8 129 + 30 61%

9 217 ₊ 32 35%

10 246 _± 14 32%

_*

11 __•

No inhibition was observed.

From the foregoing, taken in conjunction with the detailed examples presented herein, the practitioner will be able to select and synthesize βGalNAc(l→4)βGal derivatives and test them in an appropriate binding assay. Compounds that exhibit relatively high biological activity {i.e., at least two times the biological activity of βGalNAc(l-_*4)βGal) are considered as potential therapeutic compounds that can then be tested for toxicity and pharmaceutical stability according to methods known in the art and pertinentto the anticipated mode of administration, as discussed below.

1 1 V. Treatment Methods

This section describes exemplary methods for treating Candida or Pseudomonas infections. Treatment of other types of icrobial infections using compositions of the present invention will be apparentto those skilled in the art.

A. Treating Localized Candida Infection

Binding studies discussed above show that βGalNAc(1 _^4)βGal derivatives selected in accordance with the present invention are effective in blocking binding of microbes to epithelial cells, as exemplified by the studies showing blockade of C. albicans and P. aeruginos abm^' άmg. The present method exploits this finding, in a method of inhibiting C albicans infection by inhibiting binding of the infectious agent to target epithelial cells.

Local infection of the oral or vaginal mucosa is treated, in accordance with the method, by topical application of a βGalNac( 4)βGal derivative. For oral delivery, the composition may take the form of a wash or cream that can be applied, e.g., by swab or mouth rinsing at periodic intervals. Alternatively, the composition may be incorporated into a buccal formulation, such as a pellet, tablet, or other adhesive dosage form, for release from the gum region over an extended period of time. In another embodiment, the composition may be incorporated into a slow-release tablet or oral insert, or into gum. Methods for forming oral delivery .creams or devices of this type, for topical administration of incorporated composition into the oral cavity are known in the art.

For example, in experiments carried out in support of the present invention, a standard experimental model of Candida infection was used to assess the efficacy of an exemplary βGalNAc(l→4)βGal derivative, Compound 7 ("Fimbrigal-P") in preventing or reducing infection. Example 9 provides details of protocols used to measure the ability of this compound to prevent or treat Candida infection. Briefly, rats were given single dose oral inoculations of Candida albicans on three consecutive days. One group of animals, Group 1, received no further treatment; Group 2 received "preventative" treatment, in which the animals received oral inoculations of 6.25 mg Fimbrigal-P before inoculation with the pathogen. Animals in Group 3 received Fimbrigal-P in drinking water throughout the inoculation period and continuing several days after inoculation. Animals in Group 4 were subjected to a "pre-mixing" paradigm, in which they were inoculated orally with a mixture of Fimbrigal-P and Candida. Group 5 received doses of Fimbrigal-P starting after pathogen inoculation.

The results are shown in FIG. 9 and FIG. 10, where FIG. 9 shows the fungal burden in the oral cavity of the animals for each treatment group at days 5, 7, and 8 after incoculation. As can be seen, all of the treatment regimens achieved a significant decrease in fungal burden. FIG. 10 shows the fungal burden determined from tongue homogenate samples prepared from the test animals in each

12 group. The animals in each test group receiving Fimbrigal-P had a decrease in fungal burden relative to the untreated animals.

FIG. 1 1 is a dose-response curve which shows the effect of increasing amounts of Fimbrigal-P (in Carbopol Ex214, 0.4%) on fungal burden in rat oral candidiasis, as assessed in tongue homogenates 5 days after inoculation with Candida. FIG. 12 shows dose-response curves for various dosages of Fimbrigal-P in Carbopol Ex214 between 5 and 8 days following first inoculation.

Dosages and routes of administration described with respect to the foregoing animal model can be extrapolated for human and veterinary use, based on standard principles known in the art.

The composition is administered a pharmaceutically effective amount, that is, an amount effective to inhibit C albicans binding to oral or vaginal mucosal cells. As observed in studies on inhibition of C. albicans binding to BECs carried out in support of the present invention, the administering is preferably effective to produce a concentration of a βGalNac(l→4)βGal derivative of at least about 0.2 mg/ml, and preferably between about 0.01 and 100 mg/ml, over a selected period, e.g., on the order of minutes, to days and up to a week. For vaginal delivery, the composition may be administered in cream form, or by vaginal suppository or insert, accordingto known drug-delivery methods.

Administering the composition is continued until a desired reduction of infection is achieved, as monitored, for example, by the reduction of fungal organisms collected by oral or vaginal swab, and/or by an observed change in the symptomatic condition of the patient. Where the composition is employed for prophylactic purposes in an individual who may be at risk for oral or vaginal infection, the same modes of composition administration may be employed. In particular, a βGalNac(l→4)βGal derivative composition, at the dosage levels indicated above, is administered for a period of at least a few days, or until the risk of infection has passed.

B. Systemic or Disseminated Infection

In another general aspect, the infection includes treating a disseminated or systemic C. albicans infection by parenteral administration of βGalNAc(l→4)βGal derivative.

The disaccharide is administered typically in a physiological saline solution, at a pharmaceutically effective dose, i.e., a dose effective to inhibit fungal cell levels in the bloodstream or at an organ site. Administration may be by intravenous, intramuscular, subcutaneous or other parenteral route.

An effective dose level of βGalNAc(l→4)βGal can be determined from a suitable animal model system in which anti-fimbriae antibodies are screened for their ability to prevent or treat systemic infection by C. albicans. A preferred dose is effective to produce a local concentration of the compound of at least about 0.2 mg/ml. Parenteral doses may be further confirmed by animal studies, such as studies on Candida- infected mice, according to methods known in the art. Here mice

13 infected by injection with C. albicans are treated with repeated doses of the composition, in amounts ranging from 0.1 to 2.0 g compound/kg body weight, at days 2, 4, 6, 8 post-infection. The animals are then monitored at the end of this period for systemic fungal infection, as measured by fungal colony forming units in a given volume of blood. The dosage range effective to reduce infection is then taken as the range for initial clinical trials on the composition.

To monitor systemic therapy, blood samples collected during treatment of the patient can be monitored for colony forming units, and/or changes in the symptomatic condition of the patient. Methods for monitoring therapy of oral infections are provided by way of example herein.

The following examples illustrate, but in no way are intended to limit the present invention.

Materials

C albicans strain #40 was obtained from the trachea of an intubated intensive care unit patient at Toronto General Hospital. The isolate has been maintained at -70°C in 40% glycerol containing

3% trisodium citrate followingthe initial isolation and microbiological characterization of the isolate. The isolate was subsequently recovered on Sabouraud-dextrose(SAB) agar (GIBCO-BRL, Ground

Island, NY) at 37°C for 18 hours. C. albicans was then recultured on SAB agar plates for 18 hr at

37°C and harvested in 3 ml of 10 mM phosphate buffer saline (PBS) pH 7.2 and utilized to inoculate trays (30 cm _x 22 cm) of SAB agar which were then incubated for 5 days at 37°C before cells were harvested.

Example 1 Synthesis of Octyl-βGalNAcq→4)βGal Derivatives A. Quantitative Structural Measurements

Optical rotations were measured with a Perkin-Elmer 241 polarimeterat 22 + 2°C. Analytical TLC was performed on Silica Gel 60-F₂₅₄ (E. Merck, Darmstadt, Germany) with detection by quenchingof fluorescence and/or by charringwith sulfuric acid. "IATROBEADS" refers to a beaded silica gel 6RS-8060 manufactured by Iatron Laboratories (Tokyo). All commercial reagents were used as supplied and chromatography solvents were distilled prior to use. Column chromatography was performed on Silica Gel 60 (E. Merck; 40-60 _μM). 'H NMR spectra were recorded at 360 MHz (Bruker WM-360) and first order proton chemical shifts (H are referenced to either internal CHC1₃ (δH 7.24, CDC1₃) or internal acetone (δH 2.225, D₂0). Organic solutions were concentrated under vacuum at <40°C (bath). Microanalyses and Electro-spray mass (ES-MS) spectra were carried out by the analytical services, Department of Chem istry, University of Alberta.

14 B. Synthesis of 2-Q-propvl-octyl-βGalNAc( 4)βGaKCompound 7; "Fimbrigal-P"V

The step and intermediate compound designators used in this section refer to the labels shown in FIG. 4.

1. Step (a): Synthesis of compound II [Octyl β-D-galactopyranoside] Compound II was synthesized according to the procedure as described by De Bruyne and van Der Groen (1972), incorporated herein by reference. Benzene (or chloroform) (200 mL), calcium sulfate Fluka (40 g), yellow mercuric oxide (21.6 g, 0.1 mole), mercuric bromide (1.5 g), octyl alcohol (0.1 to 1 mole), and tetra-O-acetyl-β-D-galactopyranosylbromide (I; Barczai-Martos and Kδrδsy, 1950) were stirred in a closed vessel for several hours at room temperature (monitor by TLC). After filtration and thorough washing with water, the solution was evaporated in vacuo to a syrup. The crude mixture was deacetylated with sodium methoxide, and the product was recrystallizedfrom the appropriate solvent. Anal. Cal. for C,₄H₂₈0₆: C: 57.5%, H: 9.6%; found: C: 57.5%, H: 9.5%.

