WO1993007891A1 - Epidermal growth factor therapy for non-gastroduodenal lesions - Google Patents

Abstract

Compositions and methods for promoting the healing of non-gastroduodenal disorders include administering a therapeutically-effective dose of epithelial growth factor-active agent. Administration includes both per os, parenteral, and/or in situ means, with the latter including endoscopic bolus washes and/or enemas. Accordingly, damage of digestive tract tissue is inhibited and repair of any damaged tissue enhanced, thus hastening the patient's recovery from sclerotherapy treatment of inflammatory bowel disease.

Description

EPIDERMAL GROWTH FACTOR THERAPY FOR NON-GASTRODUODENAL LESIONS

FIELD OF USE

The present invention relates generally to treating alimentary tract disorders and, more particularly, to compositions and methods for treating non-gastroduodenal disorders, including varices and inflammatory bowel disease.

BACKGROUND OF THE INVENTION Liver disease is a leading cause of mortality and morbidity in the United States. There are many etiologies of liver disease, however regardless of the mechanism, hepatic injury usually has profound systemic consequences.

One of the most serious sequela of liver disease is the development of esophageal varices. These are tortuous dilatations of the veins in the esophagus. Because of their pronounced tendency to rupture, esophageal varices are frequently associated with fatal, massive upper gastrointestinal bleeding. In addition to esophageal varices, other alimentary varices are known, including gastric varices. While the following discussion focuses on esophageal varices, the discussion is applicable to other alimentary varices as well.

The immediate cause of esophageal varices is portal hypertension, a common syndrome, characterized by a chronic increase of the portal venous pressure. This is almost invariably the result of liver disease. In portal hypertension, portal blood is diverted through collateral vessels to the systemic circulation. By far the most important collateral is the gastroesophageal plexus which shunts portal blood into the azygos venous system. This collateral gives rise to varices in the lower esophagus and upper stomach.

There have been numerous attempts to treat and prevent bleeding esophageal varices. Pharmacological therapies have been aimed at using drugs which cause a sustained reduction in portal pressure and in portal-collateral blood flow. Surgical techniques have focused on either decompressing the portal venous system through shunts or attacking the esophageal varices directly. Non-surgical, invasive techniques include balloon tamponading and endoscopic injection sclerotherapy (ΕIS) , either individually or in combination.

Of the various treatment modalities, sclerotherapy is the preferred treatment for bleeding esophageal varices. Studies have shown that survival of sclerotherapy-treated patients compares favorably with other, more-invasive procedures. Unfortunately, post-sclerotherapy healing is frequently marred by complications. These complications, together with the lifelong need for endoscopic monitoring and re-sclerosis, represent the main limitations of this otherwise excellent technique. In lammatory bowel disease is another condition which, like liver disease, is characterized by substantial morbidity and mortality. Inflammatory bowel disease (IBD) is a general classification of inflammatory processes affecting both large and small intestines. Two of its most prominent forms are ulcerative colitis and Crohn's disease.

Ulcerative colitis (UC) is an inflammatory condition involving the mucosa and submucosa of the colon. The disease typically begins in the rectum and involves the bowel contiguously, with ulcers and hemorrhaging predominating in the active phase. While typically confined to the large intestine, the distal ileum may also be involved ("backwash ileitis"). The clinical hallmark of ulcerative colitis is rectal bleeding and diarrhea, with the frequency of bowel movements and the amount of blood present reflective of the activity of the disease.

Crohn's disease or regional enteritis, in comparison, is an inflammatory disease which may involve both the small and larger intestines. Unlike UC, however, Crohn's disease involves all layers of the gut; thus the term "transmural colitis" has been used to describe this disease when present in the colon. Clinically, diarrhea is the most common feature in both large and small bowel forms of this disease. Bleeding, although a prominent feature of ulcerative colitis, is present only in about 50% of patients with transmural colitis. Also noteworthy is an increased incidence of carcinoma of the small bowel in Crohn's patients; those patients with transmural colitis also have an increased incidence of colon cancer. The current therapy for both ulcerative colitis and

Crohn's disease is mainly directed to reducing the attendant inflammation. Sulfasalazine, for example, has been employed for its anti-inflammatory effect. Steroids, also well-known for their anti-inflammatory properties, are routinely employed as well. The usefulness of either agent, however, is largely limited by severe side effects. For those patients with an intractable form of IBD, surgery remains an option. Crohn's disease, unlike ulcerative colitis, usually cannot be cured by surgery however; thus, these patients are essentially without any effective method of treatment.

U.S. Patent No. 4,820,690 describes treatment of duodenal and gastric ulcers with oral urogastrone. PCT International Publication No. WO 90/12588 describes treatment of oral mucositis with growth factors. The disclosures of each of the foregoing references are hereby incorporated by reference.

What is needed is an effective prophylactic and/or therapeutic treatment for sclerotherapy-treated varices and inflammatory bowel disease. In particular, it is highly desirable to prevent sclerotherapy-induced injury and/or augment the post-sclerotherapy healing process for varices. In addition, it is desirable to provide an effective treatment for inflammatory bowel disease, including ulcerative colitis and Crohn's disease. The present invention fulfills this and other needs.

SUMMARY OF THE INVENTION The present invention provides novel compositions and methods for the prophylactic and therapeutic treatment of liver disease and inflammatory bowel disease sequelae. According to one aspect of the present invention, healing of sclerotherapy- treated varices, including esophageal and gastric varices, is promoted through the administration of a therapeutically- effective dose of epidermal growth factor-active agent. Repair of a patient's damaged digestive tract tissue is enhanced, thus hastening the patient's recovery from sclerotherapy treatment. Moreover, the total number of sclerotherapy sessions and the intervals between such sessions may be reduced.

According to another aspect of the present invention, novel compositions and methods are provided for preventing the occurrence of and promoting the healing of inflammatory bowel disorders, including ulcerative colitis and Crohn's disease, through the administration of a therapeutically-effective dose of epidermal growth factor-active agent (EGF) , such as epidermal growth factor polypeptide. Since repair of damaged alimentary tract tissue is hastened, exacerbations of these conditions are alleviated, thus leading to remission of the disease state.

The EGF dosage will vary with each particular application. Typically, the composition is administered either systemically or topically. Systemic administration includes per os and parenteral routes; topical administration includes in situ applications. The in situ means includes, for example, administering EGF-active agent by endoscopic bolus wash and/or paravenous injection, or in the case of lower GI treatments, by enema. Parenteral routes may include, for example, subcutaneous, intradermal, intramuscular, intravenous routes. The amount of EGF-active agent will range from about 2 to 7,000 μg or more, typically 10 to 1000 μg, depending on the administration interval and route, which can range from a single oral dose, parenteral dose and/or topical dose to multiple oral doses, parenteral doses, and/or topical doses over a few days or greater than 5 weeks. The dosage may also vary with the severity of the disease.

In accordance with the present invention, various administration intervals, dosages, and methods for treating varices and inflammatory bowel disease are contemplated to permit multiple treatment regimes with EGF-active agent. If desired, cocktails containing additional compositions capable of adequately enhancing healing or reducing those factors deleterious to healing may be included. For example, various compositions comprising EGF-active agent and sclerosants may also be used for treating varices.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a table of experimental data which shows the results of animal studies.

Fig. 2 is a chart showing the mean number of ulcers and mean endoscopic grade for the data of Fig. 1.

Fig. 3 is a chart showing the incidence of ulcers and the incidence of strictures for the data of Fig. 1.

Fig. 4 is a chart showing the effect of intracolonic doses of EGF on the macroscopic damage score seven days after TNBS-Ethanol induced colitis.

Fig. 5 is a chart showing the effect of intracolonic doses of prednisolone on the macroscopic damage score seven days after TNBS-Ethanol induced colitis.

Fig. 6 is a chart showing the effect of intracolonic doses of 5-ASA on the macroscopic damage score seven days after TNBS-Ethanol induced colitis. Fig. 7 is a chart showing the effect of subcutaneous injections of EGF on the macroscopic damage score seven days after TNBS-Ethanol induced colitis.

Fig. 8 is a chart showing the effect of subcutaneous injections of EGF given every twelve hours on the macroscopic damage score seven days after TNBS-Ethanol induced colitis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

I. SCLEROTHERAPY-TREATED VARICES Novel compositions and methods are provided for treating varices undergoing injection sclerotherapy through the administration of epidermal (epithelial) growth factor-active agent (EGF-active agent) . The present invention inhibits damage to tissue and enhances repair of damaged tissues of the digestive tract, thus hastening the patient's recovery from sclerotherapy injection therapy. Moreover, the total number of sclerotherapy sessions and the interval between such sessions may be reduced. The following discussion will focus on the treatment of esophageal varices which have undergone sclerotherapy. The present invention, however, is not limited to such treatments but instead may be employed advantageously in the treatment of other alimentary disorders, including gastric and rectal varices, as well as non-alimentary varices, such as spider veins and the like.

In order to understand the pathogenesis of varices, it is helpful to briefly survey esophageal varies which result from alterations in liver hemodynamics. The blood vessels conveying blood to the liver are the hepatic artery and portal vein. The hepatic artery brings oxygenated blood from the heart to the liver, while the portal vein brings venous blood rich in the products of digestion which have been absorbed from the gastrointestinal tract. Normally, the liver receives approximately 25 percent of the cardiac output, with two-thirds of this being delivered by the portal vein.

