JPH05194259A - Anti-peptic ulcer agent - Google Patents

Abstract

(57) [Summary] [Structure] An anti-peptic ulcer agent containing transforming growth factor beta (TGF-β) as an active ingredient. [Effects] The anti-peptic ulcer agent of the present application strongly promotes the healing of chronic ulcer, but has little effect on the gastric acid secretion inhibitory effect and the site protection as seen in EGF, and is different from existing anti-ulcer agents. It has a completely different mechanism of action. Further, it can be administered by any method, and is useful as a therapeutic agent for various types of digestive system diseases of a new type and as a preventive agent for their recurrence.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-peptic ulcer agent containing transforming growth factor beta (hereinafter abbreviated as "TGF-β") as an active ingredient.

[0002]

PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION TG
F (Transforming Growth Fac)
tor) is called tumor growth factor or transforming growth factor, and is secreted by cells and targeted by itself, and its receptor (EGF receptor, EGF; Epiderma)
l Growth Factor), which promotes growth, namely, as autocrine, 1979,
G. J. Reported by Todaro et al. (Cold
Spring Harbor Conf. Cell Pr
olif. 6,113,1979). Among these TGF-β
Is M. B. A substance discovered and named by Sporn et al. (Cancer Res. 42, 4776, 198).
2), a protein with a molecular weight of about 25,000 and 11 amino acid residues
It is a homodimer obtained by cross-linking two proteins having a molecular weight of 12,500 consisting of 2 and has been confirmed not to bind to the EGF receptor. Its structure was described in 1985 by R.
J. A. Reynck et al. (Na
true 316, 701, 1985).

[0003] TGF-β is known to have a contradictory activity of a growth promoting action and a suppressing action depending on the cell type and experimental system. That is, the epithelial growth inhibitory action (Science
226, 705, 1984; Cancer Cell
s 3,65, 1985), rat primary culture hepatocyte proliferation inhibitory effect (Biochem. Biophys. Res.
Commun. 133, 1042, 1985), and fibroblast proliferation promoting action (Science 233, 532).
1986) and the like. In addition, for cell differentiation, B
Suppression of differentiation of alb / C3T3 cells into adipocytes (Ex
p. Cell. Res. 165, 345, 1986),
Inhibition of differentiation of lymphocytes into antibody-producing cells (J. Immun
ol. 136, 3916, 1986), promotion of bronchial epithelial cell differentiation (Proc. Natl. Acad. Sci. U.
SA 83, 2438, 2442, 1986), promotion of renal epithelial cell differentiation (Biochem. Biophys. Re).
s. Commun. 136, 969, 1986), promotion of differentiation of fibroblasts into chondrocytes (J. Biol. Che.
m. 262, 1946, 1987) and the like, and two are known to have an promoting action and an inhibiting action.

On the other hand, among conventional growth factors, EGF is orally administered (Gut, 29, 894, 1988) and subcutaneously (Scand. J. Gastroentero).
l. Suppl. , 162, 162, 1989) have been reported to promote healing of rat acetic acid ulcers, and for rat acute ulcers, oral administration has no effect and parenteral administration such as subcutaneous administration has been reported to have an inhibitory effect (Agents and
Actions, 27.294, 1989), bFGF.
(Basic Fibroblast Growth
Factor) has been reported to have an effect of promoting the healing of chronic duodenal ulcers generated in rats by cysteamine and a suppressive effect of acute duodenal ulcers (Eu).
r. J. Pharmacol. , 183, 2090, 1
990), the relationship between TGF-β and anti-peptic ulcer action has not yet been known.

[0005]

[Means for Solving the Problems] The present inventors have TGF-β
As a result of repeated studies on new physiological activity of TGF-β
The present invention has been completed for the first time by discovering that it has an anti-peptic ulcer effect. That is, the gist of the present invention resides in an anti-peptic ulcer agent containing transforming growth factor beta (TGF-β) as an active ingredient.

The present invention will be described in detail below. The TGF-β used in the present invention can be obtained by various known methods, and specifically, it is disclosed in JP-A 61-219.
395, 62-285788, 63-2838.
No. 6, JP-A-1-120300, Mo1.
Cell. Biol. It can be easily produced by utilizing the gene recombination technology described in 7,3418,1987 and the like.