2. Step (b): Synthesis of compound III [Octyl 3-(9-benzyl-(-D-galactopyranoside|

This method is after that described by Barczai-Martos, M. and Korosy, F. (Nature 165: 369, 1950). Compound II (10.02 g, 34.32 mmol) and dibutyltin oxide (8.51 g, 34.20 mmol) were refluxed in benzene (250 mL) for 21 hours. Water was removed by passing the refluxing solvent through a column of 4A molecular sieves. To this solution was added Bu₄NI (12.87 g, 34.88 mmol) and PhCH₂Br (8.2 mL, 69 mmol), and refluxing continued for an additional 21 hours. The solution was evaporated to give a brown oil which was dissolved in CH₂C1₂ and extracted with a saturated solution ofNa₂S₂0₃. After evaporation, column chromatography of the resulting oil (1 : 1 CH₂Cl₂-EtOAc) gave III (7.8 g, 60%) as a white solid. [α]D₂₅ = -3.1° (c 1.4, CHC13). 'H-NMR (360 MHz, CDC1₃): 57.20-7.45 (m, 5 H, Ph), 4.74 (s, 2 H, PhCH₂), 4.23 (d, 1 H, J = 7.8 Hz, H-l), 3.74-4.04 (m, 5 H, H-2, H-4, H-6a, H-6b, OCH₂CH₂), 3.39-3.56 (m, 3 H, OCH₂CH₂, H-5, H-3), 2.77 (d, 1 H, J = 1 Hz, 4-OH), 2.56 (d, 1 H, J = 2.5 Hz, 2-OH), 2.44 (dd, 1 H, J = 8.5 Hz, J = 4 Hz), 1.62 (m, 2 H, OCH₂CH₂), 1.20-1.40 (10 H, octyl CH₂), 0.88 (t, 3 H, J = 7 Hz, octyl CH₃). Anal. Cal. for C₂₁H₃₄O₆ (382.50): C: 65.97%, H: 8.90%; found: C: 65.69%, H: 8.98%. "Bn" in FIG. 4 A represents C₆H₅CH₂).

3. Step (c): Synthesis of compound IV [Octyl 3-O-benzyl-4,6-di-O- benzylidene-(-D-galactopyranoside] This step is according to the method described by Lowary, T.L, and F. Korosy (Carbohydrate Research 249: 163-195, 1993). Galactoside III (6.56 g, 17.2 mmol) was dissolved in PhCHO (50 mL) and ZnCl₂ (3.79 g, 27.8 mmol) was added. After stirring for 5 hours, the solution was cooled to 0°C and water (125 mL) was

15 added. Stirring continued for 1 hour and then the mixture was diluted with CH₂C1₂ and extracted with NaHCO , water, and brine. The organic layer was dried and evaporated under reduced pressure to remove the PhCHO. Column chromatography of the resulting clear oil (3:1 hexane-EtOAc) gave IV (6.9 g, 86%) as a white solid. [α]_{D25 = +32} ^ _(c , _{4 CH} n)- Η-NMR (360 MHz, CDC1₃): 57.20-7.60 (m, 10 H, Ph), 5.46 (s, 1 H, PhCHO₂), 4.76 (s, 2 H, PhCH₂), 4.30 (dd, 1 H, J = 12.5 Hz, J = 1.5 Hz, H-6a), 4.28 (d, 1 H, J = 7.8 Hz, H-l), 4.12 (dd, 1 H, J = 3 Hz, J = 1 Hz, H-4), 4.02 (dd, 1 H, J = 12.5 Hz, J = 1.5 Hz, H-6b), 3.94 (dt, 1 H, J = 10 Hz, J = 7 Hz, OCH₂CH₂), 3.44-3.54 (m, 2 H, H-3, OCH₂CH₂), 3.34 (br s, 1 H, H-5), 2.43 (br, s, 1 H, 2-OH), 1.55-1.70 (m, 2H, OCH₂CH₂), 1.20-1.40 (10 H, octyl CH₂), 0.88 (t, 3 H, J = 7 Hz, octyl CH₃). Anal. Cal. for C₂₈H₃₈O₆ (470.61): C: 71.46%, H: 8.14%; found: C: 71.17%, H: 8.00%.

4. Step (d): Synthesis of compound V [Octyl 3-O-benzyl-4,6-di-Q-benzylidene-

2-O-propyl-β-D-galactopyranoside| To a solution containing compound IV (25.5 g, 54.3 mmol) and 1 -bromopropane (14.8 mL, 163.0 mmol) in dry DMF (100 mL), was added NaH (80% in mineral oil, 4.9 g, 170.0 mmol) in portions, the mixture was stirred at room temperature overnight. The reaction was quenched by MeOH, and diluted with AcOEt (1000 mL). The organic solution was washed with H₂O (3 _x 500 mL), dried over MgSO₄ and concentrated. The pure compound IV was obtained by column chromatography using 4:1 hexand - AcOEt as eluant (21.9 g, yield 80%). Η-NMR (360 Mhz, CDC1₃): (δ[(7.26 - 7.58 (m, 10H, Bn + Ph), 5.50 (s, IH, PhCH), 4.80 (d, IH, J=12.0 Hx, Bn), 4.76 (d, IH, J=12.0 Hz, Bn), 4.28 (d, IH. J=7.7 Hz, H-l), 1.20 - 1.70 (m, 14H, CH₂-chain), 0,94 (t, 3H, J=6.6 Hz, CH₃-propyl), 0.86 (t, 3H, J+7.4 Hz, CH3-octyl).

5. Step (e): Synthesis of compound VI [Octyl 2-O-propyl-β-D- galactopyranoside] A mixture of compound V (8.2 g, 13.0 mmol) and Pd(OH)₂ on charcoal (1.0 g) in EtOH (400 mL) was hydrogenated for 10 hours at room temperature. The catalyst was filtered off over celite and the organic solution was concentrated and co-evaporated with toluene (3 _x 100 mL) to afford triol VI (5.2 g, yield 97%). Η-NMR (360 MHz, CD,OD): δ 4.20 (d, IH, J=7.7 Hz, H-l), 3.88 (m, 1 H, OCH₂-chain), 3.25 (dd, 1 H, J=7.7 Hz, 9.7 Hz, H-2), 1.57 (m, 2H, CH₂-chain), 1.18 - 1.36 (m, 10H, CH₂-chain), 0.83 - 0.92 (m, 6H, CH₃-octyl + CH₃-propyl).

16 6. Step (f): Synthesis of compound VII Octyl 3,6-di-O-benzoyl-2-O-propyl-β- D-galacto-pyranosidej A mixture of the triol VI (5.7 g, 17.0 mmol) and bis(tributyltin) oxide (13.9 mL, 27.2 mmol) in toluene (420 mL) was heated to reflux with continuous removal of H₂O through a Dean- Stark tube. After 18 hours, the reaction mixture was allowed to cool to room temperature. Benzoyl chloride (3.95 mL, 34.0 mmol) was then added, the reaction was continued at room temperature for 12 hours. After this time TLC (AcOEt: toluene 1 :9 v/v) indicated the formation of a major product VII. Methanol (20 mL) was added to the reaction mixture and the crude reaction mixture was concentrated. The combined product of six similar reactions were combined and purified on a column of silica gel 1.5 kg using (AcOEt - toluene 1 :20 v/v) as eluent. The fractions containing compound 17 were then concentrated, pentane (300 mL) was added to induce the crystallization. The crystals were collected by filtration and washed with a small amount of pentane (44.4 g, yield 80%). [α]D₂₅=+14.7° (c 2.0, CHC13). Η-NMR (360 MHz, CDC1₃): (58.06 (m, 2H, Bz). 8.01 (m, 2H, Bz), 7.55 (m, 2H, Bz), 7.39-7.45 (m, 4H, Bz), 5.11 (dd, IH, J=3.3 Hz, 10.1 Hz, H-3), 4.60 (dd, IH, J=6.8 Hz, 11.3 Hz, H-6a), 4.51 (dd, 1 H, J=6.4 Hz, 1 1.3 Hz, H-6b), 4.43 (d, IH, J=7.7 Hz, H-l), 4.18 (br d, IH, J=2.9 Hz, H-4), 3.92 (m, 2H, H-5 + OCH₂-chain), 3.78 (m, IH, OCH₂-chain), 3.67 (dd, IH, J=7.7 Hz, 10.1 Hz, H-2), 3.52 (m, 2H, OCH₂ chain), 2.45 (br s, IH, OH-4), 1.20 - 1.70 (m, 14H, CH₂-chain), 0.86 (t, 3H, J=6.6 Hz, CH₃-propyl), 0.74 (t, 3H, J=7.4 Hz, CH₃-octyl). FABMS: Cal. for C₃₁H₄₂O₈: 542.477; found: 565.0 (M + Na⁺), Anal. Cal. for C₃₁H₄₂O₈: C: 68.61%, H: 7.8%; found: C: 68.64%, H: 8.11%.

