EP1005355A1

EP1005355A1 - Method of treating gastrointestinal ulcers and compositions therefor

Info

Publication number: EP1005355A1
Application number: EP98919780A
Authority: EP
Inventors: Sandor Szabo
Original assignee: Chemo Pathology Resour Center Inc; Radix Organization Inc
Current assignee: Chemo Pathology Resour Center Inc; Radix Organization Inc
Priority date: 1997-04-15
Filing date: 1998-04-15
Publication date: 2000-06-07
Also published as: WO1998046249A1; EP1005355A4

Abstract

The invention provides methods and compositions for providing chronic gastrointestinal ulcer healing or acute gastroprotection. In particular, administration of a pharmaceutically effective amount of VEGF, such as from about 30 ng/kg to about 1000νg/kg per day for about a week to two months results in healing of GI ulcers in a mammal, such as a human. Examples of gastrointestinal ulcers include duodenal ulcers, peptic ulcers, drug-induced ulcers, or intestinal ulcers, including but not limited to damage due to non-steroidal (NSAID) and anti-inflammatory drugs, gastro-esphogheal reflux disease or inflammatory bowel disease such as ulcerative colitis and Crohn's disease. Similarly, administration of a pharmaceutically effective amount of VEGF, such as from about 3νg/kg to about 1000νg/kg VEGF, from 0 minutes to about 60 minutes prior to potential injury to the GI tract, such as ingestion of an NSAID or ethanol, results in acute gastroprotection.

Description

Method of Treating Gastrointestinal Ulcers and Compositions

Therefor

Field of the Invention

The present invention relates to methods and compositions for treating gastrointestinal (GI) ulcers as well as for providing acute gastroprotection. Examples of GI ulcers include duodenal ulcers, peptic ulcers, drug-induced ulcers, or intestinal ulcers, including but not limited to damage due to non- steroidal (NSAID) and anti-inflammatory drugs, gastro-esphogheal reflux disease or inflammatory bowel disease such as ulcerative colitis and Crohn's disease. In particular, the present invention relates to treatment of a mammal, including a human, with vascular endothelial cell growth factor (VEGF), which is also a potent vascular permeability factor (VPF), to provide healing of GI ulcers or to provide acute gastroprotection.

Background of the Invention

The vascular system of organs and tissues develops by either of two processes, vasculogenesis or angiogenesis. Vasculogenesis is the de novo formation of blood vessels from differentiating endothelial cell precursors or angioblasts. Angiogenesis, the sprouting of new capillaries from pre-existing blood vessels, further refines the vascular network and is the principal process by which certain organs, such as the stomach, intestine or brain, become vascularized. Vasculogenesis and angiogenesis are complex multistep processes that involve remodelling of the extracellular matrix, migration and proliferation of endothelial cells, lumen formation and functional maturation of blood vessels. Multiple cell-cell or cell-extracellular matrix interactions occur, which require signalling via soluble or cell-bound molecules. A variety of molecules have been implicated in these processes, in particular angiogenic growth factors and their endothelial receptors and cell adhesion molecules. Soluble secreted molecules are likely to function as paracrine signalling molecules in these processes. Recent evidence suggests that the angiogenic factor vascular endothelial cell growth factor (VEGF) and its cellular receptors lie at the heart of the regulatory network controlling blood vessel growth and differentiation in both embryonic development and several angiogenesis- dependent diseases. While proliferation of the vascular endothelial cells is vital to angiogenesis, uncontrolled proliferation can lead to cancer and metastasis, diabetic retinopathy, atherosclerosis, rheumatoid arthritis, synovitis, psoriasis, dermatitis, endometriosis, encephalitis, and tonsilitis.

VEGF is a disulfide-linked 46-kDa dimeric glycoprotein first isolated from media conditioned by bovine folliculo stellate cells and its partial amino acid sequence determined (Gospodarowicz et al., PNAS 86(19):7311-7315; Ferrara and Henzel, BBRC 161 (2):851-858 (1989)). It has several properties expected of a candidate regulator of angiogenesis: it is secreted by normal and tumor cell lines; its is an endothelial-cell-specific mitogen; it is angiogenic in in vivo test systems such as the chorioallantoic membrane and the rabbit cornea; it is chemotactic for endothelial cells and monocytes; and it induces in endothelial cells the production of plasminogen activators, which are involved in the proteolytic degradation of the extra-cellular matrix during capillary sprouting. Several VEGF isoforms are known, which have similar biological activities but differ in their secretion from producer cells and in heparin binding (Georg Breier and Werner Risau, Trends in Cell Biology, 6:454-456( December 1996)). US Pat. Nos. 5,194,596 and 5,219,739, which are hereby incorporated by reference, disclose methods and means for producing vascular endothelial cell growth factor by means of recombinant DNA technology, such as bovine VEGF sub 120, bovine VEGF sub 164, human VEGF sub 121 and human VEGF sub 165, for use as a wound healing agent following vascular graft surgery, balloon angioplasty or to promote collateral circulation in post-myocardial infarction patients or in the treatment of wounds, such as dermal ulcers, including pressure sores, venous ulcers and diabetic ulcers, in the treatment of burns and injuries where angiogenesis is required to prepare the burn or injured site for a skin graft or flap, in cases where plastic surgery reconstruction is required and administration to the site of a bone fracture. An additional use relates in keeping wounds clean and non-infected in association with general surgery and following the repair of cuts and lacerations. Parenteral and topical compositions are disclosed.

