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  Pharmaceutical Patents  


Title:  Cholesterol-containing compounds and their use as immunogens against Borrelia burgdorferi
United States Patent: 
February 23, 2010

 Ben-Menachem; Gil (Rockville, MD), Kubler-Kielb; Joanna (Rockville, MD), Schneerson; Rachel (Bethesda, MD), Robbins; John B. (Chevy Chase, MD), Pozsgay; Vince (Rockville, MD)
  The United States of America as represented by the Department of Health and Human Services (Washington, DC)
Appl. No.:
 April 2, 2004
PCT Filed:
 April 02, 2004
PCT No.:
371(c)(1),(2),(4) Date:
 September 27, 2005
PCT Pub. No.:
PCT Pub. Date:
 October 21, 2004


Pharm Bus Intell & Healthcare Studies


Unique compounds that can be used for inducing an immune response to Borrelia burgdorferi in a subject by administering a therapeutically effective amount of the glycolipid to the subject. Such administration is particularly useful for preventing or treating Lyme disease in a subject. The compounds(s), and therapeutically acceptable salts thereof, may be formulated into pharmaceutical or immunogenic compositions.

Description of the Invention


Disclosed herein are unique compounds and their analogs such as therapeutically acceptable salts thereof. According to one aspect, disclosed herein are novel compounds of the following formula A, or a pharmaceutically acceptable salt or complex thereof, -- see Original Patent.

According to another aspect, disclosed herein is a purified compound having a formula B of -- see Original Patent.

In a further aspect, disclosed herein are conjugates that include (i) a carrier and (ii) a compound of formula A or B, wherein the compound of formula A or B is bound to the carrier.

The disclosed compounds and conjugates (both of formulae A and B) can be used for inducing an immune response to B. burgdorferi in a subject by administering a therapeutically effective amount of the compound or conjugate to the subject. Such administration is particularly useful for preventing or treating Lyme disease in a subject. The compounds(s), and therapeutically acceptable salts thereof, may be formulated into pharmaceutical or immunogenic compositions.


Native BBGL-II is referred to herein as "cholesteryl 6-O-palmitoyl-.beta.-D-galactopyranoside." The extracted native BBGL-II is isolated and purified. For instance, the purified native BBGL-II isolate is substantially free of other lipids, compounds or molecular fragments from B. burgdorferi. In one example, the purified native BBGL-II isolate includes less than one weight percent nucleic acids and less than one weight percent proteins.

Analogs of BBGL-II are also therapeutically useful as disclosed herein (see formula A). For instance, in such an analog the acyl (for example, palmitoyl) group shown for BBGL-II formula may be replaced with other saturated or unsaturated carbon-containing groups or chains containing 1 to 25 carbon atoms, particularly other acyl groups derived from organic fatty acids. The cholesteryl and the galactopyranoside ring structures may include, at any ring position, substituent groups such as alkyl (particularly an acyl group derived from a fatty acid), carboxyl, substituted carboxyl (--COR where R is alkyl or a carboxylic acid or ester), aryl, alkoxy, hetercyclic, halogen, or amino groups. The O heteroatom bridging the cholesteryl and the galactopyranoside rings may be replaced with an alkyl radical (e.g., --CH.sub.2--) or a heteroatom such as N, S or P.

In the compounds of formula A, R.sup.2 (along with the --C(O)O-- group) may be an acyl group derived from an organic fatty acid. Such acyl groups include palmitoyl, lauroyl, stearoyl, myristoyl, oleyl and linoleyl. Derivatization of the acyl group can provide a site (for example, an acyl azide) for covalently bonding to a carrier protein.

In certain variants, the R.sup.1 group is capable of forming a covalent bond to a carrier protein (in other words, conjugating to the carrier protein) as described below in more detail. Conjugation to a carrier protein may also be accomplished via derivatization of at least one of the alkyl groups or chains of the cholesteryl moiety. Such derivatization may be accomplished as described below. Alternatively, a small molecule such as adipic dihydrazide or 1,6-diaminohexane may be utilized.