Inside the liver, branches of the portal vein and hepatic artery, along with the bile duct, form portal canals. These canals are arranged such that discrete lobules of liver tissue—hepatic lobules—are discernable. At the center of each lobule is a central vein. Thus, each lobule forms a spoke-like structure with the afferent vessels (hepatic artery and portal vein) at the periphery and the efferent vessel (central vein) at the center. Blood flows from the periphery through sinusoidal channels between plates of liver cells towards the central vein. The central veins drain into the right and left hepatic veins which open directly into the inferior vena cava. Esophageal varices arise when there is portal hypertension, which is an increase in the hydrostatic pressure of the portal vein caused by the increased resistance to the blood flow through the liver. Pathophysiologically, portal hypertension may be divided into two broad types: 1) presinusoidal and 2) intrahepatic. This distinction has important clinical and therapeutic implications. The presinusoidal forms are generally associated with normal liver function. Thus, in patients with this form, liver failure is rarely a complication even among those who suffer with bleeding varices. Patients with intrahepatic forms of portal hypertension have associated hepatocellular disease, and those suffering hemorrhage frequently develop liver failure. Presinusoidal portal hypertension may be further divided into extrahepatic and intrahepatic subtypes. Extrahepatic presinusoidal portal hypertension is caused by obstruction of the main portal vein; etiologies include tumors, neonatal sepsis, pylephlebitis, and coagulopathies. Intrahepatic presinusoidal portal hypertension is due to lesions in the portal zone, including schistosomiasis, primary biliary cirrhosis, congenital hepatic fibrosis, sarcoidosis, and other lesions within the sinusoids of the liver. A large number of toxic substances have been implicated in the production of portal venous sclerosis in this form of portal hypertension.

Within the second major group, the intrahepatic form, is the most common important cause of portal hypertension— cirrhosis of the liver. Cirrhosis has been reported to account for more than 90 percent of all cases of portal hypertension. The term "cirrhosis" is derived from the Greek "kirrhos," meaning of yellow color. Cirrhosis has been defined by a recent WHO Committee as a diffuse process characterized by fibrosis and the conversion of normal liver architecture into structurally abnormal nodules. The essential histopathologic changes of cirrhosis are considered to be diffuse involvement of the liver by parenchymal necrosis, regeneration, and diffuse fibrosis, which results in a disorganization of the lobular architecture. Various classification systems have been proposed based on pathogenesis, etiology, clinical history, and microscopic morphologic appearance. An example of the latter is the Leevy, Popper, and Sherlock scheme which divides cirrhosis into the following classes: 1) Micronodular, 2) Macronodular, 3) Incomplete septal, and 4) Mixed macro- and micronodular.

Etiologically, cirrhosis associated with alcohol abuse is the most common type. Its evolution follows a fairly predictable course. The earliest response to alcohol is the accumulation of lipid in the liver cells. In some patients an isolated sclerosis about central veins or a sclerosis associated with a necrotizing process may be seen. When advanced, these lesions may obliterate central veins leading to portal hypertension. During the early stages of fatty liver, there may be little or no evidence of increased fibrous tissue or scarring. However as fibrosis progresses, the liver tends to gradually decrease in size and progressively becomes nodular.

On gross examination, the liver is generally yellow- orange, fatty, and diffusely scarred. This stage of the disease probably accounts for the original descriptive term "cirrhosis." Nodules are present which vary in size from 0.1 to 1.0 centimeters in diameter, presumably resulting from regeneration. This type of cirrhosis represents the classic form of micronodular cirrhosis. Microscopically, the normal architecture of the liver is distorted by bands of connective tissue traversing the hepatic lobules, forming pseudolobules. The alteration of hepatic blood flow is a characteristic features of cirrhosis. The total blood flow through the liver is reduced by extra hepatic portal systemic shunts. There is further reduction in hepatic blood flow by the vascularized connective tissue septa which form an anastomosis between the afferent branches of the portal vein and hepatic artery and the efferent tributaries of the hepatic veins. In cirrhosis, obstruction to portal venous blood flow occurs at all levels within the liver. The intrahepatic portal vascular bed is compromised and distorted. The hepatic veins and intrahepatic sinusoids are compressed by regenerating nodules of liver tissue. The net result is an increased impedance of blood flow through the liver by fibrosis, thrombosis, and nodular regeneration.

As a result of this altered hepatic microcirculation, up to 60 percent of people with cirrhosis have a major degree of portal hypertension. In response to the increased pressure gradient between the portal and systemic venous systems, venous collaterals are established in areas where these systems communicate. These portal-systemic collaterals are formed by opening and dilating pre-existing venous channels connecting the portal venous system with the superior and inferior vena cava; these channels are normally closed. Major sites of collateral flow involve dilated veins around the rectum

(hemorrhoids) , cardioesophageal junction (esophagogastric varices) , and periumbilical and abdominal wall collaterals (caput medusae) .

Although hemorrhoids bleed frequently and varices of the bowel rupture occasionally, massive hemorrhage from thin- walled varices in the upper stomach and lower esophagus is the major complication of portal hypertension. Variceal bleeding occurs without obvious precipitating cause and presents usually as painless massive hematemesis (vomiting blood) . Two theories have been proposed to explain variceal bleeding. The "erosion" theory purports that varices bleed because of trauma, such as from deglutition of solid food or gastroesophageal reflux, to frail variceal walls. As no causal relation has been established between bleeding and trauma, this theory has been abandoned.

On the other hand, the "explosion" theory suggests that the event precipitating bleeding is excessive hydrostatic pressure inside the varices from portal hypertension. Thus, this hypothesis suggests that portal hypertension is responsible for both the appearance and rupture of varices. Recent studies have shown that variceal pressures are substantially greater in portal hypertensive patients who have bled than in non-bleeders.

Rupture of varices is probably a ultifaceted phenomenon. In studying wall rupture in other lumenal entities (e.g. aneurysm and cecum volvulus) , researchers have noted that the critical factor in the rupture of a lumen is not the transmural pressure but the wall tension. This is expressed mathematically by Laplace's law:

Tension = (delta P) x r/w where "delta P" represents the transmural pressure gradient, "r" represents the radius of the lumen, and "w" indicates the wall thickness. It is apparent from Laplace's law that at a given pressure larger varices (greater radius) exert more wall tension. Thus, they are more likely to rupture than smaller varices.

The sequence of events leading to variceal rupture probably occur as follows. Portal hypertension promotes the opening of collaterals and formation of varices. The increased pressure, coupled with increased blood flow, leads to the dilation of varices ("r" increases) . Also upon dilation, the variceal wall becomes thinner ("w" decreases) . Because of the high hydrostatic pressure in the varies and the low (or subatmospheric) pressure in the esophageal lumen, there is a relatively high pressure gradient (delta P) . Any combinations of the above factors ("r," "w," and "delta P") which produces a tension which exceeds the bursting strength of the variceal wall, will cause rupture.

Acute esophageal variceal hemorrhage remains a catastrophic event with a high mortality from either exsanguination or hepatic coma. The latter is precipitated by shock and the stress associated with massive blood transfusion, increased protein load in the gut, and the trauma of various procedures employed in an effort to arrest the bleeding. Up to 70 percent of cirrhotic patients develop varices, and of those, up to 40 percent bleed. The mortality from initial hemorrhaging is reported to be between 30 and 80 percent. See Potzi R, et. al. Clinical Trials: Prophylactic endoscopic sclerotherapy of esophageal varices in liver cirrhosis . A multicentre perspective control randomized trial in Vienna, Gut 1989 Jun; 30(6): 873-879. Cirrhosis patients with variceal hemorrhage, especially those with cirrhosis from alcohol abuse, have a substantially higher mortality than patients with normal liver function and variceal hemorrhage secondary to non- cirrhotic etiologies.

Treatment of variceal hemorrhage has been directed along two strategies—to arrest acute bleeding and to prevent recurrence. Pharmacological modalities usually attempt to decrease portal pressure and portal-collateral blood flow. Beta-adrenergic blockers, such as propranolol, have been used with limited success. These agents reduce portal pressure by reducing portal and collateral blood flow. This results in splanchnic vasoconstriction and a decrease in cardiac output due to the antagonism of Beta-2 receptors. While there is only a moderate fall in portal pressure, there is a marked reduction in azygos blood flow. Unfortunately, over one-third of patients do not exhibit any decrease in portal pressure despite adequate Beta-blockage. Thus, this therapy is of limited value in these cases.

Another method of treatment is the selective mesenteric intra-arterial infusion of vasopressin. In this method an indwelling catheter is inserted into the superior mesenteric artery for continuous vasopressin infusion.

Vasopressin is a potent vasoconstrictor which reduces blood flow in all splanchnic organs, hence reducing portal pressure. This results in a decrease in the azygos blood flow and variceal pressure. Success has been attained in approximately 60 percent of these cases. It has been reported that there is little or no difference between selective infusion and the lesser-invasive, systemic intravenous administration. At any rate, either method is complicated by a decrease in cardiac output, and does not significantly influence the mortality of varix hemorrhage. Other drawbacks of vasopressin include heart failure, arrhythmias (particularly bradycardia) , hypertension, angina and hyponatremia.

Somatostatin has been used with mixed results for the treatment of variceal hemorrhage. Somatostatin reduces portal and collateral blood flow by causing selective splanchnic vasoconstriction. In some studies, its efficacy is comparable to vasopressin, but lacks many of its complications. Nonetheless, somatostatin does not definitively treat varices. Thus, it provides no prophylactic protection against future hemorrhaging.