US Pat. No. 5,332,671 , which is hereby incorporated by reference, discloses the production of a vascular endothelial cell growth factor and/or derivatives thereof by recombinant techniques for use in treating conditions in which a selective action on the vascular endothelial cells, in the absence of excessive tissue growth, is important, e.g., surface ulcers such as diabetic, hemophiliac and varicose ulcers and vascular injuries resulting from trauma such as subcutaneous wounds, incisions and injuries to the blood vessels or heart. Compositions for continuous infusion, sustained release, topical application or injection at empirically determined frequencies are disclosed. PCT Publication No. 9639515 discloses DNA encoding human VEGF-2 used to develop products to promote angiogenesis (e.g., stimulate growth of transplanted tissue following coronary bypass surgery) or endothelialisation in vascular graft surgery. PCT Publication No. 9639421 discloses DNA encoding human VEGF-3 used to develop products to promote angiogenesis and wound healing and to promote vascular tissue repair.

Our previous studies demonstrated that basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) which stimulate angiogenesis and granulation tissue production accelerated experimental duodenal ulcer healing that was more than two million times more potent on a molar basis that the similar effect of cimetidine (S. Szabo et al., Gastroenterology, 106:1106-1111 (1994)).

Summary of the Invention

Surprisingly, we have discovered that administration of VEGF (VPF), which is highly specific for endothelial cells, is sufficient for both acute gastroprotection and chronic GI ulcer healing in mammals. In particular, administration of a pharmaceutically effective amount of VEGF, such as from about 30 ng/kg to about 1000μg/kg, preferably about 80 ng/kg to about 800 μg/kg, more preferably about 400 ng/kg to about 700 μg/kg per day for about a week to two months, preferably two to three weeks, results in healing of GI ulcers in a mammal, such as a human. In particular, improved or enhanced mucosal restoration or prominent stimulation of angiogenesis and granulation tissue production at the ulcer edge results from this treatment.

Similarly, administration of from about 3 μg/kg to about 1000μg/kg, preferably about 8μg/kg to about 800 μg/kg, more preferably about 400 μg/kg to about 700 μg/kg VEGF, which is administered from 0 minutes to about 60 minutes prior to potential injury to the GI tract, such as ingestion of an NSAID or ethanol, results in acute gastroprotection.

Preferably, the administration is targeted to the GI tract, such as by oral administration, for example liquid solutions, immediate release dosage forms, controlled or sustained release dosage forms, or gastro-retentive dosage forms or by rectal administration, for example enemas or suppositories.

Detailed Description of the Invention

As used herein, the term "VEGF" or "vascular endothelial cell growth factor" refers to the disulfide-linked 46-kDa dimeric glycoprotein (a mammalian growth factor) first isolated from media conditioned by bovine folliculo stellate cells and its partial amino acid sequence determined (Gospodarowicz et al., PNAS 86(19):7311-7315; Ferrara and Henzel, BBRC 161(2):851-858 (1989))(hereinafter native VEGF) together with analogs and variants thereof having the biological activity of the corresponding native VEGF, including human VEGF. Analogs or variants are defined as molecules in which the amino acid sequence, glycosylation, or other features of native VEGF have been modified covalently or noncovalently, such as the analogs or variants disclosed above, and including salts thereof . An example of VEGF is the human VEGF obtainable from PeproTech, Canton, MA.