BBGL-II and/or its analogs may be isolated or purified from a B. burgdorferi culture as described below, or they can be chemically synthesized. For example, a compound of formula A may be synthesized by first obtaining a galactosyl halide in which the hydroxy groups of the galactose ring have been provided with protecting groups. The galactosyl halide then is reacted with cholesterol to provide a galactosyl-cholesterol. The protective groups on the galactose ring then are removed, and replaced with substitute protective groups. An azidoacyl acid intermediate is prepared separately. The azidoacyl acid intermediate is condensed with the galactosyl-cholesterol to provide an azidoacylated cholesterol .beta.-D-galactopyranoside.

In one example disclosed herein, a therapeutically effective amount of purified, native BBGL-II (formula B) or a chemically synthesized analog of BBGL-II (formula A) is an amount used to generate an immune response, or to treat or prevent infection with B. burgdorferi in a subject. Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of B. burgdorferi infection, or a sign or symptom of Lyme disease.

Immunogenic Compositions and their Use

In one embodiment, a method of treating a subject with a B. burgdorferi infection is provided, or preventing or inhibiting infection, or the development of clinical Lyme disease. Alternatively, the method can be used to inhibit the progress of an already existing infection. The method includes administering to the subject a therapeutically effective amount of a purified native BBGL-II or a compound of formula A, thereby treating or preventing the infection, or retarding or reversing clinical disease. In forming a composition for generating an immune response in a subject, or for vaccinating a subject, BBGL-II or a compound of formula A, is utilized.

These immunogenic compositions of the present disclosure elicit an immune response against B. burgdorferi in a mammalian host, including humans and other animals. The immune response may be either a cellular dependent response or an antibody dependent response, or both, and further the response may provide immunological memory or a booster effect or both in the mammalian host. Antigens that elicit antibodies with assistance from T cells are known as T-dependent antigens. Antigens that do not require T cell assistance to elicit antibody formation but can activated B cells directly are known as T-independent antigens. These immunogenic compositions are useful as vaccines and may provide a protective response by the mammalian subject or host to infection by a pathogenic microorganism.

More particularly, the vaccines described herein can be prepared as T-cell independent vaccines and/or as T-cell dependent vaccines. According to a particular variant, the active compound of formula A or B is primarily T-cell independent but is converted into a T-cell dependent response vaccine via conjugation with a carrier (especially a protein carrier).

A carrier may be provided for the compounds of formula A or B disclosed herein. The carrier may exist in an admixture with the immunogenic compound(s), or it may be conjugated to the immunogenic compound(s) via a chemical interaction or bond. For example, the immunogenic compound(s) may be conjugated to a macromolecular carrier. The carrier may be a polymer to which the immunogenic compound(s) is bound by hydrophobic non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the immunogenic compound(s) is covalently bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or keyhole limpet hemocyanin. The carrier should preferably be non-toxic and non-allergenic. The immunogenic compound(s) may be multivalently coupled to the macromolecular carrier as this may provide an increased immunogenicity of the vaccine preparation.

In one embodiment, a carrier is a chain of amino acids (e.g., a polypeptide or protein) or other moieties. In another embodiment, a carrier is a dimer, oligomer, or higher molecular weight polymer of a sequence of amino acids of a B. burgdorferi polypeptide. Examples of useful immunogenic carriers include keyhole limpet hemocyanin (KLH); albumins such as bovine serum albumin (BSA) and ovalbumin, PPD (purified protein derivative of tuberculin); red blood cells; tetanus toxoid; cholera toxoid; agarose beads; activated carbon; or bentonite.

The lipids disclosed herein can be attached to any protein of interest, including, but not limited to, rARU, a recombinant protein containing the repeating units of Clostridium difficile toxin A. Carriers are chosen to increase the immunogenicity of the polysaccharide and/or to raise antibodies against the carrier which are medically beneficial. Carriers that fulfill these criteria are described in the art. A polymeric carrier can be a natural or a synthetic material containing one or more functional groups that are available for conjugation, for example primary and/or secondary amino groups, azido groups, aldehydes, hydrazides, epoxides, thiols or carboxyl groups. The carrier can be water soluble or insoluble.