7. Step (g): Synthesis of compound IX [1.3A6-Tetra-O-acetyl-2-deoxy-2- phthalimido-α,β-D-galactopyranose^"l D-Galactosamine hydrochloride VIII (20.0 g, 92.7 mmol) was dissolved in H₂O (100 mL) at room temperature, solid NaHCO₃ (7.8 g) was slowly added by portions. When the CO₂ evolution stopped, phthalic anhydride ( 13.8 g) was added, and the reaction was stirred for 18 hours at room temperature. The solvent was removed under reduced pressure and the resulting mixture was dried under high vacuum for 5 hours. The reaction mixture was suspended in pyridine (80 mL) at 0°C, Ac₂O (80 mL) was added, and the reaction was allowed to warm to room temperature over 4 hours. N,N- Dimethylaminopyridine(1.0 g) was added to the solution and the reaction was stirred for 8 hours at 50 °C. The reaction mixture was concentrated under reduced pressure, redissolved in CH₂C1₂ (500 mL), and washed with water (2 _x 100 mL), dried over Na₂SO₄ and concentrated. Chromatography on silica gel using ethyl acetate - toluene (1 :3) afforded the

17 desired compound 7 as a colorless white foam α/β = 36/65, 24.0 g, 54%). 'H-NMR (360 MHz, CDC1₃): 5 7.83 (m, 2H, Phth), 7.74 (m, 2H, Phth), 6.50 (dd, 0.35H, J=3.1 Hz, 12.0 Hz, H-3α. 6.42 (d, 0.65H, J=9.6 Hz, H-lβ), 6.33 (d, 0.35H, J=3.2 Hz, H-lα).

8. Step (h): Synthesis of compound X [3A6-Tri-O-acetyl-2-deoxy-2- phthalimido-α,β-D-galactopyranosyl bromide ^"1. The anomeric α,β-mixture of IX (117 g, 0.25 mmol) was dissolved in 30% HBr-HOAc (750 mL), acetic anhydride (60 mL) was added and the reaction was stirred for 18 hours at room temperature. The volume of the reaction solution was halved by evaporation while maintaining the temperature below 40°C. Then the mixture was poured into crushed ice (1000 mL), and extracted with dichloromethane(1000 mL), the organic phase was separated and washed with 5% NaHCO₃ solution (1 x 300 mL) and H₂O (1 _x 300 mL), dried over Na₂SO₄ and concentrated. The syrupy mixture was recrystallized from a mixture of ether-hexane to afford light brown crystal of X α,β = 70/30, 67 g, 55%). Η-NMR (360 MHz, CDC1₃): 5 7.85 (m, 2H, Phth), 7.74 (m, 2H, Phth), 6.66 (d, 0.7H, J=3.7 Hz, H-lα), 6.51 (dd, 0.7H, J=3.1 Hz, 12.0 Hz, H-3α), 6.37 (d, 0.3H, J=9.6 Hz, H-lβ), 5.75 (dd, 0.3H, J=3.4 Hz, 11.1 Hz, H-3α, 5.69 (dd, 0.7H, J=0.9 Hz, 3.0 Hz, H-4α), 5.52 (br d, 0.3H, J=3.4 Hz, H-4β), 4.81 (m, 1 H, H-2α + H-2β), 4.58 (m, IH, H-5α + H-5β), 4.14 - 4.24 (m, 2H, H-6aα + H-6aβ + H-6bβ + H-6bβ, 2.21 (s, 0.9H, OAcβ), 2.15 (s, 2.1 H, OAcα), 2.05 (s, 3H, OAcα + OAcβ), 1.88 (s, 2.1 H, OAcα), 1.83 (s, 0.9H, OAcβ).

9. Step (i): Synthesis of compound XI (Octyl O-(3,4,6-tri-0-acetyl-2-deoxy-2- phthalimido-β-galacto-pyranosyl)-(l->4)-3,6-di-O-benzoyl-2-Q-propyl-β-galacto- pyranosidel. Three parallel reactions were carried out identically and the products from each were combined:

A mixture containing the acceptor alcohol VII (12.0 g, 22.1 mmol), Ag₂C0₃ (16.1 g, 58.4 mmol, 2.6 eq), AgOTf (14.4 g, 56.0 mmol, 2.5 eq), and molecular sieves 4 A (20 g) was dried under high vacuum for 3 hours. CH,C1₂ (200 mL) was added, the mixture was protected under an atmosphere of Argon and cooled to -82 °C. Another solution containing the donor 3 (22.2 g, 44.8 mmol, 2.0 eq) in CH₂C1₂ (150 mL), cooled to -82°C, was added slowly through a canula under Argon. The reaction was continued at -82 °C for 8 hours. The reaction mixture was filtered through celite, the celite was washed with CH₂C1₂.

The organic filtrates of the above three reactions were combined and washed with a 1 : 1 (v/v) mixture of saturated aqueous NaHCO₃ and aqueous 10% Na₂S₂O₃, dried over anhydrous Na₂SO₄, and concentrated. Chromatography using ethyl acetate - toluene (1 :9) gave: first, the starting acceptor VII (8.0 g), second, the disaccharide XI (46.1 g, 72.4% isolated yield, 93% based on consumed acceptor). [α]D₂₅=5.6° (c 0.5, CHC1₃). Η-NMR (360 MHz, CDC1₃): 58.03 (m, 2H, Ar), 7.88 (m, 3H, Ar), 7.53 - 7.73 (m, 9H, Ar), 5.81 (dd, IH, J=3.4 Hz, 11.3 Hz, H-3'), 5.46 (d, IH, J=8.2 Hz, H-l'), 5.36 (d, IH, J=3.2 Hz, H-4'), 4.92 (dd, IH, J=2.6 Hz, 10.3 Hz, H-3), 4.61 (dd, IH, J=5.9 Hz, 11.5 Hz, H-6a), 4.46 - 4.58 (m, 3H, H-5' + H-6b + H-2'), 4.28 (d, IH, J-7.4 Hz, H-l), 4.02 (dd, 1 H, J=6.7 Hz, 11.2 Hz, H-6a'), 3.85 - 3.92 (m, 2H, H-6b' + OCH₂-chain), 3.75 - 3.79 (m, 2H, H-4 + H-5), 3.30 - 3.40 (m, 2H, OCH₂-chain), 3.12 (dd, IH, J=7.4 Hz, 10.2 Hz, H-2), 2.90 (m, IH, OCH₂-chain), 2.14 (s, 3H, OAc), 1.93 (s, 3H, OAc), 1.81 (s, 3H, OAc), 1.45 - 1.56 (m, 2H, CH₂-chain), 1.09 - 1.29 (m, 12H, CH₂-chain), 0.86 (t, J=6.6 Hz, CH₃-propyl), 0.58 (t, J=7.3 Hz, CH₃-octyl). ES-MS: Cal. for C₅,H₆₁O₁₇N: 959.4; found: 982.4 (M + Na⁺). Anal. Cal. for C₅,H₆₁O₁₇N: C: 63.8%, H: 6.41%, N: 1.46%; found: C: 63.84%, H: 6.54%,N: 1.51%.

10. Step (]): Synthesis of compound 7 (XII) [Octyl O-(2-acetamido-2-deoxy-β-D- galactopyranosyl)-(L>4)-2-O-propyl-β-D-galactopyranoside] Several parallel reactions (total amount of starting material used: 64.0 g, 60.4 mmol) were carried out by the following typical procedure: The protected disaccharide XI (23.1 g, 24.1 mmol) was dissolved in MeOH (400 mL), hydrazine monohydrate (75 mL) was added, and the solution was refluxed for 18 hours. The reaction mixture was concentrated under reduced pressure, and dried under high vacuum for 5 hours. The resulting mixture was redissolved in MeOH (350 mL), and acetic anhydride (90 mL) was added by portions. The reaction was refluxed for 2 hours, cooled to room temperature, concentrated, and combined with the material obtained from other reactions.