In the present invention, TGF-β can be administered by any method, specifically, subcutaneous injection, intravenous injection,
Parenteral administration such as intramuscular injection or oral administration, and further, it is possible to spray or apply directly to the affected area endoscopically.
The dose is determined according to the patient's age, health condition, body weight, degree of ulcer condition, type of simultaneous treatment, frequency of treatment, desired effect, and the like. Generally one of the active ingredients
The dose per administration is 0.01 μg / human to 1800 μg
/ Human, preferably 1 μg / human to 300 μg / human, administered twice daily or less, or at intervals of several days to several weeks.

When the anti-peptic ulcer agent of the present invention is orally administered, it is in the form of tablets, capsules, powders, solutions, elixirs and the like, and when it is parenterally administered, it is a sterilized liquid such as liquid or suspension. Used in the form of. When used in dosage forms as described above, solid or liquid non-toxic pharmaceutical carriers may be included in the composition. As an example of a solid carrier, an ordinary gelatin type capsule is used. These capsules, tablets and powders generally contain a trace amount to 90%, preferably a trace amount to 10% by weight of the active ingredient.

As the liquid carrier, water, petroleum, animal and vegetable oils such as peanut oil, soybean oil, mineral oil and sesame oil, oils of mineral origin, or synthetic oils can be used. In addition, physiological saline, sucrose or similar sucrose solution, glycols such as ethylene glycol, propylene glycol, and polyethylene glycol are generally preferable as the liquid carrier. Particularly, in the case of an injectable solution using physiological saline, it is usually polar. Trace to 20%, preferably trace 10
% Active ingredient.

In the case of a liquid preparation for oral administration, a suspension or syrup containing a trace amount to 10% by weight of the active ingredient is preferable. In this case, as a carrier, a water-based excipient such as a fragrance, syrup, or pharmaceutical micelle is used. Further, the anti-peptic ulcer agent of the present invention may be TGF-β alone or other activator,
For example, EGF, TGF-α, PDGF (Platele)
t-Derived Growth Factor (platelet-derived growth factor)) and the like, and other adjuvants may be included. In addition, in order to improve the durability as a drug, the drug may be given sustained release or gastric mucosa adhesion by a conventional method. In addition, in the case of being sprayed or applied endoscopically to the affected area, a suitable polymer base or organic solvent may be used in addition to the above.

[0011]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded. Example 1 Effect on acetic acid ulcer-1 Two Donryu male rats (body weight: around 200 g) were used.
After fasting for 4 hours, the method of Takagi et al. (Japan. J. Pha
rmacol. 19, 418, 1969) to create an acetic acid ulcer. That is, pentobarbital (40 mg
/ Kg, i. p. ) Under anesthesia, the abdomen is opened and 20% acetic acid 50μ
1 was injected subgastrically in the stomach. After that, suture the abdomen,
It was returned to normal breeding. After providing a recovery period of 3 days, the drug was mixed in drinking water and administered for 7 days. The drug is 0.01
% Gelatin aqueous solution, and adjusted at the time of use. After confirming that the rat drinks approximately 30 ml per day, the daily dose of the drug was mixed in 30 ml of drinking water and changed once a day. TGF-β is 3, 10, 30 μg / kg / da.
y, hEGF as a control drug was 30 μg / kg / da
The dose was adjusted for y. After continuous casting, the stomach was removed, 10 ml of 1% formalin was injected and fixed, and under a 10x stereoscopic microscope,
The area (mm ² ) of the ulcer part was measured and used as the ulcer index.
Also, as a control, only the buffer was administered.

The results are shown in FIG. Significant difference <0.
05 and ** represent a significant difference <0.01. The numerical value in the column in the figure shows the number of rats tested, and the height of the column shows the average value of the measured values. The same applies in the following figures. Administration of TGF-β 3 to 30 μg / kg / day dose-dependently improved the acetic acid ulcer created in the rat in 7 days, and was significant at 10 μg / kg / day or more. Even at 3 μg / kg / day, no significant difference was observed, but an improvement tendency of 40% or more was observed. A significant improvement was observed with the control drug, human EGF (hEGF) 30 μg / kg / day.

Example 2 Action on Acetic Acid Ulcer-2 In the same manner as in Example 1, except that the drug was orally administered by dividing into twice a day, the ulcer index, the collagen fibrosis rate of the ulcer floor, and The vascular growth index of the ulcer floor was measured. The results are shown in Figure 2. TGF-β 3-30 μg / kg / da
Oral administration of y (divided twice a day) significantly improved acetic acid ulcers and significantly promoted the development of collagen fibers at the ulcer floor at any dose. Further, the growth of blood vessels at the ulcer floor was significantly promoted at 10 μg / kg / day or more. The collagen fibrosis rate was determined by preparing a tissue section of the ulcer site with a short diameter section passing through the central part of the ulcer, and staining it with Azan Mallory,
Take a photograph at 200x under a microscope and determine the area occupied by the blue-stained part in the entire visual field by using an image processing device (MK
It was determined by binarizing and evaluating using SIPS1000). The blood vessel growth index was determined by counting the number of cross-sections of blood vessels present in one visual field from photographs of similarly stained specimens.