Non-pharmacological methodologies of treatment include balloon tamponade, surgery, and sclerotherapy. In balloon tamponading, a Sengstaken-Blakmore tube is inserted into the esophagus for direct compression of the bleeding varix. The disadvantage of this technique includes the limited duration that the balloon can be inflated (24-36 hours) , the recurrence of bleeding upon deflation, and the high rate of potentially lethal complications such aspiration pneumonia and esophageal rupture.

Surgical techniques, which control bleeding varices and prevent recurrence, include direct suture ligation, esophagogastric devascularization procedures, and emergency portal-systemic shunting. A major problem associated with all emergency operations is hepatic decompensation; this may result when a critically ill patient, already compromised by a severely damaged liver and severe hemorrhaging, is subject to anesthesia and major surgical trauma. In addition, many patients have marked coagulopathies since the liver is responsible for producing many of the blood-clotting factors. Moreover, a majority of patients undergoing shunts procedures develop fatal hepatic encephalopathy. Perioperative mortality rates as high as 50 percent have been reported. Thus, a surgical option may be unadvisable or clinically impossible for many patients. Even in those patients with good liver function, portal-caval shunting does not appear to prolong survival and may be followed by encephalopathy and neuropsychiatric syndromes. See Conn, H. , Therapeutic portocaval anastomosis : to shunt or not to shunt,

Gastroenterology 1974; 67: 1065-71. In response to these poor results, other techniques were developed.

Endoscopic injection sclerotherapy (EIS) is a favored treatment for esophageal varices, and currently the treatment of choice for acutely hemorrhaging varices. See Terblanche et. at., Medical Progress: Controversies in the Management of Bleeding Esophageal Varices, New England Journal of Medicine, 1989 May 25; 320(21): 1393-1398. Injection sclerotherapy controls acute hemorrhage, prolongs survival, and does not predispose to encephalopathy. Injection sclerotherapy was initially reported in 1939, but was superceded by other treatment modalities at that time. With the popularization of the flexible fiberoptic endoscope in the 1970's, injection sclerotherapy has reemerged as a prominent treatment modality. See Allison J, The role of injection sclerotherapy in the emergency and definitive management of bleeding esophageal varices , Journal of American Medical Association, 1983 Mar 18; 249(11): 1484-7.

Sclerotherapy consists of the peri- and/or intravariceal injection of irritants or sclerosing solutions under endoscopic guidance. This technique stops hemorrhaging by obliterating varices, with primary control of acute hemorrhage up to 95% being reported. See Terblanche J. , et. al.. Acute bleeding varices : A five-year prospective evaluation of tamponade and sclerotherapy , American Journal of Surgery 1981; 194: 592-595. Subsequent fibrosis and scar formation helps prevent variceal recurrence. The atrophied varices are no longer able to serve as a portal-systemic shunt. The superficial submucosal varices are probably replaced by dilated intramuscular or extraesophageal collaterals, which are less prone to rupture. Allison, J. , supra at 1486.

The post-sclerotherapy histological changes of the esophagus have been characterized. Within 24 hours, thrombosis and tissue necrosis are usually present. Inadequate thrombosis is probably associated with an increased risk of re-bleeding. Necrosis, particularly that involving the muscularis propria, increases the possibility of perforation. Ulceration may occur within a week, with a concomitant increase in risk of venous perforation and subsequent bleeding; incidence of ulcer formation as high as 100% has been reported. See e .g. , Singai et al., Ulceration After Oesophageal and Gastric Variceal Sclerotherapy - Jn-flue-nce of Sucralfate and Other Factors on Healing,

Endoscopy, 20:238-240, 1988. Ulcers usually heal by re- epithelialization of the surface, but may persist as a residual ulcer, accompanied by the symptom of dyspepsia (heartburn) . After a month, fibrosis is observed but is thought to be mainly beneficial. However, it may lead to complications if present in excess.

In addition to ulcerations, the esophageal mucosa often develops erosions which when healed may result in fibrosis and stricture formation. The incidence of stricture formation has been reported as high as 59%. See e .g. , Sorensen et al._Λ Oesophageal Stricture and Dysphagia. After Endoscopic Sclerotherapy for Bleeding Varices, Gut, 25:473-477, 1984. Moreover, strictures often lead to dysphagia in many patients. See Evans D., Esophageal varices treated by sclerotherapy: a histopathological study, Gut 1982 Jul; 23(7): 615-2.0.

Numerous factors impact the sclerotherapy healing process, including patient condition, sclerosant used, operator's experience, and method of injection (peri- or intravariceal injection) . Since the obliteration/repair cycle can be highly variable, sclerotherapy is accompanied by significant risks.

In the early post-sclerotherapy period (within 30 days) , for example, the spectrum of complications includes advanced mucosal ulceration, erosions, luminal narrowing, intramural defects, sinuses, fistulae, esophageal dissection, perforation, mediastinitis, and, of course, rebleeding. Late complications include strictures, irregular contours, esophageal dys otility, luminal obstruction, and esophagitis. See Soehendra N., et al.. Morphological alterations of the esophagus after endoscopic sclerotherapy of varices, Endoscopy 1983 Sep; 15(5): 291-296; Agha, F., The esophagus after endoscopic injection sclerotherapy: acute and chronic changes, Radiology 1984 Oct; 153(1): 37-42. Moreover, no therapeutic agents, including H₂ antagonists and sucralfate, have shown efficacy in reducing these complications from sclerotherapy.

After initial obliteration of the varices, it is mandatory to perform frequent follow-up endoscopies because many patients will have recurrent varices which require repeated sclerotherapy. On average each patient receives two to five sclerotherapy sessions, performed at one to two week intervals. However, the number of sessions and interval between them is greatly in luenced by the post-sclerotherapy healing phase.

According to the present invention, there is provided the use of EGF-active agent in novel compositions and methods for treating varices by promoting post-sclerotherapy healing. According to another aspect of the present invention, there is provided the use of EGF-active agent in novel compositions and methods for treating inflammatory bowel disease. The invention provides for the manufacture of an in situ administrable medicament for promoting healing of a damaged alimentary tract of an animal. In addition, the invention provides for the use of EGF-active agent for the manufacture of systemically administrable medicaments, suitable for administration orally and/or parenterally. The invention also provides for the use of EGF-active agent for the manufacture of a medicament suitable for lower GI administration.

EGF (also known as Epidermal Growth Factor and Urogastrone) is a 53-amino acid peptide having a molecular weight of 6,045 dalton and three disulfide bridges. It has been isolated and characterized from the submaxillary glands of adult male mice (Cohen, General Biological Chemistry 1962, 237: 1555) . In humans, EGF occurs in submandibular and Brunner's glands, and is present in much lower concentration in the thyroid, jejunum, and kidney. It is acid stable and trypsin resistant.

EGF's ability to stimulate cellular growth and differentiation is thought to include epithelial, mesothelial, and endothelial cell lines. EGF also appears to play an important role in the development of fetal and neo-natal oral cavity, lungs, eyelids, and alimentary tract. Typically, the EGF is capable of binding to the epitopes of specific markers (EGF-R 170Kd) on selected cell types.

The polypeptide structure of EGF has been demonstrated to have the following amino acid sequence (SEQ ID N0:l) of the following table:

TABLE 1 Asn - Ser - Asp - Ser - Glu - Cys - Pro - Leu - Ser - His - Asp - Gly - Tyr - Cys - Leu - His - Asp - Gly - Val - Cys - Met - Tyr - lie - Glu - Ala - Leu - Asp - Lys - Tyr - Ala - Cys - Asn - Cys - Val - Val - Gly - Tyr - He - Gly - Glu - Arg - Cys -

Gin - Try - Arg - Asp - Leu - Lys - Trp - Trp - Glu - Leu - Arg By way of example and not limitation, urogastrone appears similar if not identical to EGF. Urogastrone was first identified and described in 1939 (Gray et al. , Science 1939, 89: 489) as a component in human urine. Urogastrone is probably synthesized initially in the form of a precursor macromolecule which is then cleaved to liberate the active form of the protein. This latter form,, which is composed of 53 amino acids, is known as Beta-urogastrone.

The invention is not restricted to any particular form of EGF but contemplates other related peptides, such as mouse EGF, mammalian EGF and human EGF, including polypeptide precursors and degradation products thereof, and numerous other polypeptides which include the segment containing the EGF sequence. More particularly, the present invention contemplates the use of an epidermal growth factor-active agent (EGF-active agent) . The term EGF-active agent, as defined herein, includes biologically-active molecules and compounds having binding affinity to EGF receptor, including biologically active EGF polypeptide and fragments thereof, which may be produced recombinantly, synthetically, or by other means, such as the fragmentation of biologically produced proteins and polypeptides.

The present invention also encompasses proteins or polypeptides having substantial homology with the amino acid sequence in Table 1. Homology is determined by optimizing residue matches by introducing gaps as required. This changes when considering conservative substitutions as matches. This definition is intended to include natural allelic variations in the EGF sequence. Typical homologous proteins or peptides will have from about 25% to 100% homology (if gaps can be introduced) to about 50% to 100% homology (if conservative substitutions are included) with the amino acid sequence of Table 1. Some homologous proteins or peptides, such as EGF subtypes, will exhibit some biological activity in common with the EGF polypeptide of Table 1.

As used herein, the term "biological activity" is defined as including without limitation, EGF receptor binding and cross reactivity with anti-EGF antibodies raised against EGF from natural sources. These physiologically active molecules may substantially contribute an activity characteristic of a natural polypeptide or fragment, e.g., an EGF polypeptide. Typically, an EGF polypeptide, when associated into a functional receptor, has the set of activities performed by the receptor in a biological context (e.g., in an organism or an in vitro biological context). However, a protein fragment may exhibit other specific or inherent functions, such as vital conformational constraints, serving as a substrate site for glycosylation, contributing the signal sequence for cellular targeting, or serving some presently unrecognized functions possessed inherently.