The final pure disaccharide was obtained by chromatography on reversed phase silica gel (C 18 , prepared previously in this Lab) . Reversed phase C 18 silica gel was prepared by a modification according to Evans, et al. (1980). To a slurry of dry silica gel for flash chromatography (Merck, Grade 60, 230-400 Mesh, 60 A, 300 g) in anhydrous toluene (3000 mL), was added n-octodecyltrichlorosilane(30 mL). The reaction was continued at room temperature for 5 hours. The product was filtered and washed successively with toluene (3000 mL) and dry methanol (4000 mL), and dried at 40 °C overnight. The filtrate was suspended in dry toluene (3000 mL), trimethylchlorosilane(30 mL) was added, the reaction

19 was continued for another 3 hours. After filtration, the reversed phase silica gel C18 was obtained by washing with more methanol (4000 mL) and dichloromethane (4000 mL), and air dried at room temperature. The above reaction mixture was purified on the prepared reversed phase silica gel C 18 using a stepped gradient of water and water-MeOH (1 : 1 v/v) as eluent (32 g, 92.0%). [α]D₂₅=+2° (c 0.45, H2O). Η-NMR (360 MHz, CD₃OD): 5 4.59 (d, IH, J=8.5 Hz, H-l'), 4.19 (d, IH, J=7.8 Hz, H-l), 3.96 (br d, IH, J=3.0 Hz, H-4'), 3.71 (b rd, IH, J=2.8 Hz, H-4), 3.16 (dd, IH, J=7.8 Hz, 9.6 Hz, H-2), 1.98 (s, 3H, Ac), 1.55 (m, 4H, CH₂-chain), 1.18 - 1.35 (m, 10H, CH₂-chain), 0.88 (t, 3H, J=7.4 Hz, CH₃-propyl), 0.86 (t, 3H, J=7.0 Hz, CH₃-octyl). ES - MS: Cal. for C₂₅H₄₇O_nN: 537.7; found: 538.3 (M + H⁺). Anal. Cal. for C₂₅H₄₇O,,N.H₂O: C: 54.04%, H: 8.89%, N: 2.52%; found: C: 54.53%, H: 8.89%, N: 2.52%.

A. Synthesis of Octyl (2-acetoamido-2-deoxy-β-D-galactopyranosyl-(l->4)-β-D- galactopyranoside (Compound 1, FIG. 1 and Compound XV, FIG. 5) 1. Step (k): Synthesis of Compound XIII [Octyl 2,3.6-tri-O-benzoyl-β-D- galactopyranosidej Octyl β-D-galactopyranoside (Compound II, synthesized as described in Part B, step (a), above; 2.1 g, 7.2 mmol) was dissolved in dry pyridine (25 mL), and cooled to -40°C, benzoyl chloride (2.1 mL, 18.0 mmol) was added dropwise. The reaction was continued for 3 hours at -40°C, and quenched with H₂O. CH₂C1₂ (200 mL) was added, the organic solution was washed with H₂O (2 _x 50 mL), dried over Na₂SO₄ and concentrated. The tribenzoate XIII was obtained by chromatography on silica gel using 18:1 toluene - EtOAc as eluant (1.7 g, yield 40%). [α]D₂₅=+35.3° (c 0.19, CHC13). Η-NMR (360 Mhz, CDC1₃): 5 7.93 - 8.08 (m, 6H, Bz), 7.32 - 7.58 (m, 9H, Bz), 5.75 (dd, IH, J=7.8 Hz, 10.0 Hz, H-2), 5.35 (dd, IH, J=3.0 Hz, 10.0 Hz, H-3), 4.71 (d, IH, J=7.8 Hz, H-l), 4.68 (dd, IH, J=6.8 Hz, 11.3 Hz, H-6a), 4.62 (dd, IH, J=6.4 Hz, 11.3 Hz, H-6b), 4.33 (br, IH, H-4), 4.07 (t, IH, J=6.5 Hz, H-5), 3.92 (m, IH, OCH₂-chain), 3.52 (m, IH, OCH₂-chain), 2.41 (d, IH, J=6.0 Hz, OH-4), 1.50 (m, 2H, CH₂-chain), 1.02 - 1.28 (m, 10H, CH₂-chain), 0.81 (t, 3H, J=7.4 Hz, CH₃-octyl). FABMS: Cal. for C₃₅H₄₀O₉: 604.7; found: 626.9 (M + Na⁺).

2. Step (1) Synthesis of compound XIV [Octyl O-(3,4,6-tri-0-acetyl-2-deoxy-2- phthalimido-β-D galactopyranosylH l→4)-2,3 ,6-tri-O-benzoyl-β-D-galactopyranoside| A mixture containing acceptor XIII (0.8 mg, 1.42 mmol), Ag₂CΟ₃ (1.02 g, 3.69 mmol, 2.6 eq), AgOTf (0.91 g, 3.54 mmol, 2.5 eq), and molecular sieves 4 A (1.3 g) was dried under high

20 vacuum for 3 hours. CH₂C1₂ (8 mL) was added, the mixture was protected under Ar and cooled to -82°C. Another solution containing the donorX (1.77 g) in CH₂C1₂ (7 mL), cooled to -82 °C, was added slowly through a canula under Ar. The reaction was continued at -82 °C for 8 hours. The reaction mixture was filtered off through celite, the solid was washed throughout with more CH₂C1₂. The organic solution washed with a 1 : 1 (by volume) mixture of saturated aqueous NaHCO₃ and aqueous 10% Na₂S₂O₃, dried over anhydrous Na₂SO₄, and concentrated. Chromatography using 10% ethyl acetate-toluene gave the desired disaccharide XIV (1.16 g, 80%). [α]D₂₅ = 2.9° (c 1.1, CHC13). 'H-NMR (360 MHz, CDC1₃): δ 8.06 (m, 2H, Ar), 7.95 (m, IH, Ar), 7.67 - 7.76 (m, 5H, Ar), 7.40 - 7.58 (m, 6H, Ar), 7.24 - 7.32 (m, 5H, Ar), 5.78 (dd, 1 H, J=3.4 Hz, 11.6 Hz, H-3'), 5.45 (d, IH, J=8.2 Hz, H-l'), 5.37 (d, IH, J=3.2 Hz, H-4'), 5.31 (dd, IH, J=7.7 Hz, 10.4 Hz, H-2), 5.07 (dd, IH, J=2.7 Hz, 10.5 Hz, H-3), 4.71 (dd, IH, J=5.5 Hz, 11.6 Hz, H-6a), 4.65 (d, 1 H, J=2.2 Hz, H-4), 4.53 - 4.62 (m, 3H, H-6a + H-l + H-2'), 4.02 (m, 2H, H-6a' + H-5'), 3.95 (dd, 1 H, J=6.5 Hz, 11.2 Hz, H-6b'), 3.80 (m, IH, H-5), 3.69 (m, IH, OCH₂-chain), 3.43 (m, IH, OCH₂-chain), 2.14 (s, 3H, OAc), 1.95 (s, 3H, OAc), 1.82 (s, 3H, OAc), 1.38 (m, 2H, CH₂-chain), 1.00 - 1.24 (m, 10H, CH₂-chain), 0.81 (t, 3H, J=7.0 Hz, CH₃). ES-MS: Cal. for C₅₅H₅₉O₁₈N: 1022.1; found: 1044.4 (M + Na⁺). Anal. Cal. for C₅₅H₅₉O₁₈N: C: 64.63%, H: 5.82%, N: 1.38%; found: C: 64.91%, H: 6.04%, N: 1.37%.

3. Step (m): Synthesis of compound XV [Octyl O-(2-acetamido-2-deoxy-β-D- galactopyranosyl)- ( l->4)-β-D galactopyranoside j The protected disaccharide XIV (1.02 g, 1.0 mmol) was dissolved in MeOH (20 mL), hydrazine monohydrate (6 mL) was added and the solution was refluxed overnight. The reaction mixture was concentrated under reduced pressure, and dried under high vacuum for 5 hours. The resulting mixture was redissolved in MeOH (25 mL), acetic anhydride (7 mL) was added by portions. The reaction was refluxed for 2 hours, cooled to room temperature, concentrated, and purified by chromatography on previously prepared reversed phase silica gel C18 [see Step (j)] using 100% to 40%) of water- MeOH as eluent (0.45 g, 91 %). [α]D₂₅ = + 2.2° (c 0.35, H2O). 'H-NMR (360 MHz, CD₃OD): 5 4.61 (d, IH, J=8.5 Hz, H-l'), 4.15 (d, IH, J=7.7 Hz, H-l), 3.99 (br d, IH, J=2.9 Hz, H-4'), 3.86 (br d, IH, J=3.1 Hz, H-4), 3.40 (dd, IH, J=7.8 Hz, 9.7 Hz, H-2), 2.00 (s, 3H, Ac), 1.57 (m, 2H, CH₂-chain), 1.18 - 1.36 (m, 10H, CH₂-chain), 0.86 (t, 3H, J=6.4 Hz, CH₃-octyl). ES-MS: Cal. for C₂₂H₄₁O„N: 495.6; found: 496.3 (M + H⁺). Anal. Cal. for C₂₂H₄₁O,,N-(H₂O: C: 52.04%, H: 8.39%, N: 2.78%; found: C: 52.01%, H: 8.31%,N: 2.90%.