Example 3 Action on Acetic Acid Ulcer-3 In Example 2, the dose of the drug was 0.1 to 1 μg / k.
The ulcer index was measured in the same manner except that g / day was used. Results are shown in FIG. TGF-β is 0.1 to 1 μg
It was confirmed that the oral administration of a low dose of / kg / day (divided into twice a day) significantly improved the acetic acid ulcer.

Example 4 Action on Acetic Acid Ulcer-4 In Example 1, the dose of the drug was 0.1 to 10 μg /
The ulcer index was measured in the same manner except that the value was kg / day. The results are shown in Fig. 4. TGF-β is 0.1-10 μ
The acetic acid ulcer was ameliorated in a dose-dependent manner with g / kg / day (once daily administration).

Example 5 Action on Acetic Acid Ulcers-5 In Example 1, the drug dose per administration was 1.5 μm.
The ulcer index was measured in the same manner except that the dose was g / kg and the administration frequency was changed from once per 7 days to once per day. Results are shown in FIG. TGF-β showed a remarkable improving effect at any administration frequency (interval).

Example 6 Action on Hydrochloric Acid / Ethanol Ulcer Donryu male rats (body weight: about 250 g)
It was used after 4 hours of fasting and dewatering. The drug was administered by dissolving it in a 0.01% gelatin aqueous solution (oral administration) or a 0.01% gelatin-containing physiological saline solution (subcutaneous administration) so that the dose was 2 ml / kg. The dose is TGF-β, E
GF was set to 3, 10, and 30 μg / kg. Thirty minutes after oral or subcutaneous administration of the drug, hydrochloric acid / ethanol (hydrochloric acid 1: ethanol 50) was orally administered at 1 ml / body, and 60 minutes later, the abdomen was opened and the stomach was treated with 1% formalin 10 m.
After fixing with l, the ulcer index (mm) was measured. Also, as a control, only the buffer was administered. The result is shown in Figure 6.
(Oral administration), 7 (subcutaneous administration). Oral administration of TGF-β and hEGF 3 to 30 μg / kg had no effect on hydrochloric acid / ethanol ulcer (FIG. 6). On the other hand, although subcutaneous administration all showed a dose-dependent inhibitory effect, TGF-β had a weak inhibitory effect of about 30% at 30 μg / kg, and hEGF had a strong inhibitory effect of about 80% at 30 μg / kg ( (Fig. 7).

Example 7 Action on gastric acid secretion (basic acid secretion) Male Donryu rats (body weight: about 250 g) were
Used after fasting for 4 hours. The drug was prepared as in Example 6 above. The rat was laparotomized under ether anesthesia, the pylorus was ligated, and the laparotomy was sutured. Immediately after that, the drug was orally or subcutaneously administered, and 5 hours later, it was killed with ether, and the gastric juice was collected, and the liquid volume, acidity and pepsin activity were measured. Also, as a control, only the buffer was administered. The results are shown in FIGS. TGF-β and hEGF
Oral administration of 3 to 30 μg / kg had no effect on gastric fluid volume, gastric acid secretion, or pepsin activity (FIGS. 8 and 1).
0, 12). On the other hand, in subcutaneous administration, TGF-β had no effect on any of them, but hEGF showed a dose-dependent suppressive effect (FIGS. 9, 11, and 13).

The above results are summarized as follows. Regarding the healing effect on acetic acid ulcer, that is, chronic ulcer, both TGF-β and hEGF showed extremely strong effects, but when compared at 30 μg / kg / day, the action seemed to be stronger with TGF-β. On the other hand, hydrochloric acid
The effect on ethanol ulcers, ie acute ulcers, is TG
It was not observed by oral administration of F-β, and showed a weak effect by subcutaneous administration. With hEGF, no effect by oral administration was observed, and an inhibitory effect was observed only by subcutaneous administration, and this result was reported in another report (Agents and Actions 27
294, 1989). This effect of EGF is said to be prostaglandin-mediated site protection (Gen. Pharmac. 1).
8, 647, 1987), EGF absorbed parenterally
Is said to have an effect. Although TGF-β had an action parenterally and was extremely weak as compared with EGF, it is considered that unlike EGF, cytoprotection is weak even if it is present.