It will be appreciated that the amino acid sequence of EGF polypeptide may be modified by genetic techniques. One or more amino acids can be deleted or substituted. Such amino acid changes, especially if in a region which is not within an epitope of the polypeptide, may not cause any measurable change in the serological activity of the protein or polypeptide. The resulting protein or polypeptide will have substantially the same amino acid sequence and substantially the same serological activity and is within the scope of the present invention.

Therefore, the present invention also provides for the use of other polypeptides comprising fragments of EGF and polypeptides substantially homologous thereto. The peptides employed by the present invention will generally exhibit at least about 50% homology with naturally occurring sequences of hEGF, typically at least about 80-85% homology with a natural polypeptide sequence, more typically at least about 90% homology, usually at least about 95% homology, and more usually at least about 97% homology. The length of comparison sequences will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.

Homology, for polypeptides, is typically measured using sequence analysis software, see e.g., Segue-nce Jtiialysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wisconsin 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, phenylalanine, and tyrosine.

By way of illustration and not limitation, EGF-active agent may include epidermal growth factor, fibroblast growth factor, transforming growth factors (including alpha and/or beta) , platelet-derived growth factor, insulin-like growth factor, insulin growth factor, and other agents which include the exemplary homologies and/or receptor activity set forth hereinabove.

In a preferred embodiment of the present invention, the EGF-active agent includes a human epidermal growth factor

(hEGF) polypeptide. A preferred source of polypeptides is

Upstate Biotechnology of New York. Recent technical advances have provided additional forms of polypeptides and methods of making them. The utilization of recombinant DNA technology has produced functional, assembled EGF polypeptides suitable for in vivo use. Recombinant human EGF polypeptide is available commercially and is sold, for example, by Bachem, Inc. (Catalogue No. DGRO 05, Vol. 3, Bioactivities No. 2, June 1989) .

Appropriate dosages of EGF-active agent to achieve the desired injury prevention and healing augmentation will vary depending on many factors, including the individual patient's underlying condition, the amount of esophageal damage, the patient's previous exposure to EGF or related compositions, and the particular adjuvant(s) or formulations which may be co-administered. Further, these factors will impact the different means of administration which are contemplated by the invention. The dosage should be sufficient to significantly inhibit the onset of injury and enhance healing of any injury that does occur (e.g., by decreasing the mean healing time between sclerotherapy treatments) . One preferred method of documenting this protective/augmentation response to EGF-active agent is based upon observing mucosal injury attendant with sclerotherapy, including ulceration, erosion, stricture formation, and the like. Other protocols may be substituted to assess the patient's response according to means well known by those skilled in the art. By utilizing non-treated patients as a baseline reference, positive responses to EGF-active agent can be identified. Typically, re-epithelialization and fibrosis are analyzed and a wide range of responses is seen.

To achieve a suitable inhibition of sclerotherapy- induced injury and/or augmentation of post-sclerotherapy healing, a variety of dosage routes may be followed, again depending, for example, upon any particular co-administrant utilized and the severity of the disease state. The dosage routes may be, for convenience sake, divided into three main groups— in situ, per os, and parenteral. The in situ route entails the internal, topical application of EGF-active agent, either proximally or directly to the target site, by available means including endoscopy. High dosages or bolus washes are well tolerated and may be preferably used to maximize the delivery of EGF-active agent while minimizing the invasive nature of in situ techniques. The per os route includes the oral administration of EGF-active agent by available means, including substitute means thereof, such as through a tube or syringe.

A preferable in situ administered pharmaceutical composition is one suitable for administration in unit dose form containing between about 2 and 6,000 μg of EGF-active agent. The in situ administered pharmaceutical composition will be delivered to a mammal through endoscopic means to treat a damaged alimentary tract so that each patient typically will receive a dose of between about 10 and 10,000 μg per bolus wash, usually between about 50-1,000 μg, with preferred ranges of about 10-50 μg, 50-500 μg, and 500-3,000 μg indicated for disease states of varying severity. Total daily dosages will range from between about 10 and 15,000 μg per day, with preferred ranges of about 20-100 μg, 100-1,000 μg, and 1,000- 6,000 μg, as indicated for disease states of varying severity. The amount of EGF-active agent administered by bolus washes may range from about 10 up to about 10,000 μg or more. Larger amounts by bolus washes may be tolerated if the administration schedule calls for a single or a few washes. Lower amounts per bolus may be administered over longer time periods of up to 5 weeks or more. In one dosage protocol, between about 3 and 300 μg/kg/day, more preferably between about 10 and 30 μg/kg/day, with preferred ranges of 20-50 μg/kg/day, 50-100 μg/kg/day, and 100-300 μg/kg/day indicated for disease states of varying severity.

Alternatively, EGF-active agent may be administered in a single bolus wash of about 10 and 10,000 μg per bolus wash, preferably about 100 and 6,000 μg per bolus wash with preferred ranges of 10-100 μg, 100-300 μg, 300-600 μg, and 600- 3,000 μg indicated for disease states of varying severity, simultaneously, before, and/or after sclerotherapy. In an preferred embodiment, EGF-active agent is administered just prior to sclerotherapy (e.g., 10 minutes before). Yet another schedule entails administering about 2-5 daily bolus washes, each between about 100 and 2,000 μg per wash, most usually between 200 and 500 μg per wash. Still another schedule entails the continuous delivery (e.g., by nasogastric tube) of EGF-active agent to achieve a dosage of about 3 and 500 μg/kg/day, usually between 10-100 μg/kg/day.

The bolus washes will typically include a liquid solution or suspension having the EGF-active agent. Longer contact with the mucosal surface can be attained by selecting a suitable vehicle which is capable of adhering to mucosa. Exemplary vehicles include pectin-containing formulations such as Orabase® (Colgate-Hoyt Laboratories, Norwood, MA) , sucralfate suspensions, Kaopectate®, sodium alginate, and milk of magnesia. Other suitable carriers include gels, lotions, and ointments having a pharmaceutically acceptable non-toxic carrier. When sustained-release delivery is desired, a liposome carrier may be employed; a liposome carrier suitable for use with the present invention is described in U.S. Patent o. 4,944,948, which is hereby incorporated by reference. Hydrogels, available from several vendors, may also be employed as a suitable carrier for EGF-active agent.

In situ administration may also include intraesophageal injections of EGF-active agent. In this instance, EGF-active agent is administered by single or multiple paravenous injections of about 10 and 100 μg/kg, typically performed during a sclerotherapy session. , EGF-active agent may also be combined with the sclerosants of interest, thereby forming an enhanced sclerosant; in this formulation, the need for separate injections is alleviated. Regardless of route, however, a therapeutically effective dose of EGF-active agent would be an amount sufficient to inhibit sclerotherapy- induced injury and/or augment the patient's post-sclerotherapy healing significantly. The invention further provides a method for promoting healing a damaged alimentary tract of a mammal which comprises the endoscopic bolus administration of EGF-active agent to the mammal. The method of the present invention is of particular interest in promoting the healing of post-sclerotherapy injury.

This method comprises placing the patient in a suitable position, such as a lateral decubitus position. Appropriate venous access and necessary hemodynamic monitoring are secured. Appropriate pre-medication, such as diazepam, meperidine and atropine, is given. The patient's oropharynx is anesthetized with a topical agent, such as lidocaine. A flexible fiberoptic endoscope (such as that available from Olympus of Japan) is inserted into the oropharynx and passed into the esophagus. After identification of a distended varix, the endoscopic needle is directed into the varix and/or into a paravenous region. Between 0.5 and 5 ml of a sclerosing solution are injected; the sclerosing solution may also contain a therapeutically effective dose of EGF-active agent. After completion of the injection, the needle is withdrawn. A bolus wash of therapeutically effective concentration of EGF-active agent may then be delivered to the treatment region through the liquid port of the endoscope. An endoscopically administered pharmaceutical composition may be formulated by means known to the art. The procedure is repeated as necessary.

As another feature of the present invention, there is provided a pharmaceutical composition for per os administration which comprises EGF-active agent in association with a pharmaceutical carrier. The oral pharmaceutical composition may be formulated by means known to the art. A preferable oral pharmaceutical composition for promoting healing of non- gastroduodenal digestive tract injury in animals, the composition being in dose unit form adapted for administration to animals, is a dosage unit containing from about 1.0 to about 3.0 mg of EGF-active agent. The oral pharmaceutical composition will be administered to a mammal for the treatment of damaged alimentary tract so that each patient receives an oral dose of between about 10 μg per day to 200 mg per day, typically between about 100 μg to 50 mg per day, with preferred ranges of 10 μg-200 μg, 200 μg-1,000 μg, 1 mg-10 mg, and 10 mg to about 100 mg indicated for disease states of varying severity. A preferable orally administered pharmaceutical composition is one suitable for administration in unit dose form containing between 10 and 7,000 μg, more preferably between about 10 and 100 μg of EGF-active agent in a controlled-release delivery medium. The orally administered pharmaceutical composition will be delivered to a mammal through per os means to treat a damaged alimentary tract so that each patient will receive a oral dose of between about 5 and 500 μg/kg/day, more preferably between about 10 and 35 μg/kg/day, with preferred ranges of about 10-50 μg/kg/day, 50- 200 μg/kg/day, and 200-500 μg/kg/day indicated for disease states of varying severity. Larger amounts per os may be tolerated if the administration schedule calls for a single or a few doses. Lower amounts per os may be administered over longer time periods of up to 5 weeks or more. The per os doses are preferably administered on the first day of treatment with sclerotherapy, with the remaining per os doses administered three to five times daily thereafter. Alternatively, EGF-active agent may be administered in a single per os dose of between about 100 μg and 5,000 μg preferably between 0 to 3 hours or more before sclerotherapy. Yet another schedule entails administering about a single dose of between about 2,000 to 7,000 μg per dose. Again, a therapeutically effective dose of EGF-active agent would be an amount sufficient to inhibit sclerotherapy-induced injury and/or augment the patient's post-sclerotherapy healing significantly and will typically range from 100-500 μg, 500- 2,000 μg, 2,000-5,000 μg per dose depending upon the severity of the disease state.