21 Example 2 Purification of C. albicans Fimbriae Fimbriae were purified from yeast phase of C. albicans. C. albicans cells were harvested from the agar surface by gentle scraping with a bent glass rod. Harvested cells were suspended in a minimal volume (50 ml/tray) of Preparation Buffer (10 mM sodium phosphate saline pH 7.2, containing 1 mM CaCl, and 1 mM phenylmethylsulfonyl fluoride). Harvested cells were washed three times with 500 ml of Preparation Buffer by centrifugation (12,000 x g for 20 min at 4°C). Fimbriae were sheared from the cell surface by gentle homogenization (4 _x 45 second cycles) using a Brinkmann Homogenizer. The cells were removed by centrifugation (12,000 _x g for 20 min) and by subsequent filtration of the supernatant through a 0.45 μm filter (Millex-PF, Millipore). The supernatant was concentrated approximately 10-fold with polyethyleneglycol, PEG (M.W. 8,000). The concentrated fimbriae preparation was dialyzed overnight at 4°C against Preparation Buffer. This material was termed crude fimbriae (CF). The CF preparation was fractionated by size- exclusion high performance liquid chromatography using an isocratic gradient (flow rate = 0.5 ml/min; column = Waters Protein-PAK 300 SW 10 μm) with Preparation Buffer as the solvent. The material that was eluted in the void volume was collected, concentrated with PEG and dialyzed against Preparation Buffer. This material was termed semi-enriched fimbriae and was rechromatographied under identical conditions. The peak which corresponded with the void volume of the column was again collected, concentrated and dialyzed against Preparation Buffer. This fraction was termed enriched fimbriae (EF).

A typical fimbriae preparation from 200g wet weight of C. albicans yielded about 70 mg of crude fimbriae (CF), which then yielded 5 mg of enriched fimbriae (EF).

Protein concentrations of CF and EF were determined using a bicinchoninic acid (BCA) protein assay (Pierce) described by Smith, et al. (1985) with bovine serum albumin (BSA) as protein standard.

22 Example 3 Purification of Adhesin Protein from Fimbriae

A. Isolation of purified Fimbrial Protein from Candida

Fimbriae were removed from washed cells by shearing, separated from cells by centrifugation and filtration and then subjected to HPLC size-exclusion chromatography (SEC) as detailed in Example 1. The fimbriae eluted as two peaks (the first peak eluting at the void volume of the column). Fimbriae were mainly associated with the first peak, as determined by SDS-PAGE and electron microscopy, carried out accordingto the methods detailed in Example 4. The first peak was collected and rechromatographed under identical conditions with the fimbriae eluting at the void volume of the column.

CF and EF preparations obtained as described above were analyzed by SDS-PAGE, as described in Example 4. The molecular weight of the purified fimbrial subunit present in the EF fraction was approximately 66 kDa, as visualized by SDS PAGE.

To purify the fimbrial protein, gel slices containingthe 66 kDa protein were cut out of the gel. The gel slices were washed (2 _X 5 min) with elution buffer (20 mM ammonium bicarbonate) by gently shaking at 50 rpm on a Gyrotory shaker model G2 (New Brunswick Scientific Co., New Brunswick, NJ) for 30 min at room temperature. The gel slices were put into dialysis tubing (M.W. cut-off of 6000 to 8000) and suspended in water. The proteins were electroeluted from the gel slices in 20 mM ammonium bicarbonate using an electroelution apparatus (Elutrap™; Schleicher & Scheull, Keene, NH) by applying a constant voltage of 200V for 5 hours or 80V overnight. The eluate was collected and dialyzed against deionized water.

The eluted protein fraction was further purified by reversed-phase HPLC (Aquapore C₄ column 100 _x 4.6 mm, 7 μm internal diameter; Brownlee Laboratories, Santa Clara, CA) using a linear AB gradient (where solvent A is 0.05% aqueous trifluoroacetic acid [TFA] and solvent B is 0.05% TFA in acetonitrile ) of 2% B/min gradient at a flow rate of 1 ml/min. The eluate containing the peak was collected and lyophilized.

B. Amino Acid Composition

Amino acid analysis of Candida fimbrial protein was carried out on the protein fraction purified by reversed-phase chromatography, as described in Part A. A small amount of the lyophilized fraction was hydrolyzed in a glass tube with 200 μl of 6N HC1, containing 0.1% (w/v) phenol at 1 10°C for 24 hours in vacuo. The acid from the hydrolysate was removed by evaporation, resuspended in citrate buffer pH 2.2 and the amino acid content was analyzed with a Beckman Model 6300 amino acid analyzer. No attempt was made to analyze for total 1/2 Cys or Trp, nor were the values for Ser and Thr corrected to take into account losses during hydrolysis. Table 3 shows the amino acid composition as number of residues/fimbrial subunit determined for C. albicans strain 40,

23 using this method.

Amino acid values are given as the number of residues determined to be present per protein molecule, with the integer value calculated in parentheses. This analysis, in which no correction was made for cysteine and tryptophan or for the destructive loss of serine and threonine during hydrolysis, indicated that the most frequent amino acid residues of the protein portion of fimbriae were valine (Val), aspartic/asparagine(Asx), glutamic acid/glutamine(Glx), serine (Ser), threonine (Thr), glycine (Gly), leucine (Leu), isoleucine (He), lysine (Lys) and alanine (Ala), while little methionine (Met) or histidine (His) was detected. (Standard 3-letter amino acid codes are used in the table.)

Table 3

Amino Acid Compositions of Fimbrial Subunits from C. albicans Strain #40

Amino Acid Residues Residues/FimbrialSubunit

Asx 8.24'(8)²

Thr^* 4.73(5)

Ser' 5.73(6)

Glx 8.17(8)

Pro 2.79(3)

Gly 7.02(7)

Ala 6.00(6)

Cys ND

Val 5.57(6)

Met 0.82(1)

He 4.73

Leu 6.82(7)

Tyr 2.55(3)

Phe 3.21(3)

His 1.48(1)

Lys 5.91(6)

Trp ND

Arg 3.81(4)

No. of Residues (79)

Estimated MW 8644

1. Number of residues determined experimentally.

2. Integervalue. ND Not determined.

No correction was made for Cys and Trp, or for the destructive loss of Ser and Thr during hydrolysis.

24 A. Carbohydrate Composition

A phenol-sulfuric acid carbohydrate assay described by Dubois, et al. (1956) was used to determine the amount of carbohydrate present in the EF preparation. EF was diluted 1 :10 with 2N H₂SO₄. Diluted EF (0.5 ml) was added to 0.5 ml of a 5% solution of aqueous phenol and 2.5 ml of H₂SO₄ reagent (2.5 g hydrazine sulfate in IL of concentrated sulfuric acid) and mixed vigorously. The mixture was incubated in the dark for 1 h at room temperature. The absorbance at 490 nm (A₄₉₀) of the reaction mixture was recorded. D-mannose (Sigma Chemical Co., St. Louis, MO) was dissolved in 2N H₂SO₄ and employed as a standard (0 to lOO μg/ml). Based on the known amount of EF used for the carbohydrate analysis, both the protein and carbohydrate content in the C. albicans fimbriae were used to determine the ratio of carbohydrate and protein.

The carbohydrate composition of the EF was determined using standard methods known in the art. Briefly, lyophilized carbohydrate samples were methanolyzed with dry 2 M HCl/methanol for 16 hours at 85 °C. The derivatization mixture (2 μl) was used directly. Samples were analyzed with a Varian Vista 6000 equipped with a Varian CDS 401 data station and a flame ionization detector (Varian, Santa Clara, CA), and employed a J & W DB-5 (95% methyl-, 5% phenylpolysiloxane) 30 cm long _x 0.25 mm internal diameter column using helium carrier at a flow rate of 1 ml/min. The column was held isothermal for the initial 4 min at 90 °C; the temperature was then allowed to rise 8°C/min to a maximum of 270°C. Authentic carbohydrate samples (Sigma Chemical Co.) were derivatized and utilized as a standards.

B. Purification of P. aerusinosaFAK pili Pili from P. aeruginosa strains PAK were purified as previously described by

Paranchych, et al. (1979), incoφorated herein by reference.

C. Biotinylationof P. aeruginosaV AK pili

Biotinylationof P. aeruginosa pili was carried out accordingto the procedure of Wong, et al. (1995). Briefly, 1 ml of the PAK pili (1.76 mg/ml) was mixed with 30 μl of 20 mg/rnl solution of biotinamidocaproate N-hydroxysuccinimide ester (Sigma Chemical Co.) in a dialysis tubing (MW cutoff: 12,000 - 14,000). The dialysis tubing was placed inside a 50 ml conical tube and incubated under constant shaking for 45 min at room temperature. Glycine

25 (10 mM) was added to quench the reaction, and excess biotin ester was removed by extensive dialysis with 4 changes of phosphate-buffered saline (PBS, 10 mM phosphate and 150 mM NaCl, pH 7.4) at 4°C.

Example 4

Physical Properties of Purified Fimbriae

A. Molecular Weight Determination

Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was performed with 12.5% acrylamide gels in a mini-gel apparatus (Mini-protein® II Dual Slab Cell; Bio-Rad Laboratories, Richmond, CA) according to standard procedures. Samples were electrophoresedfor 50 min at a constant voltage of 200V with a power supply model 1420A (BioRad Laboratories). Gels were stained with Coomassie blue (R-250; Bio-Rad Laboratories) or with silver stain.