EGF has no effect on basal gastric acid secretion when administered orally (Agents and A
(actions 27, 294, 1989), and it has been reported that it exhibits an inhibitory effect on the secretion of fluid volume, acid and pepsin upon parenteral administration (Gastroenterology 8
1, 438, 1981, Gut 22, 927, 198.
1, J. Physiol. 325, 35, 1982),
Our results this time also showed a similar effect. The mechanism is currently unknown. TGF-β
It has no effect on oral administration or subcutaneous administration, and is therefore considered to have no effect on gastric acid secretion.

From the above, TGF-β obviously promotes the healing of chronic ulcers, but its mechanism is that gastric acid secretion inhibitory action and cytoprotection are scarcely involved and that existing anti-ulcer agents are used. It is considered that the mechanism of action is completely different from. From the known facts, it is considered that TGF-β suppresses the proliferation of epithelial cells, whereas EGF promotes it. Further, both of them are considered to act in a promoting manner on fibroblasts. In the case of acetic acid ulcer, both epithelial regeneration and regeneration of the deep ulcer bottom (such as collagen fibers) underneath are conditions for healing. In addition, one of the causes of recurrence of ulcer is considered to be that the regenerated epithelium grows before the lesion of the deep stomach wall is healed (Medical P
ractice 7, 1995 1990). In such a case, if the well-balanced healing is performed by slowing the fast-growing epithelial regeneration and promoting the slow-regenerating deep layer, the healing without recurrence can be realized.
TGF-β is considered to have a favorable profile as a future drug for treating or preventing ulcer.

[0022]

INDUSTRIAL APPLICABILITY The anti-peptic ulcer agent of the present invention is further used as a therapeutic agent for gastrointestinal diseases such as gastric ulcer, duodenal ulcer, esophageal ulcer, esophageal injury, acute gastritis, chronic gastritis, duodenitis, gastrointestinal perforation and the like. It is considered to be useful as a drug for preventing their recurrence.

[Brief description of drawings]

FIG. 1 is a diagram showing the relationship between the oral dose of an active ingredient and the area of the ulcer part in the acetic acid ulcer model of Example 1.

FIG. 2 is a diagram showing the effect of the acetic acid ulcer model of Example 2. In the figure, (a) represents the relationship between the oral dose of the active ingredient and the area of the ulcer part, (b) represents the relationship between the oral dose of the active ingredient and the development of collagen fibers at the ulcer floor,
(C) shows the relationship between the oral dose of the active ingredient and the vascular development of the ulcer floor.

FIG. 3 is a graph showing the relationship between the oral dose of the active ingredient and the area of the ulcer part in the acetic acid ulcer model of Example 3.

FIG. 4 is a graph showing the relationship between the oral dose of the active ingredient and the area of the ulcer part in the acetic acid ulcer model of Example 4.

FIG. 5 is a graph showing the relationship between the administration interval of the active ingredient and the area of the ulcer part in the acetic acid ulcer model of Example 5.

FIG. 6 is a graph showing the relationship between the oral dose of the active ingredient and the length of the ulcer part in the hydrochloric acid / ethanol ulcer model of Example 6.

FIG. 7 is a graph showing the relationship between the subcutaneous dose of the active ingredient and the length of the ulcer portion in the hydrochloric acid / ethanol ulcer model of Example 6.

FIG. 8 is a graph showing the relationship between the oral dose of an active ingredient and the amount of gastric juice in the basal gastric acid secretion model of Example 7.

FIG. 9 is a graph showing the relationship between subcutaneous dose of active ingredient and gastric juice volume in the basal gastric acid secretion model of Example 7.

FIG. 10 is a diagram showing the relationship between the oral dose of an active ingredient and the gastric acid secretion amount in the basic gastric acid secretion model of Example 7.

FIG. 11 is a graph showing the relationship between the subcutaneous dose of the active ingredient and the gastric acid secretion amount in the basal gastric acid secretion model of Example 7.

FIG. 12 is an oral dose of the active ingredient and gastric secretion (pepsin activity) in the basic gastric acid secretion model of Example 7.
It is a figure which shows the relationship of.

FIG. 13 is a subcutaneous dose of the active ingredient and gastric secretion (pepsin activity) in the basal gastric acid secretion model of Example 7.
It is a figure which shows the relationship of.

Claims (1)

[Claims] 1. An anti-peptic ulcer agent containing transforming growth factor beta (TGF-β) as an active ingredient.