A preferable parenterally administered pharmaceutical composition is one suitable for administration in unit dose form containing between 0.5 and 5,000 μg of EGF-active agent in a suitable delivery medium. Depending upon the severity of disease, preferred compositions of EGF-active agents will be administered in the unit dosage forms of 1-10 μg, 10-50 μg, 50- 200 μg, 200-500 μg, 500-2,000 μg, and 2,000-5,000 μg. The parenterally administered pharmaceutical composition will be delivered to a mammal through continuous infusions and/or periodic doses to treat a damaged alimentary tract so that each patient will receive between 5 and 1,000 μg/kg/day, more preferably between about 10 and 100 μg/kg/day of EGF-active agent per day. A preferred dosage range for subcutaneous administration to treat patients with diseases such as colitis are 25 to lOOμg/kg/day.

The parenterally administered EGF-active agent dose may range from about 10 up to about 30 μg/kg or more, frequently between 25-100 μg/kg, and may be administered by intradermal, intramuscular, intravenous, and/or subcutaneous techniques. Larger amounts may be tolerated parenterally if the administration schedule calls for a single or a few doses. Lower parenteral amounts may be administered over longer time periods of up to 5 weeks or more. The initial parenteral doses are preferably administered on the first day of treatment of sclerotherapy, with the remaining parenteral doses administered at B.I.D. to Q.I.D. intervals, more preferably T.I.D., thereafter. Alternatively, EGF-active agent may be administered in a single parenteral dose of about 0.01 mg and 5.0 mg preferably within about 0 to 5 hours or more of sclerotherapy. Yet another schedule entails administering single daily parenteral doses, each between about 10 and 2,000 μg/kg. Still another schedule entails administering single weekly parenteral doses of between about 50 and 5,000 ug/kg. Again, a therapeutically effective dose of EGF-active agent would be an amount sufficient to inhibit sclerotherapy-induced injury and/or augment the patient's post-sclerotherapy healing significantly.

Various combinations of EGF-active agent, including compositions in conjunction with adjuvants, are acceptable for use with susceptible diseases, such as esophageal ulcers, esophagitis, and other esophageal lesions. EGF-active agent also finds uses in other human therapies. By way of example and not limitation, EGF can be used to treat other alimentary tract injuries, such as mucositis.

Depending on the intended application, EGF-active agent may be used alone or with other gastrointestinal protective agents such as H₂ blockers, prostaglandins, and proton-pump inhibitors. These "cocktails" can be designed to universally and safely augment the healing response in a variety of treatments. Any of a variety of gastrointestinal protective agents known to the skilled artisan can be combined in the cocktail. For example, ranitidine may be utilized with EGF-active agent at concentrations ranging from about 150 mg to 300 mg/day, preferably about 300 mg/day. Similarly, proton pump inhibitors (e.g., omeprazole) may be utilized with EGF-active agent at dosages of about 20 to 40 mg, preferably about 20 mg/day.

Other agents may be advantageously employed. For example, other growth factors (including their active fragments) may be employed, alone or in combination with other growth factors, as the co-administrant(s) ; exemplary growth factors include, without limitation, epidermal growth factor, fibroblast growth factor, transforming growth factor (alpha and/or beta) , platelet-derived growth factor, insulin-like growth factor, and insulin growth factor.

Still other agents may be co-administered. Analgesic and anesthetics, for example, can be co-administered to alleviate the pain that accompanies lesions. Antimicrobial agents, including antibacterial agents, antifungal agents, and the like, may be co-administered to prevent or treat secondary infections.

Typically EGF-active agent and the additional gastrointestinal protective agent will be given coincidentally, such as both at day 1 with subsequent daily administrations, or the like, but alternating administrations may be utilized. Actual methods for preparing and administering bolus washes and oral compositions will be known or apparent to those skilled in the art and are described in detail, e.g., in Remington's Pharmaceutical of Science, 16th Edition, Mack Publishing Company, Pennsylvania (1982) , which is incorporated herein by reference.

The period of administration of EGF-active agent with adjuvants or other synergistic agents should ideally be coincided. In humans, the wound healing response commonly can be detected about 5 to 15 days after exposure to EGF-active agent and peaks at about two to three weeks or more thereafter. To maximize this response, for example, EGF-active agent is preferably administered within the first few days after initial exposure to sclerotherapy. Subsequent doses of EGF-active agent may be administered to further increase this and other aspects of the healing process. In this manner, prolonged or multiple treatment therapy regimes with the EGF-active agent can be utilized to provide an increased level of efficacy.

II. INFLAMMATORY BOWEL DISEASE Also of interest to the present invention is inflammatory bowel disease (IBD) . Inflammatory bowel disease, which is a general classification of inflammatory processes affecting both large and small intestines, is typified by ulcerative colitis and Crohn's disease. Ulcerative colitis is an inflammatory condition involving the mucosa and submucosa of the colon, which is characterized by periods of acute attacks and remissions. The disease typically begins in the rectum and involves the bowel contiguously. Macroscopically, the mucosa may have a granular appearance if the disease is mild; in the active phase, however, ulcers and hemorrhage predominate. When fulminant, stripping and sloughing of the mucosa occur. The clinical hallmark of ulcerative colitis is rectal bleeding and diarrhea, with the frequency of bowel movements and the amount of blood present reflective of the activity of the disease. Patients also experience abdominal bloating, fatigue and malaise, and a perfuse mucoid diarrhea (which may lead to electrolyte imbalance) . Other aspects of ulcerative colitis include extraintestinal manifestations and risk of carcinoma. Skin manifestations include erythema nodosum, which is characterized by red, swollen nodules on the thighs and legs; other secondary complications may involve the eyes, joints, and liver. An increased risk of carcinoma occurs in those patients who develop the disease during childhood, with the risk increasing by about 10% each decade.

The current therapy for ulcerative colitis is directed to reducing the attendant inflammation. Sulfasalazine is used to initiate and maintain a remission in UC patients. Its active moiety, 5-amino salicylate, has a direct anti- inflammatory effect in dosages of about 2-8 grams a day. The drug is not without side effects, however. Nausea and abdominal distress often result. In addition, severe skin reactions may occur including exfoliative dermatitis and photosensitive reactions. Additional side effects include hemolytic anemia, agranulocytosis, pancytopenia, and thrombocytopenia.

For those with severe disease or who do not respond satisfactorily to sulfasalazine, corticosteroids and corticotrophin (ACTH) are employed. The steroids, which have an anti-inflammatory effect on the bowel, are typically administered in high dosages and are adjusted as the patient improves. However, the well-known serious side effects of steroids limit their usefulness. For example, hypertension, diabetes, osteoporosis, cataracts, and steroid-induced myopathy are all are side effects common in steroid-treated patients. Steroids also increase a patient's susceptibility to many infections, including oral candidiasis.

Surgery for ulcerative colitis is indicated in those patients with an intractable form of the disease. In addition, the presence of a perforation, intra-abdominal abscess, stricture, or premalignant biopsy also mandates surgery. Total proctocolectomy with ileostomy is a standard approach.

Crohn's disease or regional enteritis, on the other hand, is an inflammatory disease of both the small or larger intestine. The inflammation involves all layers of the gut, and thus the term "transmural colitis" has been used to describe this disease of the colon. Macroscopic examination typically reveals mucosal ulceration: aphthous ulcers within mucosa that appears normal, deep ulcers within areas of swollen mucosa, and long linear serpiginous ulcers. Punctate areas of mucosal hemorrhage may alternate with areas of gross hemorrhage. Perianal fistulas may occur with either colonic or small bowel forms of the disease.

Clinically, diarrhea is the most common feature in both large and small bowel forms of the disease. Bleeding, although a prominent feature of ulcerative colitis, is present only in about 50% of patients with transmural colitis. Weakness, fatigue, anorexia, and fever are common symptoms of active disease. An intra-abdominal abscess or enterocutaneous fistula may be the initial manifestation of the disease. With involvement of the small intestine, severe alabsorption may occur, often leading to a deficiency of dietary fats and fat- soluble vitamins.

As with ulcerative colitis, extraintestinal and neoplastic sequelae are known. Secondary manifestations may include arthritis, pyoderma gangrenosum, erythema nodosum, uveitis, and liver disease. In patients with long-standing disease, amyloidosis may develop. The incidence of carcinoma of the small bowel is increased in Crohn's patients with small bowel forms of the disease, while there is also an increased risk of colonic cancer in patients with transmural colitis.