B. Electron Microscopy

Fimbriae were diluted 1 :100 with 10 mM sodium phosphate buffer pH 7.2. A 20 μl drop of diluted fimbriae solution was placed on a freshly prepared carbon/formvar coated 3 mm 200 mesh copper electron microscope grid (Fisher Scientific, Orangeburg, NY). The grid was blotted with Whatman ^#\ filter paper, then negatively stained with 1% (w/v) phosphotungsticacid at a pH 7.0 for 10 seconds. The stain was removed by blotting and the sample was examined with a Philips model 410 transmission electron microscope operating at an accelerating potential of 80 kV (Eindhoven. Netherlands). Micrographs were recorded on Kodak electron microscope film #4489 (Eastman Kodak Co., Rochester, NY).

Scanning electron micrographs of fimbriae of yeast phase C. albicans bound to human BECs were made as follows: Specimens (3 ml) were fixed with a 2.5% (v/v) glutaraldehyde (J.B. EM Services Inc., Point Claire, Dorval Quebec, Canada) in 0.1M phosphate buffer pH 7.3 and incubated overnight at 4°C. Samples were aliquoted in 1.5 ml eppendorf tubes centrifuged at 120 rpm for 10 minutes and washed 3 _x 20 min with 1.0 ml phosphate buffer pH 7.3. Samples were post-fixed in 2% (w/v) osmium tetroxide in 0.1 M phosphate buffer pH7.3 for 1 hour. The cells were washed by centrifugation as described above. Specimens were then resuspended in 1.0% (w/v) tannic acid in distilled water and incubated for 30 minutes at room temperature. The solution was removed by aspiration and the cells were washed with water and then resuspended in 2% (w/v) aqueous osmium tetroxide for 1 hour

26 and then washed with water. Specimens were then dehydrated in a graded series of ethanol io 100%. Samples were critical point dried and subsequently salted onto a standard Cambridge scanning electron microscope stub pre-coated with double-sided adhesive tape. Specimens were then directily examined in a Hitachi (Tokyo, Japan) S 4000 field emission scanning electron microscope at an accelerating potential of 2.5 kV.

Example 5

Binding of Fimbriae and Fungal Cells to Epithelial Cells A. Direct Fimbrial Binding Assay

Human buccal epithelial cells (BECs) were collected from 10 healthy, non-smoking male volunteers by gentle scraping of the buccal mucosal surface with wooden applicator sticks. These sticks were then agitated in 40 ml of PBS pH 7.2 to remove the BECs. BECs were washed 3 _x 10 min with 10 ml of PBS by centrifugation at 2,000 x g. Cell clumps were removed by filtration through a 70 μm nylon mesh (Spectrum, Cole-Parmer Instrument Co., Niles, IL). The cell concentration was determined directly with a hemocytometer and BECs were resuspended in PBS to a concentration of 2.0 _x 10^D BECs/ml.

The fimbrial adherence assay was performed using a Manifold filtration apparatus equipped with individual vacuum stopcocks (Model FH 225V; Hoefer Scientific Instruments, San Francisco, CA). Polycarbonate filters, 12 μm pore size (Nucleopore Costar Corporation, Pleasanton, CA), were pre-incubated overnight at 4°C with 50 ml of PBS pH 7.2, containing 0.45%) (v/v) Tween 20. The pretreated filters were placed into each chamber and washed with 2.5 ml of PBS. 1 ml containing 2.0 _x 10^D BECs in PBS was added to each chamber. Enriched fimbriae (100 μl /chamber, ranging from 0 to 80 μg fimbriae protein /ml) in PBS containing 0.05% (v/v) Tween-20 was added to each chamber and incubated with the BECs for 1.5 hours at room temperature. Unbound fimbriae were removed with washes of 2.5 ml of PBS. Mouse anti-C. albicans fimbriae monoclonal antibody prepared according to standard methods. (Mishell and Shiigi, 1980). Following removal, ascites fluid diluted 1:3000 with PBS) was added to the BECs (1.2 ml/chamber) and incubated for 1.5 hour at room temperature. BECs were washed five times with 2.5 ml of PBS. Goat anti-mouse IgG(H+L) -peroxidase conjugates (Jackson Laboratories, Bar Harbor, ME) diluted 1:5000 with PBS was added (1.0 ml/chamber) and incubated for another hour at room temperature. The cells were then washed seven times with 2.5 ml/chamber of PBS. The polycarbonate filters containing BECs were removed from the filtration manifold and placed into glass

27 scintillation vials. The horseradish peroxidase substrate solution (ABTS) was added to each vial (1 ml/vial) and incubated for 30 min at room temperature on a shaker at 100 rpm. The reaction was stopped by the addition of 4 mM sodium azide (200 μl/vial). The substrate solution was pipeted into Eppendorf tubes and centrifuged at 5,000 g for 3 minutes. Aliquots of the supernatants were pipetted into microtiter wells (200 μl/well) and the resulting absorbance at 405 nm was recorded with a Titertek Multiskan Plus microplate recorder.

B. Inhibition of C. albicans binding to BECs

C. albicans cells were radiolabelled with ³⁵S-methionine, as described by McEachran and Irvin (1985). Briefly, a loopful of culture from Sabouraud-dextrose agar (GIBCO-BRL) was used as a source of inoculum for 10 ml of M9 medium supplemented with 0.4% (w/v) glucose. Cultures were incubated at 25 °C for 12 hours with 150 rpm agitation in G25 Gyrotory shaker (New Brunswick Scientific Co.). Cultures were supplemented with 5 μCi/ml of [^3DS]-L-methionine (New England Nuclear, Boston, MA) after 10 hours of incubation. Cells were harvested by centrifugation (12,000 _x g for 10 min) and washed 3 times with 10 ml of PBS pH 7.2 to remove unincorporated methionine. Washed cells were resuspended in PBS. No clumping was observed during the assay. The amount of [³³S]-L- methionine incorporated by the C. albicans cells was determined by filtering 1.0 ml of a 1 :100 dilution of washed C. albicans culture through a 0.2 μm polycarbonate filter (Nucleopore Corporation) in triplicate, washing with 15 ml of PBS, and placing the filter in scintillation vials with 5.0 ml of Aquasol (New England Nuclear, MA). The counts per minute were determined with a Beckman LS-150 liquid-scintillation counter. The specific activity of [³⁵S]-C. albicans cells was generally 0.2 cpm/CFU and this remained stably associated with the C. albicans cells throughout the assay. BECs (0.5 ml) were preincubated with EF at varying concentrations (from 0 to 18 μg protein ml) in polystyrene tubes at 37 °C for 1 hour (final concentrations: 2.0 _x 10⁵ BECs/ml). An equal volume of radio-labelled yeast suspended in PBS pH 7.2 was added to the BECs and incubated at 37°C for 2 hours, shaking at 300 rpm. Triplicate aliquots were removed after the assay and filtered through 12 μm polycarbonate filters pretreated with 3%> (w/v) BSA in PBS. BECs were washed with 15 ml of PBS. The filters were then placed in scintillation vials and the cpms were determined as described above. Yeast binding to BECs was corrected for nonspecific binding of yeast to the 12.0 μm filter (nonspecific binding was generally less than 15% of the experimental value). The BEC concentration was determined

28 at the end of the assay to correct for cells lost during incubation.

Total and viable cell counts were performed before and after the adhesion assay. Total cell counts were determined using a hemocytometer. Viable counts were determined by serially diluting C. albicans in PBS pH 7.2 and plating appropriate dilutions on SAB agar which were incubated at 37°C until visible and countable colonies formed (usually 24 to 48 hours).

In a similar manner, experiments were carried out testing the ability of βGalNac(l-,4)βGal derivatives to inhibit binding of C. albicans to BECs. Here, increasing concentrations of a test compound were added to the reaction mixture, to determine a concentration of derivative effective to inhibit binding by 50%

Example 6 Binding of Candida fimbriae to Glycosphingolipids

A. Thin-layer Chromatography Plate Binding Assay The thin-layer chromatography (TLC) plate binding assay was performed as described by Baker, et al (1991) with minor modifications. Aluminum-backed silica gel Si60 high performance TLC plates (Merck Kieselgel Si60, no fluorescence indicator; E. Merck) were cut to produce 8 _x 2.5 cm plates which were chromatographed with 100%> methanol to the top of the plate to remove impurities and the plates were air dried. Glycosphingolipids (GSLs) (10 μg of each GSL) were loaded 1.0 cm above the base of the plate. The following glycosphingolipids purchased from Sigma Chemical Co. were used: mono-sialoganglioside (M-GM,), asialoganglioside GM, (asialo-GM,), asialoganglioside GM₂ (asialo-GM₂), lactosylcerebroside (LCS), ceramide trihexoside (CTH). GSLs were separated on the TLC plates in chloroform-methanol-water(65:35:8, v/v/v) and air dried. One set of plates was sprayed with 10%) sulphuric acid in ethanol and heated at 100-150°C for 5-10 min to char the GSLs for visual detection, and the other set was used for the fimbrial binding assay. The four corners of the plate were bent to 90° and the remainder of the assay was done with the TLC plates inverted in all solutions and at room temperature in an incubator shaker (model G25 Gyroshaker; New Brunswick Scientific.) at 20 rpm agitation. The TLC plate was blocked with 50 mM tris-hydroxy-methyl aminomethane pH 7.5 containing 150 mM NaCl (TBS), 0.25% (w/v) gelatin, 3% (w/v) BSA, 5 mM EDTA and 0.05% (v/v) Nonidet P40 in a glass petri dish for 2 hr at room temperature. The blocking solution was aspirated and 10 ml of EF (100 μg EF/ml in 100 mM TBS, pH 7.5) was then added. The fimbriae were allowed to bind