Water soluble peptide carriers are preferred, and include but are not limited to natural or synthetic polypeptides or proteins, such as bovine serum albumin, and bacterial or viral proteins or non-toxic mutants or polypeptide fragments thereof, e.g., tetanus toxin or toxoid, diphtheria toxin or toxoid, Pseudomonas aeruginosa exotoxin or toxoid, recombinant Pseudomonas aeruginosa exoprotein A, pertussis toxin or toxoid, Clostridium perfringens and Clostridium welchii exotoxins or toxoids, mutant non-toxic Shiga toxin holotoxin, Shiga toxins 1 and 2, the B subunit of Shiga toxins 1 and 2, and hepatitis B surface antigen and core antigen.

Alternative carriers are some substance, animal, vegetable, or mineral in origin, that is physiologically acceptable and functions to present the BBGL-II lipid to the immune system. Thus, a wide variety of carriers are acceptable, and include materials which are inert, or which have biological activity and/or promote an immune response. For example, an example of a protein carrier includes, but is not limited to, keyhole lympet protein, and hemocyanin. Polysaccharides can also be used as carriers, and include those of molecular weight 10,000 to 1,000,000, such as starches, dextran, agarose, ficoll, or it's carboxyl methyl derivative and carboxy methyl cellulose.

Polyamino acids are also contemplated for use as carriers, and these polyamino acids include, among others, polylysine, polyalanyl polylysine, polyglutamic acid, polyaspartic acid and poly(C.sub.2-C.sub.10) amino acids.

Organic polymers can be used as carriers, and these polymers include, for example, polymers and copolymers of amines, amides, olefins, vinyls, esters, acetals, polyamides, carbonates and ethers and the like. Generally speaking, the molecular weight of these polymers will vary dramatically. The polymers can have from two repeating units up to several thousand, e.g., two thousand repeating units. The number of repeating units will be consistent with the use of the immunizing composition in a host animal. Generally speaking, such polymers will have a lower molecular weight, say between 10,000 and 100,000 (the molecular weight being determined by ultracentrifugation).

Inorganic polymers can also be employed. These inorganic polymers can be inorganic polymers containing organic moieties. In particular, silicates and aluminum hydroxide can be used as carriers. It is preferred that the carrier be one which is an immunological adjuvant. In such cases, it is particularly contemplated that the adjuvant be muramyl dipeptide or its analogs.

The carrier can also be the residue of a crosslinking agent employed to interconnect a plurality of synthetic peptide containing chains. Crosslinking agents which have as their functional group an aldehyde (such as glutaraldehyde), carboxyl, amine, amido, imido or azidophenyl group. In particular, there is contemplated the use of butyraldehyde as a crosslinking agent, a divalent imido ester or a carbodiimide.

The present disclosure further includes methods for preparing the immunogenic composition that involves conjugating the lipids disclosed herein to a carrier. Examples of such carrier include those mentioned above as well as a polypeptide or non-peptide moiety that could act as a carrier or adjuvant or have other biological activity in combination with the lipids. The compounds of formula A or B may be conjugated to a protein carrier by mixing the compounds with the protein carrier in the presence of a reagent that allows covalent bond formation between the compound and the protein carrier. Illustrative coupling reagents include glutaraldehyde, hydroxysuccinimides, and carbodiimides. Alternatively, a small chemical molecule may be attached to either the compound of the protein carrier, and this molecule, because of its reactivity, serves as a linker molecule between the compound and the protein carrier. Illustrative linkers include adipic dihydrazide, aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone and p-nitrophenyl amine.

For example, the compounds of formula A or B may be conjugated to a polypeptide by one of a number of means, such as by first derivatizing the polypeptide by succinylation and then conjugating the lipid component to the polypeptide through a reaction of the polypeptide and compound with 1, ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Additionally the activation of the lipid component can be accomplished by the use of any of several reagents, but preferably cyanogen bromide.