Like ulcerative colitis, Crohn's disease is a protean illness characterized by exacerbations and remissions. Similarly, the management of Crohn's disease mirrors that of ulcerative colitis, with both sulfasalazine and steroids typically employed. Unlike ulcerative colitis however, surgery is not curative in Crohn's disease. Therefore, surgery is reserved for those patients who do not respond to medical treatment or who have disorders such as abscess, free perforation, or unremitting obstruction that does not respond to medical management. Despite current medical and/or surgical intervention, about 5-10% of Crohn's patients will die of the illness. To achieve a suitable therapeutic response to treating inflammatory bowel disease, a variety of dosage routes may be followed. As described hereinabove for treatment of sclerotherapy-induced injury, the dosage routes may depend, for example, upon any particular co-administrant utilized and the severity of the disease state. Exemplary dosage routes may be divided into two main groups: in situ and parenteral. The in situ route entails the internal, topical application of EGF- active agent, either proximally or directly to the target site, by available means including endoscopy; in situ delivery may also be achieved by enemas or other colonic irrigating techniques.

A preferable in situ administered pharmaceutical composition is one suitable for administration in unit dose form containing between 100 and 2,000 μg of EGF-active agent. The in situ administered pharmaceutical composition will be delivered to a mammal through endoscopic and/or enema means to treat a damaged alimentary tract so that each patient will receive a dose of between 60 and 2,000 μg per application, and between 100 and 7,000 μg per day. Larger amounts applications may be tolerated if the administration schedule calls for a single or a few applications. Lower amounts per application may be administered over longer time periods of up to 5 weeks or more. In one dosage protocol, between dosages between about 10 and 30 μg/kg/day are employed.

Alternatively, EGF-active agent may be administered in a single enema or application of about 100 μg to 10,000 μg, with preferred dosage ranges of 100-500 μg, 500-2,000 μg, and 2,000-10,000 μg indicated for disease states of varying severity, given in intervals of between one to five times a day. Yet another schedule entails administering about three daily applications, each about 5 to 30 μg/kg. Treatment may also be coincided with other therapeutic modalities. In another protocol, for example, EGF-active agent may be administered simultaneously with a steriod and/or sulfasalazine. Alternative protocols may be adopted, for example, giving steriod on even days and EGF-active agent on odd days.

The enemas will typically include a liquid solution or suspension having the EGF-active agent. Longer contact with the mucosal surface can be attained by selecting a suitable vehicle which is capable of adhering to mucosa. Exemplary vehicles include, as set forth hereinabove, pectin-containing formulations such as Orabase® (Colgate-Hoyt Laboratories, Norwood, MA) , sucralfate suspensions, Kaopectate®, sodium alginate, and milk of magnesia. Other suitable carriers include gels, lotions, and ointments having a pharmaceutically acceptable non-toxic carrier. When sustained-release delivery is desired, a liposome carrier or hydrogel may be employed. For treatment of rectal lesions, retention techniques (e.g., retention enemas) may be advantageously employed.

In situ administration may also include intramural injections of EGF-active agent at or proximal to the region of interest. In this instance, EGF-active agent is administered by single or multiple intramural injections of about 10 and 1000 μg/kg, typically performed via endoscopy. Regardless of route, however, a therapeutically effective dose of EGF-active agent would be an amount sufficient to inhibit the sequelae of IBD and/or augment the patient's healing significantly. EXPERIMENTAL DATA The following experimental data are offered for purposes of illustration and not limitation.

EXAMPLE I An animal model for studying sclerotherapy-induced erosions and ulcerations in the esophagus was developed by Dr. Lone S. Jensen at the University of Aarhus, Aarhus, Denmark, who has also developed and published similar models. See e.g., Jensen et al., Endoscopic sclerotherapy of esophageal varices in an experimental animal model. , Scand. J. Gastro. 21: 725- 732, 1986.

In the model, portal venus hypertension was produced in eighteen Gottingen minipigs by prehepatic banding of the portal vein with an ASC-band, as previously described by Jensen et al., Effect of acute portal hypertension on hepatosplanchnid hemodynamics and liver function. Scand. J. Gastro. 21: 568-575, 1986. The animals were allowed to recover from the surgical procedure, with appropriate post-operative nursing care and housing provided. Four weeks post-op, the minipigs were examined for size and number of esophageal varices and were categorized according to the Degradi System, as described by Jensen et al. The animals were then randomized into the following three groups. Group I: Sclerotherapy control group, animals received only sclerosant (polidocanol 2%) injected paravenously.

Group II: Sclerotherapy and EGF (20 μg/kg) administered concomitantly, with the EGF and the sclerosant injected paravenously.

Group III: Same as Group I, but with the additional EGF was administered subcutaneously at the dose of 10 μg/kg three times daily for five weeks.

After randomizing the minipigs, sclerotherapy of esophageal varices was performed at weekly intervals as follows. Under endoscopic guidance, employing a 4 millimeter long needle (Olympus NM-3) , polidoconol 2% was injected paravenously in small deposits of 0.5 ml. along each variceal column. Group II and III minipigs received concomitant EGF doses, with the latter group also receiving the maintenance dose. Supplemental sclerotherapy was conducted at follow-up endoscopy until the varices were eradicated; if varices recurred during the observation period, sclerotherapy was repeated.

The EGF dosages employed were prepared from stock solutions as follows. Stock EGF was dissolved in a saline solution containing 0.01% polysorbate 80 to produce a concentration of about 1.0 milligrams per ml. Polysorbate 80 functioned to minimize the adsorption of EGF to experimental glass vessels. The stock EGF solutions were kept refrigerated at -20 C°. Dosages were prepared daily by dilution of stock solution with appropriate amounts of the saline solution. The minipigs were examined at 5, 6, 7, 8, and 12 weeks after surgery, as follows. Under light anesthesia with Ketamine™ (10 mg/kilogram) and Midazolam (0.5 mg/kg), endoscopic and gastroscopic examinations were performed using a flexible endoscope (Olympus GIF-K2) . For each minipig, esophageal and gastric mucosae were visualized and graded for ulcerations, erosions, and strictures by a scientist who was blinded as to the treatment.

Assessment of mucosal injury was performed by visualization of the mucosae, post embolization of the variceal veins. The following four-point endoscopic score for erosions, ulcerations, and strictures was employed for grading: Grade 0: Normal mucosa.

Grade 1: Red and swollen mucosa with superficial erosion. Grade 2: Extensive edema, ulcerations, and fibrin coding (irrespective of its size) .

Grade 3: Same as Grade 2, but with stricture formation observed.

Esophageal stricture formation was also assessed in all animals, even in the absence of edema, erosions, or ulcerations.

Referring to Figs. 1-3, data for the three groups are presented. The number of ulcers and endoscopic grades were averaged for all three groups over an eight week period. It was found that groups receiving EGF (Groups II and III) had a decreased mean number of ulcers as well as a decreased overall incidence of ulcers; additionally, these groups had lower overall mean endoscopic scores (grades) . It was also noted that the incidence of strictures was eliminated in the EGF groups. The best results were obtained in Group III, the group receiving the maintenance dose of EGF. While not illustrated, it was additionally noted that the obliterating effect of sclerotherapy appeared enhanced in the EGF-receiving groups; furthermore, preliminary results suggested that a single weekly dosage had signigicant efficacy. These data establish that EGF reduced the incidence and severity of sclerotherapy-induced injury, including ulcers, erosions, and strictures. Moreover, these data establish that EGF promotes healing* of sclerotherapy-treated varices.

EXAMPLE II Background Several years ago, Kirkegaard et al. demonstrated that intraduodenal administration of epidermal growth factor (EGF) inhibited the development of cystamine induced duodenal ulcers in the rat. See, Kirkegaard, P., Olsen, P.S., Poulsen, S.S., Nex, E. Epidermal Growth Factor Inhibits Cysteamine- Induced Duodenal Ulcers, Gastroenterology 1983:85:1277-1283. In addition, Ulshen et al. investigated the effect of intraluminal administration of EGF on the small bowel mucosa of adult rats. See, Ulshen, M.H., Lyn-Cook, L.E., Raasch, R.H. Effects of Intraluminal Epidermal Growth Factor on Proliferation in the Small Intestine of Adult Rats, Gastroenterology 1986:91:1134-

1140. EGF infused into the small bowel had a trophic effect on the mucosa at the site of infusion as well as unexposed mucosa distant from the site of EGF infusion. These observations suggest that exogenous EGF may have usefulness as a pharmacologic agent in the treatment of mucosal damage.

Morris et al. recently described a model of chronic inflammatory bowel disease in the rate. See, Morris, G.P., Beck, P.L., Herridge, M.S., Depew, W.T. Szewczuk, M.R. , Wallace, J.L. Hapten- Induced Model of Chronic Inflammation and Ulceration in the Rat Colon , Gastroenterology 1989:96:795-803. The intracolonic administration of trinitrobenzenesulfonic acid (TNBS) dissolved in ethanol induces inflammatory lesions in the

^*5 distal colon that persist for 5-6 weeks. The tissue insult includes ulcerations of the mucosa, transmural inflammation and

^* the development of granulomas. This model shows both chronicity and morphological features of inflammatory bowel disease. It is therefore, an appropriate model to investigate

10 the potential effects of EGF to either protect or enhance the healing of experimental colonic inflammation.

Objective The aim of this study was to determine if Epidermal 15 Growth Factor (EGF) can protect or reverse colonic damage induced by trinitrobenzenesulfonic acid in the rat colon.