29 to GSLs for 2 hours at room temperature. The plates were gently washed (2 _x 5 min) with 40 ml of 100 mM TBS containing 0.1% (v/v) Tween 20 (TBST). The murine anti-EF monoclonal antibody, Fml6, was diluted 1 :200 with TBST and 10 ml was added to the TLC plates. The solution was incubated for 1 hour at room temperature. Unbound antibodies were removed by washing the plates with 10 ml of TBST (2 _x 5 min). The plate was then incubated with 10 ml of goat anti-mouse immunoglobulin G alkaline phosphatase conjugate (Jackson Laboratories) diluted 1 :5,000 with TBST for 1 hour at room temperature. The plates were washed (2 _x 5 min.) with 10 ml of TBST. The alkaline phosphatase activity was localized with Nitro Blue Tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) dissolved in 100 mM Tris buffer, pH 9.5 containing 100 mM NaCl and 5 mM MgCl₂. Color development was quenched by rinsing the TLC plate with deionized water and submerging the plate into a 150 mM EDTA solution pH 8.0 for 3-5 min. The plates were air dried, stored in plastic in the dark until they were photographed.

B. Plate Binding Assay

Polystyrene microtiter plate wells (Nunc) were coated with asialo-GM, or ceramide trihexoside (CTH) (Sigma Chemical Co.). Aliquots of the GSLs (5 μg/ml resuspended in methanol) were added into the wells (100 μl/well) and the plates were incubated overnight at 4°C. The wells were washed three times with 250 μl/well of 10 mM phosphate buffered saline pH 7.4 containing 150 mM NaCl (PBS) supplemented with 0.05% (w/v) bovine serum albumin (Buffer A). Excess binding sites were blocked by the addition of 200 μl/well of 5% (w/v) BSA in PBS pH 7.4 and incubation at 37°C for 1 hour. Wells were washed three times with 250 μl/well of buffer A. Enriched fimbriae ranging from 0 to 40 μg protein/ml in Buffer A were added to the wells (100 μl/well) and incubated for 2 hour at 37 °C. Aliquots of mouse anti-EF monoclonal antibodies, Fml6 (diluted 1 :500), was added to each well (100 μl) and incubated at 37°C for 2 hours. Wells were washed 5 times with 250 μl/well of Buffer A. Antibody binding to EF was assessed by the addition of a goat anti-mouse heavy and light chain [IgG(H+L)] immunoglobulin G-peroxidase conjugates (Jackson Laboratories) to each well (100 μl/well) and incubated for 1 hour at 37°C. The wells were washed 5 times with 250 μl/well of Buffer A and a substrate solution containing 1 mM 2,2'-azido-di-[3- ethylbenzthiazoline sulfonic acid] (ABTS) in 10 mM sodium citrate buffer (pH 4.2) containing 0.03% (v/v) hydrogen peroxide was added (125 μl/well). The reaction was stopped by an addition of 125 μl/well of 4 mM sodium azide and the absorbance at 405 nm

30 was recorded.

Inhibition of binding of Candida fimbrial binding was carried out as described above, except that C. albicans fimbriae were preincubated with asialo-GM, and CTH, respectively, for 1 hour at 37 °C prior to their addition into the wells. A fixed concentration of EF (50 μg/ml) was incubated with varying GSL concentrations (0 to 15 μg/ml) in these assays. The remaining of the protocols were as described above with the direct binding assays.

Example 7

Binding of Candida albicans Fimbriae to Epithelial Cells

Human buccal epithelial cells (BECs) were collected from 10 healthy, non-smoking male volunteers and washed as described in Example 5.

Both GSLs (asialo-GM, and CTH) and βGalNAc(l→4)βGal-methylesterwere used to inhibit C. albicans fimbriae binding to BECs. The binding assay was performed using 12 μm polycarbonate filter membranes (Nucleopore Costar Corp.) placed in chambers in a Manifold filtration apparatus equipped with individual vacuum stopcocks (Model FH 225 V; Hoefer Scientific Instruments). The protocols described in Example 5 were employed with the following modifications: C. albicans fimbriae (50 μg) were preincubated either βGalNAc( 4)βGal-methylester, asialo-GM„ or CTH (total volume of 1 ml PBS pH 7.2 containing 0.05%) [v/v] Tween-20) at 37 °C for 1 hour. The mixtures were added to BECs (2.0 x 10⁵ BECs in 1 ml PBS pH 7.2) and incubated at room temperature for 1.5 hours. The assay mixture consisted of 2.0 _x 10⁵ BECs, fimbriae (50 μg) and varying concentrations of competitors in a total volume of 1.0 ml of 10 mM phosphate buffer, pH 7.2, containing 150 mM NaCl.

Example 8

Competitive inhibition of P. aerusinosa Pili Binding to Immobilized Asi lo-GM_!

Polystyrene microtitre plates (Costar, Cambridge, MA) were coated with 100 μl/well of asialo GM, (Sigma Chemical Co.; 5 μg/ml) in methanol. The solvent was evaporated at room temperature inside a fumehood. Non-specific binding sites in the wells were blocked with

100 μl/well of 5% (wt/vol) BSA in PBS. After incubating at 37 °C for 1.5 h, the wells were washed three times with PBS (250 μl) supplemented with 0.05%> (wt/vol) BSA (Buffer A).

Aliquots (100 μl) of biotinylated P. aeruginosa PAK pili (0.88 mg/ml, diluted 1:1000 in

31 Buffer A) containing varying concentration of sugar analogs (in the range of 0.6 μM to 300 μM) were added to each well. After 1.5 h incubation at 37°C, the wells were washed three times with 250 μl of Buffer A. One hundred microliters of streptavidin-alkaline phosphatase conjugate (Gibco-BRL) at 1 :1500 dilution with Buffer A was added to each well. After incubation for 1.5 h at 37°C, the plate was washed three times with 250 μl/well of Buffer A. Then, 125 μl of p-nitrophenylphosphate substrate solution ( 1 mg/ml in 10% diethanolamine, pH 9.8) was added to each well. The optical density readings at 405 nm were recorded after 1 and 2 h incubation at room temperature. The inhibition results were expressed as the percent inhibition resulting from the addition of sugar analogs as compared with the one without the sugar analog. All incubations were done in triplicates and at least four times repeated. Data were reported as means ₊ standard deviations of 4-6 separate experiments.

Example 9 Treatment of Oral Candidiasis An animal model of oral candidiasis in rats was used to evaluate various treatment regimens and dosages of Compound 7 ("Fimbrigal-P") in preventing and/or reducing infection by Candida albicans.

A. Formulation Fimbrigal-P (Compound 7, herein) was formulated in a polymeric mucoadhesive dosage form to prolong the presence of the drug in the oral cavity. The polymeric mucoadhesive dosage form found to be most effective for this purpose is a Carbopol, which is a synthetic high molecular weight polymer of acrylic acid (The Thornley Co., Wilmington,

DE). Various polymeric forms (934P, 940P, 981, 1342, 1382, and Ex214, all BFGoodrich grade) were used. A small amount of alkali was added to formulations when need to bring the pH to neutrality. This adjustment results in ionization of carboxyl groups of the acrylic acid and gel formation.

Formulations of the above-listed molecular weight forms were mixed to form a concentration of 0.1 % (wt/vol) in water and were tested for viscosity. The formulations were tested by mixing equal volumes of the Carbopol mixture and artificial saliva (24 mM

Na₂HPO₄, 25 mM K₂HPO₄, 150 mM KHCO₃, 100 mM NaCl and 1.5 mM MgCl₂).

Carbopols exhibiting good flow and viscosity characteristics included 0.1 % Carbopol 1342 and 0.2% Carbopol Ex214.

32 Adhesive properties of the Carbopol formulations were evaluated by application to the subcutaneous side of a piece of human skin (3x3 cm²) obtained from breast reduction surgery, as a model for mucosa. Carbopol Ex214 and Carbopol 1342 exhibited superior adhesion, as well as viscosity properties, for purposes of mucosal application and were used for further experiments.

A formulation of 25 mg/ml Fimbrigal-P in 0.4%) Carbopol Ex214 was made by adding 0.4%) Carbopol Ex214 (wt/vol water) to an equal volume of Fimbrigal-P (50 mg/ml in water). The resulting mixture formed a clear gel that was stable to storage in a refrigerator. For certain studies, however, Fimbrigal-P (25 mg/mL) was added to 0.2% Carbopol 1342.