The present invention encompasses administration of a pharmacologically effective amount of VEGF, such as from about 30 ng/kg to about 1000 μg/kg, preferably about 80 ng/kg to about 800 μg/kg, more preferably about 400 ng/kg to about 700 μg/kg per day for about a week to two months, preferably two to four weeks, to treat duodenal ulcers or other GI ulcers in a mammal, such as a human. Examples of GI ulcers include but are not limited to duodenal ulcers, peptic ulcers, drug-induced ulcers, or intestinal ulcers, including but not limited to damage due to non-steroidal (NSAID) and anti-inflammatory drugs, gastro- esphogheal reflux disease or inflammatory bowel disease such as ulcerative colitis and Crohn's disease. The treatment regimen may consist of once daily administration or dividing the daily dose into multiple administrations, such as administration from 2 to 4 times per day.

The present invention also encompasses administration of from about 3 μg/kg to about 1000 μg/kg, preferably about 8 μg/kg to about 800 μg/kg, most preferably 400 μg/kg to about 700 μg/kg VEGF to provide for acute gastroprotection. Typically, the administration of VEGF occurs from about 0 minutes to about 60 minutes, preferably from about 0 minutes to about 30 minutes prior to insult to the GI tract, such as ingestion of NSAIDS or ethanol. Preferably, the administration is targeted to the GI tract. For instance, oral administration is a preferred administration route. In particular, liquid solutions, immediate release dosage forms, controlled or sustained release dosage forms or gastro-retentive dosage forms are preferred. Additionally, oral buccal dosage forms are useful for the delivery of VEGF.

Oral immediate release, sustained release, controlled release, oral buccal and/or gastro-retentive dosage forms can be prepared from nano- or microparticulates including but not limited to biodegradable matrix nano- or microparticles, nanocapsules, microcapsules or spray-dried vehicles. Emulsions, capsules or tablets incorporating these nano- or microparticulate forms can be prepared. Additionally, solid dosage forms can be prepared as tablets, mini- tablets, beads, capsules, etc., including forms with a controlled/sustained release coating. In these forms, the VEGF is typically released by erosion, diffusion, osmotic pressure, or by a combination of these mechanisms. Oral solutions, including elixirs, emulsions and effervescent solutions, can also be employed in this invention.

Typical controlled, sustained, or gastro-retentive excipients may include, but are not limited to, biodegradable polymers or biopolymers, such as poly- lactide, poly-glycolide, poly(lactide-co-glycolide), other polymers or copolymers of poly(α-hydroxycarboxylic acids), poly(lactones), poly(acetals), poly(orthoesters), poly(alkylcyanoacrylates) or poly(orthcarbonates). Additional excipients useful for oral administration forms include, but are not limited to, polysaccharides, for example cellulose derivatives such as etherified cellulose derivatives, including alkyl celluloses, hydroxyalkyl celluloses, and alkylhydroxyalkyl celluloses, for example, methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, and hydroxypropyl cellulose; starch and fractionated starch; agar; alginic acid and alginates; gum arabic; agarose; carrageenan; dextrans; destins; fructans; chitosans; glycogens; and glucans. Similarly, rectal administration is also a preferred administration route. In particular, enemas or rectal suppositories are preferred.

While the compositions for administering VEGF are preferably those which target the VEGF to the GI tract, such as oral or rectal administration, less preferred but still useful are compositions adapted for other routes of administration, such as vaginal or parenteral administration.

Example 1

Chronic duodenal ulcers were induced in Sprague-Dawley female rats (160-200g) by administering cysteamine-HCI (25 mg/100g) 3 times at 4-hour intervals by gavage and cortisone (5 mg/100g) s.c. 4 hours after the last dose of cystemine. To randomize rats with equally severe penetrating or perforated duodenal ulcers, the animals were anesthetized and laparatomized on the third day following treatment with cysteamine-HCI. After randomization, treatment was started with either vehicle saline (controls) or human VEGF (PeproTech, Canton, MA) at a dose of 1μg/100g once daily by gavage for 21 days.

At autopsy, the size of duodenal ulcers was measured, evaluated by stereomicroscopic planimetry and histologic sections taken. The results revealed that the size of duodenal ulcers in controls was 7.4 ± 1.6 mm². In contrast, the size of the ulcers in the VEGF-treated group were 1.9 ± 0.9 mm² (p<0.05), histologically accompanied by complete healing or prominent angiogenesis and granulation tissue production. The density of blood vessels per x400 magnification field in the granulation tissue at the ulcer edge was 8.0 ± 0.5 in the controls as opposed to 16.9 ± 1.9 (p<0.001) in the VEGF-treated rats. Thus, oral treatment of rats with VEGF (VPF) accelerated the healing of cysteamine-induced chronic duodenal ulcers. In addition, the healing was accompanied by complete mucosal restoration and stimulation of angiogenesis and granulation tissue production. Example 2

To investigate whether VEGF provides acute gastroprotection, groups of fasted rats were given by gavage human VEGF (PeproTech, Canton, MA) at 0.1μg/100g [VEGF group 1] or 1μg/100g [VEGF group 2] followed by 1 ml of 75% ethanol 30 minutes later. The control group was treated with 1 ml of 75% ethanol only.

