Method of treating inflammatory bowel disease with tributyrin

A method for treating inflammatory bowel disease in a patient by inhibiting the production of interleukin-8 in intestinal epithelial cells by administering to said patient an enema of an effective amount of tributyrin.

BACKGROUND OF THE INVENTION 
Short chain fatty acids (SCFA) are normal products of anaerobic bacterial 
fermentation of carbohydrates in the colon and are the major energy source 
for the colonic epithelium. Approximately 90% of the total SCFA content in 
the colon is composed of acetic, propionic, and n-butyric acids. Cummings, 
J. H. Lancet (1983) 1:1206-1209; Roediger, W. Gut (1980) 21:793-798; 
Roediger, W. Gastroenterology (1982) 83:424-429. It has been suggested 
that a lack of luminal SCFAs leads to mucosal atrophy in the short term 
and nutritional colitis after prolonged periods. Roediger, W. Dis. Col. 
and Rectum (1990) 33:858-862. This is particularly evident in diversion 
colitis which develops after diversion of the fecal stream and resolves 
with restoration of colorectal continuity. Glotzer et al. Gastroenterology 
(1981) 80:438-441. SCFA enemas have been shown to be effective in the 
treatment of diversion colitis. Harig et al. N. Engl. J. Med. (1989) 
320:23-28. 
It is not known whether or not ulcerative colitis has the same cause as 
diversion colitis. While some investigators have shown that SCFA levels 
are decreased in the stool of patients with ulcerative colitis (Vernia et 
al. Dig. Dis. Sci. (1988) 33:1353-1358) and that mitochondrial fatty acid 
oxidation is abnormal in colon cells isolated from patients with active 
disease (Roediger, W. Lancet (1980) 2:712-715), it is not known whether 
these alterations are responsible for or a result of active ulcerative 
colitis. The histologic appearance of active ulcerative colitis includes 
an intense lymphoplasmocytosis limited to the mucosa and submucosa often 
notable for a neutrophilic infiltrate invading the colonic epithelium, 
referred to as a crypt abscess. Ulcerative colitis may be, therefore, 
classified as a disorder of the colonic mucosa. Several investigators have 
shown that the colonic epithelium is in a hyperproliferative state with 
the expansion of the proliferative compartment from the lower crypt to the 
upper crypt extending to the surface epithelium of the colon. Biasco et 
al. Cancer Res. (1984) 44:5450-5454; Serafini et al. Gut (1981) 
22:648-652. This proliferative state is independent of the degree of 
inflammation as well as the duration of disease and exists even when the 
disease is in a quiescent state. These findings suggest an intrinsic 
abnormality of the colonic epithelium in ulcerative colitis. Similar 
hyperproliferative states have been observed in patients at risk for 
colonic malignancy such as in familial polyposis coli, sporadic colon 
adenomas and familial nonpolyposis colon cancer. Risio, M. J. Cell 
Biochem. (1992) 16G:79-87. 
Butyrate enemas have been used to reduce inflammation in patients with 
distal ulcerative colitis. Breuer et al. Dig. Dis. Sci. (1991) 36:185-187; 
Scheppach et al. Gastroenterology (1992) 103:51-56; Steinhart et al. Am. 
J. Gastro. (1994) 89:179-183. In two studies, butyrate enemas were shown 
to result in a significant clinical response in patients whose disease did 
not respond to traditional forms of treatment including use of 
corticosteroids and 5-amino salicylic acid compounds. The basis of this 
response is unknown. Scheppach et al. observed that the labeling index of 
clonocytes in the upper crypt of patients with ulcerative colitis fell to 
that of normal healthy controls after treatment with butyrate enemas. 
Irrigation of the colon with short chain fatty acids also resulted in 
improvement in patients with diversion colitis. However, the use of 
butyrate enemas in these diseases is severely limited due to its extremely 
strong odor which leads to patients refusing to continue treatment. 
Butyric acid has also been shown to induce cytodifferentiation in vitro of 
a wide variety of neoplastic cells. Chen, Z. and Breitman, T. R. Cancer 
Research (1994) 54:3494-3499. The potential clinical utility of butyric 
acid, however, is limited by the apparent difficulty of achieving 
effective concentrations because of rapid metabolism and short plasma 
half-life. Results in such studies have been variable. For examples, 
butyric acid, 500 mg/kg body weight per day, as the sodium salt, was given 
parenterally to a child with acute myelogenous leukemia and induced 
partial remission. However, sodium butyrate provided at 1% to 2% in the 
drinking water enhanced development of colonic neoplasia in 
1,2-dimethylhydrazine-initiated rats. In further studies Deschner et al. 
demonstrated dietary butyrate in the form of tributyrin was non-toxic when 
fed at the 5% level, allowing for normal weight gain and good health. 