B. Animal Studies

Sprague-Dawleyrats (250-300 gm; Charles-River, Montreal, Canada) were fed for one week with 0.1% tetracycline HCL, then changed to 0.01 % tetracycline feedings until the end of the experiment. On day one of the experiment, animals were given 150 mg/kg cyclophosphamideintraperitoneally(i.p.) 14-24 hours before infection.

Animals were inoculated by oral administration on three consecutive days of 2 _x 10⁷ blastospores of Candida albicans (American Type Culture Collection #90028) in a volume of 200 microliters of normal saline, unless otherwise indicated. Animals were treated with βGalNAc(l→4)βGal derivative (Fimbrigal-P) according to the treatment regimens presented in Table 4 and described below.

Table 4 - Rat Oral Candidiasis Study

Group 1, Control Group Day of Experiment

(n = 15)

1 2 3 4 5 6 7 8 9

Cyclophosphamide 150 mg kg i.p. X

Candida- Inoculation X X X

Sacrifice 5 rats for hsitopathology and fungal burden X X X

Group 2, Preventative,Fimbrigal-P,25 mg/mL in Carbopol Day of Experiment

E_x2140.4%

(n = 15)

1 2 3 4 5 6 7 8 9

Cyclophosphamide 150 mg/kg i.p. X

Cα/?fifo/α-Inoculation X X X

Fimbrigal-P 6.25 mg O ^L b.i.d. X X X

Sacrifice 5 rats for histopathology and fungal burden X X X

33 Group 3, Drinking Water Treatment, 0.5 mg/mL Day of Experiment (n = 15)

1 2 3 4 5 6 7 8 9

Cyclophosphamide 150 mg/kg i.p. X

Cαnώ^'efa-Inoculation X X X

Fimbrigal-P 0.5 mg mL X X X X X X X X

Sacrifice 5 rats for histopathologyand fungal burden X X X

Group 4, Premixing Treatment Day of Experiment (n = 15)

1 2 3 4 5 6 7 8 9

Cyclophosphamide 150 mg/kg i.p. X

Candida Inoculation: Candida albicans 2_X 10⁷ blastospores÷ X X X Fimbrigal-P 6.25 mg in 200 μl normal saline

Sacrifices rats for histopathologyand fungal burden X X X

Group 5, Treatment Day of Experiment (n = 15)

1 2 3 4 5 6 7 8 9

Cyclophosphamide 150 mg/kg i.p. X

Candida Inoculation X X X

Fimbrigal-P 6.25 mg/250μl orally, b.i.d. X X X

Sacrifice 5 rats for histopathologyand fungal burden X X X

Candida inoculation in all Groups: Candida albicans 90028 (ATCC), 2 _x 10⁷ blastospores/200μL of normal saline, orally.

Groups 2 and 5: Fimbrigal-P (25 mg/mL in 0.4% of Carbopol Ex214) 250 μL/animal, orally, using tuberculin syringes.

Group 3 : Fimbrigal-P (0.5 mg/mL in water) was given to the animals as drinking water from day 2 to end of experiment.

Group 4: 100 μL Fimbrigal-P (50 mg/mL) was mixed with 100 μL of normal saline containing 2 _x 10' blastosporesthen immediately applied orally to animals.

In all test Groups except Group 3, the animals were fed for one week with 0.1% tetracylin HCL and then changed to the 0.01% tetracyclin solution until end of experiment. Group 3 animals received water solution containing 0.5 mg/ml of Fimbrigal-P.

Group 1 represents the control group, treated only with cyclophosphamide, and inoculated with Candida, as indicated.

Rats in Group 2 (Preventative Group) were given a dose of Fimbrigal-P (6.25 mg/250μl Carbopol Ex214 0.4% gel dosage form dose) one hour prior to each Candida inoculation and once each 12 hour interval for a total of 6 doses into the oral cavity.

34 Group 3 (Continuous Treatment Group) were given Fimbrigal-P in continuously available drinking water (0.5 mg/ml). The medicated water was provided to the animals from 2 hours prior to the first inoculation until the day of sacrifice.

Group 4 (Premix Group) animals were inoculated with a suspension of Candida (2x10⁷ 5 blastospores) that was mixed with 6.25 mg Fimbrigal-P in 200 μl normal saline (100 μL Fimbrigal-P (50 mg/mL) was mixed with 100 μL normal saline containing 2x10⁷ blastospores; the mixture was immediately applied to the oral cavity).

Animals in Group 5 (Treatment Group) received various concentrations of Fimbrigal-P (0.05, 0.25, 1.25 or 6.25 mg/250 _μL Carbopol Ex214 0.4%gel dosage form/dose) starting 1

10 hour after the last Candida inoculation, twice a day for three days (total of 6 doses) into the oral cavity.

Following Candida inoculations for three consecutive days, the treatment regimen for each test Group (Table 4) was initiated. Animals were tested daily for the presence of oral Candida by microcurette sampling from the oral cavity. Animals were anesthetized using

15 methoxyflurane and placed in a lumbar supine position on a surgical pad. A retractor was used to keep the mouth open, and the oral cavity was checked with an otoscope. Five individual regional samples were then taken from each animal by striking the end of a 1.5 mm diameter microcurette five times on the surface of each of five specific regions of the oral cavity: surface of tongue, right buccal mucosa, left buccal mucosa, margin of gingiva and

20 palate. Each individual regional sample was dispersed in 100 microliters of sterile normal saline, and the regional samples were pooled into a single sample for each animal. Samples were refrigerated a maximum of 24 hours before further processing. Sample tubes (Eppendorf™) were mixed on a vortex mixer, and dilutions were made prior to inoculation of Inhibitory Mold Agar (IMA; Becton Dickinson) agar plates and incubation for 24-48 hours at

25 30°C. Colonies were counted to determine the amount of Candida albicans present in each sample and the results are shown in FIG. 9.

Following inoculation and treatment, if any, five animals from each group were sacrificed to quantitate the amount of Candida albicans present in the tongues. Animals were anesthetized using methoxyflurane and placed in a lumbar supine position on a surgical pad.

30 A blood sample (3.5 mL) was taken from each animal by cardiac puncture. The tongue was cut using scissors and divided into two equal parts. One half was fixed with formalin (10%>) for histopathological (HP) examination, and the other half was immersed in 3 mL sterile normal saline for homogenization. Prior to homogenization using a Cyclon Virtishear

35 homogenizer, tongues were weighed and minced into very small pieces, using scissors_^ Dilutions of homogenate samples were made in saline, and aliquots were inoculated on IMA agar plates at 30°C for 24-48 hours. The number of colonies were counted to determine quantitatively the amount of Candida albicans in each sample and the results are shown in FIG. 10. Values are reported as mean number of blastospores/mL ("fungal burden") for each treatment group.

While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.

36

Claims

IT IS CLAIMED:

1. A βGalNac( l→4)βGal derivative having the formula

OR²

wherein R¹ is an alkyl group containing 1 to 4 carbons, R² is independently H or a lower alkyl group containing 1 to 4 carbons, R³ is an alkyl group containing between about 5 and about 12 carbons, R⁴ is independently H or a lower alkyl group containing 1 to 4 carbons.

2. The ╬▓GalNac( 4)╬▓Gal derivative of claim 1 , wherein R³ is a heptyl, octyl, nonyl or decyl straight chain hydrocarbon group and wherein R² and R³ are independently H or lower alkyl groups selected from the group consisting of CH₃, CH₂CH₃, C₃H₇, and Hj.

3. The ╬▓GalNac(l-4)╬▓Gal derivative of claim 2, wherein R¹ is COCH₃, R² is independently H or CH₃, R³ is (CH^CHj, and R⁴ is independently H or (CH^CHj.

4. The βGalNAc(l-4)βGal derivative of claim 3 having the formula octyl O-(2- acetamido-2-deoxy4β-D-galactφyranosyl)-(l-.4)-2-0-propyl-β-D-galactopyranoside as shown in FIG.3G.

5. The ╬▓GalNAc(l-4)╬▓Gal derivative of claim 3 having the formula octyl O-(2- acetamido-2-deoxy-4- -methyl-╬▓-D-galactopyranosyl)-(L┬╗4)-╬▓-D-galactopyrancside as shown in FIG. 3C.

6. The ╬▓GalNAc(l-4)╬▓Gal derivative of claim 3 having the formula octyl O-(2-deoxy- 2-propionamido4╬▓-D-galactopyranosyl)-(l-4)-╬▓-D-galactopyranoside,as shown in FIG.3H.

37

7. A method of treating a microbial infection in an individual in need of suGh treatment, comprising administering to the individual, a pharmaceutically effective amount of a composition of a βGalNac(l→4)βGal derivative according to any of claims 1-6.

8. The method of claim 7, wherein said infection is a Candida albicans infection.

9. The method of claim 8, wherein said infection is an oral or vaginal infection and said administering is by topical application to the infected site.

10. The method of claim 9, wherein said infection is oral candidiasis, and said administering is to the buccal cavity after onset of Candida infection.

11. The method of claim 9, wherein said infection is oral candidiasis, and said administering is commenced prior to or concomitant with exposure to Candida.

12. The method of claim 7, wherein said infection is a Pseudomonas infection.

38