Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
1998-09-02
2003-08-19
Solola, Taofiq (Department: 1626)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C549S400000, C549S415000
Reexamination Certificate
active
06608102
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to isolation and identification of plant proanthocyanidin extracts. These extracts can be obtained from any of a variety of proanthocyanidin-containing plants including members of the plant families Ericaceae, Rosaceae, Pinaceae and Vitaceae, and preferably are from cranberry plants (especially
Vaccinium macrocarpon
), other Vaccinium spp. and grapes (Vitus spp.) These extracts are useful for prevention and treatment of urinary tract infections caused by P-type fimbriated
Escherichia coli
as well as other uses. Extracts containing proanthocyanidins with A-type interflavanoind bonds have been found to have potent bioactivity for inhibiting adherence of P-type
E. coli.
BACKGROUND OF THE INVENTION
Millions of women each year are diagnosed with cystitis (bladder infections) and pyelonephritis (kidney infections). Countless numbers of dogs and cats also suffer from chronic urinary infections and die from renal infections.
E. coli
bacteria is the most common pathogen associated with these infections, causing over 80% of urinary tract infections. Over 30% of women suffer recurrent infections within a 6 to 12-month period and are forced to resort to extended use of antibiotics to treat these infections. Recurrent use of antibiotics can lead to pathogen resistance and result in deleterious side effects and toxicity reactions. Consequently there exists a need for safe alternative medications (e.g., non-antibiotics) that can be used to prevent or treat urinary tract infections in both animals and humans.
Cranberry juice has been shown to reduce bacteriuria associated with urinary tract infections in humans (Avorn et al., 1994, J. Am. Med. Soc. 271:751-754). The effect appears to be due to the ability of certain cranberry compounds to inhibit adhesion of type 1 (implicated in bladder infections) and P-type (implicated in kidney infections)
E. coli
bacterial phenotypes to human bladder epithelial cells (Sobota, 1984, J. Urol. 131:1013-1016; Schmidt & Sobota, 1988, Microbios. 55:173-181; Zafriri et al., 1989, Antimicrob. Agents Chemo. 33:92-98). Zafriri et al. (1989) reported that fructose was responsible for the inhibition of type 1
E. coli
to uroepithelial cells. Zafriri et al. also reported that cranberry juice contained a non-dialyzable substance (or substances) which inhibited binding of P-type
E. coli
but failed to define the chemical nature of this inhibitor. Cranberry juice has also been shown to cause immediate inhibition of agglutination as well as loss of fimbriae after long-term exposure of bacteria to the juice (Ahuja et al., 1998, J. Urol. 159:559-562).
A partially-purified anti-adherence activity from cranberry has also been described (U.S. Pat. Nos. 5,474,774; 5,525,341; and 5,646,178, all to Walker et al.). This activity was obtained using acidified alcohol as an extraction solvent with whole cranberry fruit followed by separation of the activity from monomer and dimer sugars by precipitation with a metal acetate or sulfate. Upon further manipulation, the reported activity consisted of a fraction enriched for polyphenol and flavonoid compounds that contained as much as 10% anthocyanins. The specificity of this anti-adherence activity for type 1 or P-type
E. coli
was not determined.
Walker et al. (WO 96/30033; and U.S. Pat. Nos. 5,646,178 and 5,650,432) described a series of proanthocyanidin monomers, dimers, polymers, flavonoid derivatives thereof and related compounds purported to have the ability to interfere with bacterial adherence to a surface. The dimers and polymers of Walker were limited to compounds having B-type interflavanoid linkages. However, Walker failed to provide any experimental data correlating biological activity with a specifically-identified compound. The Walker method involved alkalinizing a plant material homogenate to a pH greater than 10, a treatment which causes degradation of proanthocyanidins, and precipitating the polyphenolic compounds (together with other materials) by addition of alcohol. This precipitate contained the proposed anti-adherence activity and was further fractionated to yield the purified active compound. Using this process with an aqueous solution of commercially-available Ocean Spray cranberry powder, Walker reported obtaining a single active compound and partially characterized the compound but failed to provide a complete (or any) chemical structure for this compound. Walker also failed to characterize the biological activity of this compound with respect to inhibition of adherence of type 1 or P-type
E. coli
. In fact, the Walker assay methods could not distinguish between these two biological activities.
Thus, prior to the present invention, there had been no identification of the class of bioactive compounds that inhibit P-type
E. coli
from adhering to surfaces such as uroepithelial cells. In accordance with the invention, it has been discovered that extracted mixtures of proanthocyanidins and purified proanthocyanidins are the bioactive compounds present in cranberry and other plants that possess anti-adherence activity against P-type
E. coli.
A large variety of plants are known to contain proanthocyanidins and methods for isolating small amounts of proanthocyanidins from several different plant species have been reported. Various purification methods for proanthocyanidins from plant material have been described by Thompson et al. (1972, J. Chem. Soc., Perkins Trans. I. 11:1387); Jones et al. (1976, Phytochem. 15:1407-1409); Wang et al. (1978, J. Food Sci. 43:1402-1404); Czochanska et al. (1980, J. Chem. Soc., Perkin Trans. I:2278-2286); Foo & Porter (1981, J. Sci. Food Agric. 32:711-716); Marwan & Nagel (1986a, J. Food Sci. 51:1009-1013); Marwan & Nagel (1986b, J. Food Sci. 51:1069-1070); Morimoto et al. (1988, Chem. Pharm. Bull. 36:33-38); Devlin, U.S. Pat. No.4,309,207; and Bomser et al. (1996, Planta Med. 62:212-216).
Accordingly, the present invention identifies proanthocyanidins as the compounds that mediate inhibition of adherence of P-type
E. coli
to cellular surfaces and further provides an improved method of obtaining substantially pure mixtures of proanthocyanidins as well as individual proanthocyanidin compounds from the plant material of cranberries and other plants known to contain proanthocyanidins. These proanthocyanidin mixtures and individual proanthocyanidin compounds are thus useful in the prevention and/or therapeutic treatment of urinary tract infections, particularly those infections mediated by P-type
E. coli
or other microorganisms that contain structurally-related fimbriae or molecules involved in microbial adherence.
There also exists a need for an inexpensive and rapid test to identify the type of urinary tract infection that a person has contracted. Current clinical diagnosis is based on counting the number of bacteria present in a patient's urine without attempting to distinguish the bacterial strain as type 1
E. coli
, P-type
E. coli
or a mixture of both. Since kidney infections (associated more with P-type
E. coli
) are usually more serious than bladder infections (associated more with type 1
E. coli
) and require different treatment regimes, having a test to distinguish between these phenotypic strain variations would be beneficial. Thus, an inexpensive, rapid diagnostic kit to diagnose P-type infection would allow patients to receive the proper treatment in a more timely manner and avoid the use of ineffective medications. Accordingly, the invention also addresses this problem by providing proanthocyanidin mixtures which specifically bind to P-type
E. coli
as receptor analogs or prevent P-type fimbrial induction for assay kits designed to aid in the clinical diagnosis of pyelonephritis-type infections.
SUMMARY OF THE INVENTION
The present invention is directed to proanthocyanidin extracts substantially free of anthocyanins and flavonols. These extracts are also shown to be free of hydrolyzable tannins, alkaloids, lipids, carbohydrates, simple sugars, protein and amino acids, alcohols and organic
Howell Amy B.
Vorsa Nicholi
Hale and Dorr LLP
Rutgers, the State University of New Jersey
Solola Taofiq
LandOfFree
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