Experimental Design The effectiveness of EGF as a cytoprotective agent

20 was determined by EGF's inhibition of macroscopic damage to the colon after TNBS induced colitis. Colonic damage was assessed macroscopically by two independent observers and assigned a number according to the criteria in Table 2. The severity of colonic damage was scored on a 0

25 (normal) to 5 (severe) scale. Animal body weight changes, colon wet weights and mortality were determined, however these results are insignificant and not detailed in this report. Male Sprague-Dawley rats weighing between 270-290 g were used in this study and the colonic damage was evaluated after seven

30 days. TABLE 2 MACROSCOPIC COLONIC DAMAGE ASSESSMENT

Score Appearance

0 No Damage

1 Slight bowel wall thickening, no localized hyperemia

2 Hyperemia and/or bowel wall thickening

3 Single erosion with hyperemia and bowel wall thickening

4 Two or more erosions with hyperemia and bowel wall thickening 5 Two or more major sites of damage or major damage > 1 cm along the length of colon

All scoring of damage was performed by two independent observers who were unaware of the treatment.

This study was conducted in three phases:

1) development of the TNBS model of colonic inflammation

2) screen EGF's usefulness as a cytoprotective agent by intra colonic and subcutaneous routes of administration

3) determine if EGF's cytoprotective effect is dose and time dependent

4) examine the effect of EGF on the chronic inflammatory changes induced by TNBS.

Phase 1. Establish experimental model of colonic inflammation.

The severity of colonic damage varied with the concentration of TNBS and the percentage of ethanol in the diluent. As shown in Table 3, the colonic damage with 15 mg, 25 mg and 30 mg TNBS was dependent upon the percentage of ethanol in the diluent. A solution of 30 mg TNBS dissolved in 0.25 ml of 30% ethanol was selected for all subsequent studies. At this concentration, one to two erosions, hyperemia and bowel wall thickening were consistently produced. Erosions of the mucosa were located 3-5 cm proximal to the anal verge and the bowel wall thickening involved the entire distal colon from the site of TNBS-Ethanol instillation (8 cm proximal to anal verge) .

TABLE 3

EFFECTS OF THE CONCENTRATION OF TNBS AND THE PERCENTAGE OF COLONIC DAMAGE

Macroscopic colonic damage was scored on a 0 (normal) to 5 (severe) scale by two independent observers. Rats were sacrificed seven days after intracolonic administration of 0.25 ml of varying concentrations of TNBS and percentages of ethanol in the diluent. Data are expressed as the mean ± SEM. The intracolonic administration of 25 mg TNBS in 0.25 ml normal saline or administration of 0.25 ml 30% ethanol alone resulted in damage scores of 0.67 ± 0.44 and 1.00 ± 0.58 respectively. Thus, indicating that the combination of TNBS and the ethanol as the "barrier breaker" is necessary to induce damage. The total volume of TNBS-Ethanol administered to induce colitis also was evaluated. A volume of 0.25 ml containing 30 mg TNBS in 30% ethanol when given intracolonically resulted in higher colonic damage scores than a 0.50 ml volume. The erosions that were present in the 0.50 ml group were less frequent and located 2 cm from the anal verge.

The effect of fasting the animals for 48 hours prior to administration of 30 mg TNBS in 30% ethanol was examined. Animals that were fasted 48 hours had a 10-20% decrease in body weight before the TNBS-Ethanol was given intracolonically. Half of the fasted animals died before seven days and those that did survive had severe colonic damage, a large loss in body weight and appeared to be in poor health.

Therefore in all subsequent studies, 0.25 ml containing 30 mg TNBS in 30% ethanol was administered intracolonically 8 cm proximal to the anal verge in the non- fasted and unanesthetized rat. All drug treatment protocols also were performed in the non-fasted and unanesthetized rat.

Phase 2. Determine the ability of EGF to prevent TNB induced colitis.

This study investigated the cytoprotective ability of EGF by two different routes of delivery. EGF was given either intracolonically or subcutaneously one-half hour to one hour before TNBS inducted colitis and on a once daily interval for seven days.

Intracolonic Delivery of EGF Results of daily dosing.

As shown in Figs. 4-6, EGF given intracolonically (0.50 ml) one hour before and once daily for seven days had no effect on the TNBS-Ethanol induced colonic damage.

Prednisolone (0.50 ml) also was ineffective in reducing the colonic damage. However, there was a dose dependent reduction in the damage score by 5-aminosalicylic acid (0.50 ml) when administered at a dose of 200 mg per kilogram body weight. Results of a single predose.

A single pretreatment dose without any further drug treatment for seven days was tested. EGF (100 ug/kg) and prednisolone (50 mg/kg) given intracolonically one hour prior to TNBS-Ethanol administration did not prevent or protect against the TNBS-Ethanol induced colitis.

Results of EGF alone.

The effect of intracolonic administration of EGF alone on the normal colon was evaluated. EGF (0.50 ml) was given intracolonically for seven days in non TNBS-Ethanol treated rats. EGF had no effect on the macroscopic appearance and wet weight of the distal 8 cm of colon. Subcutaneous Delivery of EGF EGF was given subcutaneously (0.25 ml) one-half hour before administration of TNBS-Ethanol and once daily for seven days. In Fig. 7, there was a dose dependent reduction in colonic damage by EGF. EGF administered at a dose of 100 ug per kilogram body weight reduced the damage score from 3.63 ± 0.22 to 2.3 ± 0.17 SEM. The 25 ug/kg dose of EGF showed a tendency to reduce the damage score, however it was not significant.

Phase 3. Examination of the dose and time dependency of EGF's cytoprotective effect.

Macroscopic Assessment Since EGF given once daily subcutaneously had a cytoprotective effect that was dose dependent, the effect of EGF given at more frequent intervals was evaluated. EGF was subcutaneously injected (0.25 ml) one-half hour before TNBS- Ethanol and then every twelve hours thereafter. As shown in Fig. 8, EGF reduced the colonic damage score at doses of 25 ug and 100 ug per kilogram body weight.

Microscopic Assessment The histologic appearance of the colon from a representative sample of animals that had macroscopically visible erosions seven days after TNBS-Ethanol were examined. The colon exhibited marked ulceration of the mucosa and inflammation. There was a loss of the glandular mucosa, edema and extensive infiltration by polymorphonuclear leukocytes, eosinophils, mast cells and lymphocytes. There was occasional invasion of the smooth muscle by polymorphonuclear leukocytes extending through the smooth muscle to the serosa. The colonic wall thickness from serosa to luminal surface of the mucosa was increased.

Colonic tissue was examined from a representative sample of animals that were injected with EGF subcutaneously for seven days and had a macroscopic damage score of 2. The glandular mucosa was intact and areas of ulceration were not seen. In these tissues, the colonic wall thickness was still increased and many inflammatory cells were present in the submucosa.

Phase 4. The effect of EGF on the chronic inflammatory changes induced by TNBS.

The colonic damage produced by TNBS-Ethanol was evaluated 21 days after intracolonic administration. Two concentrations of TNBS 30 mg and 50 mg both in 50% ethanol were administered. After 21 days, colonic damage scores were 3.00 ± 0.01 and 3.5 ± 0.50 respectively. The rationale for testing EGFs ability to reverse the colonic damage after 21 days was based upon the possibility that long term treatment may be necessary before any beneficial effects would be observed. Since EGF given subcutaneously for seven days reversed the colonic damage by TNBS-Ethanol, EGF was not further evaluated in the 21 day model.

Discussion The results of this study demonstrate that intracolonic administration of trinitrobenzenesulfonic acid in ethanol results in ulceration and inflammation of the rat colon. This damage was characterized by marked thickening of the colonic wall, hyperemia and areas of marked erosion of the mucosa. There was a loss of the glandular mucosa, edema and extensive infiltration by polymorphonuclear leukocytes, eosinophils, mast cells and lymphocytes. There was occasional involvement of the smooth muscle to the serosa and the colonic wall thickness from serosa to lumenal surface of the mucosa was increased. The concentration of TNBS and the percentage of ethanol used in the present study were based upon the results of a dose-response study. The dose selected consistently produced ulceration of the mucosa and inflammation that is similar to results obtained by other investigators. See, Morris, G.P., Beck, P.L. , Herridge, M.S., Depew, W.T. Szewczuk, M.R., Wallace, J.L. -ffapten-∑nduced Model of Chronic Inflammation and Ulceration in the Rat Colon, Gastroenterology 1989:96:795-803; Wallace, J.L., MacNaughton, W.K., Morris, G.P., Beck, P.L. Inhibition of Leukotriene Synthesis Markedly Accelerates Healing in a Rat Model of Inflammatory Bowel Disease, Gastroenterology 1989:96:29-36; Rachmilewitz, D., Simon, P.L., Schwartz, L.W. , Griswold, D.E., Fondacaro, J.D., Wasserman, M.A. T-n-fla-Tunatory Mediators of Experimental Colitis in Rats , Gastroenterology 1989:97:326-337; Vilaseca, J. Salas, A., Guarner, F., Rodriguez, R. , Malagelada, J. Participation of Thromboxane and Other Eicosanoid Synthesis in the Course of Experimental Inflammatory Colitis , Gastroenterology 1990:98:269-277. The morphological and histological features of the model share features that are found in human inflammatory bowel disease. See, Sharon, P., Ligu sky, M. , Rachmilewitz, D. , Zor, U. -Role o-f Prostaglandins in Ulcerative Colitis . Enhanced Production During Active Disease and Inhibition by Sulfasalazine , Gastroenterology 1978:75:638-640; Ligumsky, M. , Karmeli, F., Sharon, P., Zor, U. , Cohen, F., Rachmilewitz, D. Enhanced Thromboxane A₂ and Prostacyclin Production by Cultured Rectal Mucosa in Ulcerative Colitis and Inhibition by Steroids and Sulfasalazine , Gastroenterology 1981:81:444-449; Hawkey, C.J., Karmeli, F. , Rachmilewitz, D.