Cancer Letters (1990) 52:79-82. The dietary butyrate in this study did not 
enhance colonic neoplasia, however, the tributyrate also did not exert a 
significant protective effect against chemically induced colonic 
neoplasia. 
Chen and Breitman suggest use of tributyrin as a prodrug for butyric acid 
either as a sole agent or in combination with other agents, for 
cytodifferentiation therapy of human leukemia and other malignancies. 
Cancer Research (1994) 54:3494-3499. Diets high in short chain 
triglycerides such as triacetin and tributyrin have been demonstrated to 
enhance colonic mucosal adaptation and significantly increase jejunal 
mucosal mass as compared with chemically defined diets containing 
equivalent calories in the form of carbohydrate or medium chain 
triglycerides in rats with surgically created short-bowel syndrome. Kripke 
et al. Am. J. Clin. Nutr. (1991) 53:954-62. From these studies it was 
suggested that short chain triglycerides may be a useful new enteral fuel 
in patients with short-bowel syndrome or other disorders of fat absorption 
in which medium chain triglycerides represent the current standard of 
nutritional care. Such diets have also been suggested for patients with 
neurological injuries to supply systemic caloric and protein requirements 
without the adverse effects of conventional high glucose diets. Robertson 
et al. Stroke (1992) 23:564-568. However, Nudelman et al. examined a 
number of possible prodrugs for butyric acid in cytodifferentiation 
experiments including glyceryl tributyrate and did not find tributyrates 
to increase efficacy. J. Med. Chem. (1992) 35(4):687-694. Glyceryl 
tributyrate, which is capable of releasing three butyrate units per 
prodrug molecule and has a lipophilicity similar to methyl butyrate, which 
was shown to inhibit cell proliferation 90%, was only as active as butyric 
acid with a maximum inhibition of 35%. This lack of activity of the 
tributyrate was suggested to be due to lower penetration rates into the 
cells, slow hydrolysis, or a combination of both factors. 
It has now been unexpectedly found that tributyrin can be used 
therapeutically in the treatment of inflammatory bowel disease. 
SUMMARY OF THE INVENTION 
An object of the invention is to provide a method of treating inflammatory 
bowel disease in a patient comprising administration of an effective 
amount of tributyrin to the patient.

DETAILED DESCRIPTION OF THE INVENTION 
The incidence of inflammatory bowel disease, a term used to designate two 
chronic inflammatory gastrointestinal disorders, Crohn's disease and 
ulcerative colitis, is estimated to be between 4% and 10% with 25,000 new 
cases occurring annually. Crohn's disease is a subacute and chronic 
inflammation that extends through the intestinal mucosa resulting in the 
formation of fistulas, fissures and abscesses. Granulomas occur in 50% of 
the cases. As the disease advances, the intestinal lumen narrows, causing 
obstruction. Ulcerative colitis, a chronic inflammatory disease of the 
superficial mucosa of the colon affects approximately 500,000 individuals 
in the United States alone. This disease is characterized by multiple 
ulcerations and friability of the colonic mucosa associated with diffuse 
inflammations. In addition to the morbidity associated with intestinal 
inflammation, patients with ulcerative colitis for greater than 8 to 10 
years duration have a dramatically increased risk for developing colon 
cancer. Medical treatment for both Crohn's disease and ulcerative colitis, 
collectively referred to as chronic inflammatory bowel disease, is aimed 
at reducing inflammation. 
Butyrate enemas have been found to be an effective treatment for ulcerative 
colitis unresponsive to conventional treatment. However, the utility of 
these enemas is severely limited due to their intensely unpleasant odor 
which leads to patients refusing to use the treatment. It has now been 
found that tributyrin, a prodrug of butyrate which does not have an 
unpleasant odor, is an even more potent inhibitor of epithelial 
inflammatory response than sodium butyrate. These studies indicate that 
tributyrin is a more effective and better tolerated anti-inflammatory 
agent in the treatment of chronic inflammatory bowel disease. Tributyrin 
was initially synthesized during the 1920's. It is commercially available 
through a number of chemical distributors including Sigma Chemical 
Company, St. Louis, Mo. and Aldrich Chemical Company Inc., Milwaukee, Wis. 
