WO1994020132A1

WO1994020132A1 - A method of treating a mammal with a biologically active compound

Info

Publication number: WO1994020132A1
Application number: PCT/DK1994/000094
Authority: WO
Inventors: Henrik Christensen
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1993-03-09
Filing date: 1994-03-04
Publication date: 1994-09-15
Anticipated expiration: 1995-09-09
Also published as: AU6256594A; DK25493D0

Abstract

A method of treating an inflammation in intestinal tissue in a mammal wherein growth hormone is administered to the mammal is disclosed. The administration of growth hormone has been shown to enhance the recovery from Crohn's disease and thus to reduce the mortality. The mammal to be treated is preferably a human being, and the growth hormone is preferably human growth hormone.

Description

TITLE

A Method of Treating a Mammal with a Biologically Active Com- pound. i

The present invention relates to a method for treating coli- 5 tis or Crohn's disease in a mammal by treating the mammal with human growth hormone, and the use of human growth hor¬ mone for the preparation of a pharmaceutical preparation for treating colitis or Crohn's disease.

BACKGROUND OF THE INVENTION

10 Crohn's disease is a condition involving colitis, diarrhoea, inflammation of the small intestine and strictures. This con¬ dition often involves chronic anorexia, malnutrition, loss of weight, and failure of linear growth.

Crohn's disease is considered a chronic condition which still 15 cannot be cured, and, at present, in case of a rupture colon must be removed and a permanent sto i established. To some degree it is possible to relieve the patient by treating some of the symptoms. Thus, nutrial supplementation has been shown to diminish symptoms and the activity of the disease and lead

20 to a partial or full restoration of linear growth failure

(Gastroenterology 1979, 76:720-727 & Gastroenterology 1976, 70:1017-1021)

Insulin-like Growth Factor I or somatomedin C (IGF-1) level in serum is considered a useful index of the nutritional sta- 25 tus in critically ill patients (Horm.Res. 1986, 24:166-176 & Crit Care Med 1987, 15:732-736). The IGF-1 level is also re¬ duced in serum from children and adolescents having a chronic inflammatory bowel disease, and normalization of the IGF-1 level by increasing the caloric intake has been reported to

30 improve the growth velocity and reduce the symptoms of acti- vity of the disease in patients having inflammatory bowel disease (Gastroenterology 1986, 91:830-836). In O91/12018 it is disclosed that administration of IGF-1 to patients suf¬ fering from gut diseases such as colitis or Crohn's disease may increase the weight of the gut.

In spite of these results stated above with respect to dimi¬ nishing symptoms of the disease there has until now been no disclosure or indication of how to diminish the inflammatory activity

It is the object of the present invention to provide a means for reducing the inflammatory activity in a mammal suffering from colitis or Crohn's disease.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention it has now surpri- singly been found that the administration of human growth hormone to a patient suffering from inflammation in intesti¬ nal tissue reduces the acroscopical and microscopical damage of the tissue.

The present invention relates, according to a first aspect thereof, to a method of treating an inflammation in intesti¬ nal tissue in a mammal wherein growth hormone is administered to the individual.

Such inflammation may be a colitis. A preferred aspect of the invention is a method of treating Crohn's disease by admini- stration of growth hormone to the individual suffering from this disease which still is considered chronical.

According to a preferred aspect of the invention, the mammal to be treated is a human being, the condition to be treated is Crohn's disease, and the growth hormone is human growth hormone.

According to a third aspect, the invention provides the use of a growth hormone for the preparation of a pharmaceutical preparation for treating an inflammation in intestinal tissue in a mammal.

The inflammation to be treated using such pharmaceutical pre- pertations may be a colitis, e.g. being a part of Crohn's disease.

In accordance with still another preferred aspect of the in¬ vention, human growth hormone is used for the preparation of a pharmaceutical preparation to be used for treating colitis in a human being.

In accordance with a most preferred aspect of the invention, human growth hormone is used for the preparation of a pharma¬ ceutical preparation to be used for treating Crohn's disease in a human being.

According to a still further aspect, the invention provides a method for preparing a pharmaceutical composition for trea- ting colitis comprising mixing human growth hormone, optio¬ nally in combination with a pharmaceutical acceptable carri¬ er, with excipients for lyophilizing and lyophilizing the resulting mixture, said human growth hormone being pure so as to yield a single major band on a polyacryl amide gel.

The invention may be worked using any growth hormone such as human growth hormone or an animal growth hormone such as bo¬ vine, porcine, equine, canine, avian or an ovine growth hor¬ mone. However, the growth hormone used is preferably the one corresponding to the species of the individual to be treated, e.g. human growth hormone for treating human beings etc. The administration of growth hormone according to the inven¬ tion may be carried out by subcutaneous, intravenous or in¬ tramuscular injection or by intravenous infusion of a solu¬ tion comprising the growth hormone. It is also considered as being within the scope of the invention to carry out the ad¬ ministration by any other method such as nasal, buccal, or pulmonary administration, in the form of an implant having a sustained release of the growth hormone or by implanting an osmotic pump delivering a solution comprising growth hormone over, a span of time.

The dosis may vary from 0.1 to 4 mg GH/kg bw per day pre¬ ferably from 1 to 3 mg GH/kg bw per day, more preferred about 2 mg GH/kg bw per day which is preferably given in di¬ vided doses two times a day

The administration of growth hormone according to the inven¬ tion may be carried out for a period of from 1 day to three weeks, preferably from 4 to 14 days and most preferred from 7 to 10 days.