The compounds of formula A or B also may be administered to a subject via a liposome delivery system in order to enhance their stability and/or immunogenicity. Delivery of the compounds via liposomes may be particularly advantageous because the liposome may be internalized by phagocytic cells in the treated subject. Such cells, upon ingesting the liposome, would digest the liposomal membrane and subsequently present the polypeptides to the immune system in conjunction with other molecules required to elicit a strong immune response. The liposome system may be any variety of unilamellar vesicles, multilamellar vesicles, or stable plurilamellar vesicles, and may be prepared and administered according to methods well known to those of skill in the art, for example in accordance with the teachings of U.S. Pat. Nos. 5,169,637, 4,762,915, 5,000,958 or 5,185,154.

The present disclosure involves administering to a subject a therapeutically effective dose of a pharmaceutical composition containing at least one compound of formula A or B. The pharmaceutical composition may include a pharmaceutically acceptable carrier. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. By subject is meant any mammal, including a human.

The pharmaceutical compositions are preferably prepared and administered in dose units. Solid dose units are tablets, capsules and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the patient, different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals. The administration may also be accomplished via a continuous dose for a longer period of time such as, for example, 1-10 weeks.

The pharmaceutical compositions are in general administered orally or parenterally. Parenteral administration routes include, but are not limited to, subcutaneous injections (SQ and depo SQ), intravenous (IV), intramuscular (IM and depo-IM), intrasternal injection or infusion techniques, intranasal (inhalation), intrathecal, transdermal, topical, and ophthalmic. The compound may also be administered from implanted reservoirs or pumps. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference. Inocula are typically prepared as solutions in physiologically tolerable (acceptable) diluents such as water, saline, phosphate-buffered saline, or the like, to form an aqueous pharmaceutical composition. Adjuvants, such as aluminum hydroxide, may also be included in the compositions.

The pharmaceutical compositions can be administered locally or systemically. Amounts effective for therapeutic use will, of course, depend on the severity of the disease and the weight and general state of the patient. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. Various considerations are described, e.g., in Gilman et al., eds., Goodman and Gilman: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.

Effective doses of the compounds disclosed herein will vary depending on the nature and severity of the condition to be treated, the age and condition of the patient and other clinical factors. Thus, the final determination of the appropriate treatment regimen will be made by the attending clinician. Typically, the dose range for a compound disclosed will be from about 0.1 .mu.g/kg body weight to about 100 mg/kg body weight. Other suitable ranges include doses of from about 1 .mu.g/kg to 10 mg/kg body weight. The dosing schedule may vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to a lipid. In the case of a more aggressive disease it may be preferable to administer doses such as those described above by alternate routes including intravenously or intrathecally. Continuous infusion may also be appropriate.

For administration to animals, purified therapeutically active molecules are generally combined with a pharmaceutically acceptable carrier. Pharmaceutical preparations may contain only one type of therapeutic molecule, or may be composed of a combination of several types of therapeutic molecules. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

As is known in the art, protein-based pharmaceuticals may be only inefficiently delivered through ingestion. However, pill-based forms of pharmaceutical proteins may be administered subcutaneously, particularly if formulated in a slow-release composition. Slow-release formulations may be produced by combining the target protein with a biocompatible matrix, such as cholesterol. Another possible method of administering protein pharmaceuticals is through the use of mini osmotic pumps. As stated above a biocompatible carrier would also be used in conjunction with this method of delivery.

The pharmaceutical compositions can be administered by any means that achieve their intended purpose. Amounts and regimens for the administration of the therapeutic molecules can be determined readily by those with ordinary skill in the clinical art of treating Lyme disease and any other condition associated with B. burgdorferi infection. For use in treating these conditions, molecules are administered in an amount effective to inhibit B. burgdorferi replication. Typical amounts initially administered would be those amounts adequate to achieve tissue concentrations at the site of action which have been found to achieve the desired effect in vitro. The compounds of formula A or B can be administered to a host in vivo, for example through systemic administration, such as intravenous or intraperitoneal administration. Also, the compounds of formula A or B can be administered intralesionally: i.e., the peptide or protein is injected directly into the lesion. In order to increase the immune response, a subsequent or booster dose may be administered approximately 4 to 6 weeks after the initial injection. Subsequent doses may be administered as indicated herein, or as desired by the practitioner.

Claim 1 of 12 Claims

1. A compound of formula A below, or a pharmaceutically acceptable salt thereof, wherein the compound of formula A comprises -- see Original Patent.

If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.


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