Tributyrin has been traditionally used as an analytical reagent in the 
study of the enzymatic hydrolysis of triglycerides. More recently 
tributyrin has been suggested as a possible nutritional agent. 
Tributyrin is an ester of butyrate, a short chain fatty acid. Short chain 
fatty acids are normal products of anaerobic bacterial fermentation of 
carbohydrates in the colon and are the major source of nutrition for the 
human colonic epithelium. Butyrate, the short chain fatty acid most avidly 
metabolized by colonocytes, has recently been demonstrated to be an 
effective treatment for several intestinal inflammatory diseases including 
ulcerative colitis and diversion colitis. In these studies, the butyrate 
was administered as sodium butyrate, a water soluble, polar compound. It 
has now been found that tributyrin, an apolar and hydrophobic compound, 
can also penetrate colonic epithelial cells at least as effectively as 
sodium butyrate, resulting in a 3-fold increase in effect as predicted by 
the molar ratio of butyrate delivered. 
In vitro data demonstrate that sodium butyrate is capable of inducing 
intestinal epithelial cell differentiation concurrent with inhibiting gene 
expression for the proinflammatory cytokine IL-8. It has now been found 
that tributyrin leads to an identical biological response in vitro at 
concentrations one third (1/3) that of sodium butyrate. These results were 
unexpected in light of in vitro experiments related to cytodifferentiation 
wherein it was found that tributyrin was only as effective as sodium 
butyrate in inhibiting cell proliferation at the same dose. Nudelman et 
al. J. Med. Chem. (1992) 35(4):687-694. Furthermore, these results suggest 
that colonic epithelial cells contain intracellular esterases that allow 
tributyrin to be metabolized to butyrate. 
No toxicity has been observed in mice treated with tributyrin either orally 
or intraperitoneally with a dose of 26.5 mmole/kg. Planlchon et al. J. 
Pharm. Sci. (1993) 82:1046-1048. In addition, tributyrin has been well 
tolerated in humans. For example, no detectable side effects were seen 
after six premature infants were fed butyrates for 4 days at doses of 
about 20 mmol/kg/day. Snyderman et al. Arch Dis. Child. (1955) 30:83-84. 
In the present invention, a method is provided for treating inflammatory 
bowel disease in a patient which comprises administering to a patient an 
effective amount of tributyrin. By "effective amount" it is meant a 
concentration sufficient to inhibit inflammation in the colon associated 
with this disease. Effective concentrations of tributyrin can be easily 
determined based upon the data provided in the instant disclosure and 
knowledge of those of skill in the art. By "patient" it is meant an 
individual suffering from inflammatory bowel disease. 
Treatment of inflammatory bowel disease with tributyrin will be 
demonstrated in Phase I and Phase II clinical trials. In these studies, 
tributyrin will be administered as an enema at 25, 50 and 100 mM 
concentrations. In a preferred embodiment, an aqueous emulsion of 
tributyrin is prepared in 0.9N saline. A 60 cc tributyrin enema is 
administered BID with at least a 30 minute retention. Efficacy of the 
treatment will be determined by endoscopic appearance of the mucosa by 
flexible sigmoidoscopy before and after treatment with tributyrin. Random 
biopsies of the colonic mucosa will be obtained during these procedures 
for histologic analysis. These biopsies will be used to determine the 
degree of inflammation present. In addition, the biopsies will be used to 
determine if there is alteration in colonocyte proliferation and/or 
differentiation by quantitatively scoring the presence of 
immunohistochemical markers for cell proliferation such as proliferating 
cell nuclear antigen (PCNA) (Yu et al., Histochemical Journal (1992) 
24:121-131) and cellular differentiation such as down regulated adenoma 
(DRA) (Schweinfest et al. Proc. Natl. Acad. Sci. USA (1993) 90:4166-4170). 
The pattern of staining will be referenced to 6 zones that divide the 
colonic epithelium from the crypt base, the proliferative undifferentiated 
zone in normal colon, to the surface epithelium, the nonproliferative 
differentiated zone in normal colon. In addition, clinical symptoms 
including frequency of bowel movements, sense of well being and the 
presence or absence of diarrhea, visible blood in the feces, abdominal 
pain or tenesmus, or fever will be recorded. 