Imbalance of Prostacyclin and Thromboxane Synthesis in Crohn ' s Disease , Gut 1983:24:1176-1182; Boughton-Smith, N.K. , Hawkey, C.J. , Whittle, B.J.R. Biosynthesis of Lipoxygenase and Cyclo- oxygenase Products from [14] C-arachidonic Acid by Human Colonic Mucosa , Gut 1983:24:1176-1182; Sharon, P., Stenson, W.F. Enhanced Synthesis of Leukotriene B₄ by Colonic Mucosa in Inflammatory Bowel Disease , Gastroenterology 1984:86:453-460; Peskar, B.M. , Dreyling, K.W. , Peskar, B.A., May, B., Goebell, H. Enhanced Formation of Sulfidopeptide-leukotrienes in Ulcerative Colitis and Crohn ' s Disease : Inhibition by Sulfasalazine and 5-aminosalicylic Acid, Agents Actions 1986:18:381-383. Thus, the model is relevant in the assessment of potentially therapeutic agents for the treatment of colonic inflammation. The potential therapeutic benefit of epidermal growth factor was investigated in this model. Intracolonic delivery of EGF was unsuccessful in reversing the colonic damage produced by TNBS-Ethanol. It is possible that the ulceration and inflammation produced by TNBS-Ethanol may have been to severe for any potential benefit of EGF to be observed. However, this is unlikely since 5-aminosalicylic acid had a dose dependent reversal of colonic damage. The ineffectiveness of EGF may be the result of its poor bioavailability, since it may be poorly absorbed and have a short half-life in the colon.

However, when EGF is given subcutaneously, colonic damage is reversible. Although EGF injected subcutaneously reverses erosions of the mucosa, the colonic wall is moderately thickened. We observed a difference in the dose of EGF and frequency of subcutaneous injections of EGF on reversal of the colonic damage induced by TNBS-Ethanol. Single daily subcutaneous injections of 100 ug/kg EGF reverses colonic damage whereas 25 ug/kg does not. More frequent injections of EGF reverses colonic damage at doses of 25 ug/kg and 100 ug/kg. This difference in dose and frequency of EGF administration may be due its short half-life and mechanism of action.

Summary Epidermal growth factor was evaluated as an agent that could protect or reverse colonic damage in a rat model of colonic inflammation. Trinitrobenzenesulphonic acid in ethanol was administered intracolonically and colonic damage assessed after seven days. Daily intracolonic treatment with EGF and prednisolone for seven days did not protect or reverse the colonic damage by TNBS-Ethanol, however 5-aminosalicylic acid was successful. A single daily subcutaneous injection of EGF for seven days reversed colonic damage at a dose of 100 ug/kg but not at 25 ug/kg. Subcutaneous injections of EGF every 12 hours for seven days reversed colonic damage at doses of 25 ug/kg and 100 ug/kg. It was concluded that subcutaneous delivery of EGF is effective in reversing colonic damage in the experimental model of colitis.

Moreover, these data suggest that the efficacy of EGF in reversal of colonic damage may be enhanced by continuous delivery of EGF. Continuous infusion of EGF both lumenally and systemically stimulates mucosal proliferation and absorption in the small intestine. See, Schartz, M.D., Storozuk, R. , Influence of Epidermal Growth Factor on Intestinal Function in the Rat: Comparison of Systemic Infusion Versus Luminal Perfusion, Am J. Surgery 1988:155:18-22; and Ulshen et al. supra . While the invention is described in some detail with specific reference to a preferred embodiment and certain alternatives, there is no intent to limit the invention to that particular embodiment or those specific alternatives. Those skilled in the art will appreciate other alternatives within the scope of the present invention. Therefore, the true scope of the invention is defined not by the foregoing description but by the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Reher- John M.

(ii) TITLE OF INVENTION: Epidermal Growth Factor Therapy for Non-Gastroduodenal Lesions

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: William M. Smith

(B) STREET: One Market Plaza, Steuart Tower, Suite 2000

(C) CITY: San Francisco

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 94105

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk ^' (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/777,827

(B) FILING DATE: 15-OCT-1991 CC) CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Smith, William M.

(B) REGISTRATION NUMBER: 30,223

(C) REFERENCE/DOCKET NUMBER: 13954-2

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 415-326-2400

(B) TELEFAX: 415-326-2400

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 53 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His 1 5 10 15

Asp Gly Val Cys Met Tyr lie Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30

Cys Val Val Gly Tyr lie Gly Glu Arg Cys Gin Tyr Arg Asp Leu Lys 35 40 45

Trp Trp Glu Leu Arg 50

Claims

WHAT IS CLAIMED IS:

1. An improved method for treatment of non- gastroduodenal alimentary disorders, the improvement comprising administering a therapeutically effective dose of epidermal growth factor-active agent.

2. The method for claim 1, wherein the administering includes systemically administering a therapeutically effective dose of epidermal growth factor-active agent.

3. The method of claim 2, wherein the systemically administering step includes subcutaneously administering epidermal growth factor-active agent at a dose of about 3 - 30 mg/kg/day.

4. The method of claim 2, wherein the systemically administering step includes parenterally administering epidermal growth factor-active agent.

5. The method of claim 4, wherein the parenterally administering step includes intravenously administering epidermal growth factor-active agent at a dose of about 1 - 100 μg/kg.

6. The method of claim 2, wherein the parenterally administering step includes intramuscularly administering epidermal growth factor polypeptide at a dose of about 3 - 30 μg/kg/day.

7. The method of claim 1, wherein the alimentary disorder includes gastro-esophageal varices.

8. The method of claim 1, wherein the alimentary disorder includes inflammatory bowel disease.

9. The method of claim 8, wherein the alimentary disorder is ulcerative colitis.

10. The method of claim 8, wherein the alimentary disorder is Crohn's disease.

11. The method of claim 1, wherein the epidermal ^*5 growth factor-active agent includes fibroblast growth factor polypeptide.

12. The method of claim 1, wherein the epidermal growth factor-active agent includes transforming growth factor

10 polypeptide.

13. The method of claim 1, wherein the epidermal growth factor-active agent includes platelet-derived growth factor polypeptide.

15

14. The method of claim 1, wherein the epidermal growth factor-active agent includes nerve growth factor.

15. A composition for promoting healing of non- 0 gastroduodenal digestive tract injury in animals comprising a pharmaceutical carrier having incorporated therein from about 0.01 to 30.0 μg of epidermal growth factor-active agent.

16. The composition of claim 15, wherein said

25 pharmaceutical carrier is in a controlled-released delivery medium.

17. The composition of claim 15, wherein the epidermal growth factor-active agent is reconstituted epidermal 0 growth factor polypeptide.

18. The composition of claim 15, wherein the pharmaceutical carrier includes a liposome carrier.

5 19. A composition for promoting healing of non- gastroduodenal digestive tract injury in animals, said composition being in dosage unit form adapted for administration to animals, each dosage unit containing from about 0.1 to about 3.0 mg/kg of epidermal growth factor-active agent.

20. The composition of claim 19, wherein said dosage unit form is in a controlled-released delivery medium.

21. The composition of.claim 19, wherein said epidermal growth factor-active agent includes epidermal growth factor polypeptide.

22. The composition of claim 19, wherein the dosage unit is in a form adapted for in situ delivery to animals.

23. The composition of claim 19, wherein the dosage unit is in a form adapted for per os delivery to animals.

24. The composition of claim 22, wherein the in situ delivery is endoscopic.

25. The composition of claim 22, wherein the in situ delivery by enema.

26. An improved method for promoting healing of non- gastroduodenal digestive tract injury in animals, the improvement comprising administering in situ a therapeutically effective dose of epidermal growth factor-active agent proximal to said injury.

27. The method of claim 26, wherein said non- gastroduodenal digestive tract injury includes esophageal lesions with epithelial denuding.

28. The method of claim 26, wherein said non- gastroduodenal digestive tract injury is colitis.

29. The method of claim 26, wherein the administering in situ is performed endoscopically.

30. The method of claim 26, wherein the administering in situ includes injecting at a paravenous location.

31. The method of claim 26, wherein the administering in situ includes delivering bolus washes.

32. The method of claim 26, wherein said epidermal growth factor-active agent is in a controlled-release medium.

33. An improved method for promoting healing of non- gastroduodenal digestive tract injury in animals, the improvement comprising administering per os a therapeutically effective dose of epidermal growth factor-active agent in a sustain-released formulation.

34. The method of claim 33, wherein said non- gastroduodenal digestive tract injury is an esophageal lesion with epithelial denuding.

35. A method for promoting healing of post- sclerotherapy esophageal injury, the method comprising: administering in situ a therapeutically effective bolus dose of epidermal growth factor polypeptide proximal to said injury; and administering per os a therapeutically effective dose of epidermal growth factor polypeptide in a sustain-released formulation to said injury.

36. An improved method for treating inflammatory bowel disease in animals, the improvement comprising administering a therapeutically effective dose of epidermal growth factor-active agent in a suitable pharmaceutical formulation.

37. The method of claim 36, wherein the epidermal growth factor-active agent is selected from the group consisting of epidermal growth factor polypeptide, fibroblast growth factor polypeptide, transforming growth factor polypeptide, and platelet-derived growth factor polypeptide.

38. The method of claim 36, wherein the epidermal growth factor-active agent is administered subcutaneously.

39. The method of claim 36, wherein the epidermal growth factor-active agent is administered topically.