Autopsy was performed 1 hour after treatment with the alcohol and the area of hemorrhagic mucosal lesions (HML) was measured by computerized stereomicroscopic planimetry. In the controls, HML involved 7.1 ± 1.4% of glandular stomach. VEGF group 1 showed HML which involved 7.7 ± 2.7% while VEGF group 2 showed HML which involved 4.0 ± 1.6% (p<0.05) of the grandular stomach. Thus, VEGF (VPF) exerted gastroprotection which might be associated with the known acute vascular effects of the peptide.

Example 3

Normally fed female Sprague-Dawley rats (170-200 g) were given 0.1 ml of 6% iodoacetamide (Sigma) in 1% methyl-cellulose intracolonically (i.e.) with Nelston catheter 7 cm from the anus to induce ulcerative colitis-like lesions. From the second day, the rats were treated with vehicle saline or VEGF

(rhVEGF, 38kDa, 165 AA, PeproTech) 10, 100 or 500 ng/100g i.e. once daily while the clinical signs of the illness, such as diarrhea, lethargy and weight loss were monitored. On the 10^th day, the animals were killed and the area and severity of colonic lesions were evaluated macroscopically, with stereomicroscopic planimetry ,and histologically. The results of this evaluation are tablulated in Table I.

Histologic examination revealed extensive granulation tissue production with partial or complete colonic re-epithelialization. The VEGF (10-500ng/100g) dose-dependently reduced the severity of diarrhea in the iodoacetamide-induced colitis. The VEGF also decreased the area and severity of the colonic lesions. On a molar basis, these small doses of VEGF indicate that this peptide is very potent in the healing of not only upper but also lower GI tract lesions as well.

The skilled artisan in the field would expect that the examples given above as practiced in the rat are predictive of healing in other mammals, particularly humans.

Claims

What is Claimed is:

1. A method of treating a GI ulcer in a mammal comprising administering a pharmacologically effective amount of VEGF to the mammal.

2. A method of providing acute gastroprotection in a mammal comprising administering a pharmacologically effective amount of VEGF to the mammal.

3. The method of claims 1 or 2, wherein the mammal is a human.

4. The method of any of the previous claims, wherein the administration of the pharmacologically effective amount of VEGF is by oral administration.

5. The method of any of the previous claims, wherein the administration of the pharmacologically effective amount of VEGF is by rectal administration.

6. The method of any of the previous claims, wherein the pharmacologically effective amount of VEGF ranges from about 30 ng/kg to about 1000 ╬╝g/kg per day.

7. The method of any of the previous claims, wherein the administration of the pharmacologically effective amount of VEGF is once daily.

8. The method of any of the previous claims, wherein the GI ulcer is selected from the group consisting of duodenal ulcers, peptic ulcers, drug-induced ulcers, intestinal ulcers, GI damage due to NSAID ingestion, GI damage due to anti- inflammatory drugs, GI damage due to gastro-esphogheal reflux disease or GI damage due to inflammatory bowel disease.

9. The method of claim 1 or claims 3 through 8, wherein the VEGF is administered daily from about 1 week to about a month to provide for healing of the GI ulcer.

10. The method of any of claims 2 through 8, wherein the VEGF is administered from about 0 minutes to about 60 minutes prior to insult to the GI tract to provide acute gastroprotection.

11. A composition for treating a GI ulcer or for providing acute gastroprotection in a mammal comprising a pharmacologically effective amount of VEGF in combination with a pharmaceutically acceptable carrier.

12. The composition of Claim 11 , wherein the composition is adapted to target release to the GI tract.

13. The composition of either claim 11 or 12, wherein the pharmacologically effective amount of VEGF ranges from about 30 ng/kg to about 1000 ╬╝g/kg.

14. The composition of any of claims 11 through 13, wherein the pharmacologically effective amount of VEGF ranges from about 80 ng/kg to about 800 ╬╝g/kg.

15. The composition of any of claims 11 through 14, wherein the composition is adapted for oral or rectal administration.

16. The composition of any of claims 11 through 15, wherein the pharmaceutically acceptable carrier comprises a liquid solution selected from the group consisting of elixirs, emulsions and effervescent solutions.

17. The composition of any of claims 11 through 15, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an immediate release dosage form, a controlled release dosage form, a sustained release dosage form, a gastro-retentive dosage form, an oral buccal dosage form, an enema and a rectal suppository dosage form.