The following nonlimiting examples are provided to further illustrate the 
present invention. 
EXAMPLES 
Example 1 
Induction of IL-8 mRNA in Caco-2 cells 
Caco-2 cells (American Type Culture Collection, Rockville, Md.) were plated 
at a density of 4.times.10.sup.4 cells per cm.sup.2 in 10 cm dishes 
containing Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine 
serum and penicillin/streptomycin as described by Wu et al. J. Biol. Chem. 
267:7863-7870 and Traber et al. Mol. Cell. Biol. 12:3614-3627. At day 5 
the cells were stimulated with complete medium containing 20 ng/ml of 
IL-1.beta. (R and D Systems, Minneapolis, Minn.). Total RNA was isolated 
from these cells 2, 4 and 6 hours after the addition of IL-1.beta. 
containing medium as well as from a control cell population not treated 
with IL-1.beta. in accordance with procedures described by Chomczynski, P. 
and Sacchi, N. Anal. Biochem. (1987) 162:156-159. Ten micrograms of RNA 
for each sample was electrophoretically separated, transferred to a nylon 
membrane, and UV crosslinked in accordance with procedures described by 
Traber et al. Am. J. physiol. (1992) 262:G123-G130. A cDNA probe for IL-8 
was prepared by RT-PCR of total RNA from Caco-2 cells stimulated with 
IL-1.beta. for 2 hours. Random hexamers were used for reverse 
transcription of the RNA using reaction conditions similar to those 
described by Wu et al. Gastroenterology (1993) 105:837-844. A 277 bp cDNA 
was amplified from the RT reaction by PCR using the 5' primer IL-8(+103) 
and the 3' primer IL-8(EX4), cloned using a TA-vector (Marchuk et al. Nuc. 
Acids Res. (1991) 19:1154), and labeled with .sup.32 P using a Random 
Primers DNA labeling System (Gibco BRL, Gaithersburg, Md.). Hybridization 
of the Northern blots were performed using conditions as described by 
Huang et al. Mol. Endo. (1993) 7:1391-1398. 
IL-8(+103) 5'-GTGGGATCCATGACTTCCAAGCTGGCC-3' (SEQ ID NO: 1) 
IL-8(EX4) 5'-GTGGGATCCGAATTCTCAGCCCTCTTC-3' (SEQ ID NO: 2) 
GGATCC indicates the BAM HI site. 
No IL-8 mRNA was detectable in control cells (see FIG. 1). However, 
following stimulation with IL-1.beta., there was a dramatic increase of 
IL-8 mRNA with peak levels at 2 hours which decreased at later time points 
(see FIG. 1). Rehybridization of the same blot with GAPDH demonstrated the 
loading and integrity of the RNA in each lane (see FIG. 1). 
EXAMPLE 2 
Inhibition of IL-1.beta. stimulated IL-8 gene expression with tributyrin 
Approximately 18 hours post-plating, Caco-2 cells were placed in complete 
medium containing various concentrations of tributyrin (Sigma, St. Louis, 
Mo.) diluted in ethanol (0.1 to 1.0 mM). Caco-2 cells were also plated 
that were not treated with this compound. The medium was changed on a 
daily basis for 3 days. On day 5 all the cells (except for control) were 
stimulated with IL-1.beta. (5 ng/ml) for 2 hours. Total RNA was then 
isolated and Northern blots performed as described in Example 1. The blots 
were hybridized to the IL-8 cDNA probe also described in Example 1. 
Rehybridization of the same blot with a probe for 7S ribosomal RNA 
demonstrates the loading and integrity of the RNA in each lane. 
Increasing concentrations of tributyrin from 0.1 to 1.0 mM tributyrin lead 
to a dose dependent reduction in steady state levels of IL-8 mRNA (see 
FIG. 2). These concentrations were well tolerated by Caco-2 cells without 
any evidence of cell death. Treatment of Caco-2 cells at concentration 
higher that 1.0 mM, which caused significant toxicity and cell death, led 
to increased expression of IL-8 mRNA. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(iv) ANTI-SENSE: No 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
GTGGGATCCATGACTTCCAAGCTGGCC27 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(iv) ANTI-SENSE: No 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
GTGGGATCCGAATTCTCAGCCCTCTTC27 
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