The total dosis given to a human being for a period of 14 days may vary from 50 to 150 IU growth hormone.

The physician supervising the administration will decide on the most appropriate dose regimen which may be repeated periods of administration alternating with periods without administration of a long period of "continous" treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is explained below with reference to the dra¬ wings, in which Fig. 1 shows rating of the macroscopical damage according to a scale from 1 to 10, and

Fig. 2 shows the MPO Activity in TNB-treated rats having bhGH, or NaCl and untreated rats, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The invention is explained more in detail in the below Exam¬ ple illustrating the invention. The examples presents embodi¬ ments of the invention but are, however, not to be considered as limiting the scope of the invention being defined by the appending claims.

EXAMPLE

Animals

Three-month-old male ister rats (Møllegaard, LI. Skensved, Denmark) 217-260 g b.w. were used. The animals were kept three in each cage in a room having constant temperature of 21°C and a 12 h light/dark cycle and acclimated for a minimum of three days before start of the experiment. They had access to standard rat pellet food (Altromin diet 1324, Chr. Peter- sen Ltd., Slagelse, Denmark) and tap water ad libitum. The body Weight of each rat and food intake for each cage were recorded daily.

Induction of colonic inflammation

The rats were anaesthetized (pentobarbital, 50 mg per kg body weight IP) and a 4-cm long midline laparotomy was performed. Fecal contents of left colon were gently milked out and a rubber katheter (OD=3mm) having marks at the outside, visible through the bowel wall, was inserted via anus into the left colon. The catheter was placed at a distance of 9 cm from the anus and the site of the catheter tip was marked by one anti- mesenteric suture of 6-0 polypropylene (Prolene R, Ethicon Ltd.) to ensure exact and identical localisation of the sec¬ tions later sampled from left colon for further analyses. The catheter was then withdrawn 1 cm, and in 48 rats 30 mg 2,4,6- trinitrobenzene sulfonic acid (TNB) (Fluka Chemie AG, Buchs, Switzerland) dissolved in 50% ethanol (vol/vol) in a total volume of 0.25 ml per rat was instilled into the lumen of the colon (Gastroenterology (1989), 96, 795-803. The instillation procedure required about 5 sec to complete. Isotonic NaCl (0.154 mol/1) was instilled in 18 rats and these sham-instil- led rats were used as reference to intact colon. The sham instilled rats received no treatment and were killed either on day 0 immediately after skin closure (6 rats) , after 4 days (6 rats) , or after 7 days (6 rats) . The abdominal inci¬ sion were closed standarized by a continuous suture of 4-0 monofilament nylon (Dermalon R, Davis & Geek) in muscle and metallic agraffs in skin.

Treatment

Immidiately after skin closure the TNB-instilled rats were randomised into 4 groups of 12 rats. 50% of the rats were used as controls and injected with isotonic NaCl (0.154 mol/1) and the remaining 50% received 1.0 mg biosynthetic human growth hormone (bhGH; Norditropin^®, Novo Nordisk A/S, Gentofte, Denmark; specific activity: 1 mg=3 IU) per kg body weight twice a day (one daily injection between 8.00-9.00 a.m. and the second between 3.00-4.00 p.m.). All injections were given subcutaneously in the nape of the neck and the administration was continued until death at day 4 og day 7.

Macroscopical damage score and collecting of specimens for further analyses After 4 or 7 days the rats were anaesthetized with pentobar¬ bital and the abdominal cavity was reopened through the pre¬ vious incision. After exsanguination the colon was dissected from the peritoneal reflection to the ileo-coecal junction and opened longitudinally along the mesenteric border and gently rinsed in Ringer's solution (pH=7.4, 20°C) . Each colon was pinned out on a cork plate and photographed. Considering the area and the presence or absence of ulcers, the macrosco¬ pic damage was scored on a 1-10 scale (see Am. J. Physiol. (1990), 258, G527-G534, the below Table) on a coded dias of each colon to avoid observers bias. Colon was divided at the suture and the section from the suture to theperitoneal re¬ flection was weighed and cut distal to the suture by a multi- bladed cutting instrument with parallel razor blades (5 mm spacing) .

The macroscopical colonic damage score of the TNB-instilled bhGH rats (•) was significantly lower on day 4 than the score of the TNB-indstilled control rats (Δ) (p<0.005) (Figure 1), whereas no difference between the groups was found 7 days after the TNB-instillation. Sham-instilled rats are marked with (A) . Histological assessment as described below of the samples taken from rats killed 4 days after TNB-instillation confirmed the macroscopic scoring, since the microscopical score on this day of TNB-instilled control rats was 21% hig¬ her (mean score = 4.6) than TNB-instilled bhGH rats (mean score = 3.8) (p<0.05). On day 7 there was no difference bet¬ ween the two TNB-instilled groups (mean score of controls = 3.5, mean score of bGH rats = 3.8).

Myeloperoxidase (MPO)

The segment 1 cm distal to the suture was used for determina- tion of the MPO activity. For analysis in duplicate the seg¬ ment was parted into two samples of approximately 75 mg each, weighed, frozen immediately and stored at ÷20°C. The MPO ac¬ tivity was determined within 10 days as the rate of decompo¬ sition of hydrogen peroxide using o-dianisidine as hydrogen donor, determined by measuring the rate of color development by a spectophotometer (Bechman DU-2) at 460 ran, using the method described by Bradley et al. (J. Invest. Dermatol (1980), 78, 206-209). One unit of MPO activity was defined as that converting 1 μmol of hydrogen peroxide to water per mi¬ nute at 25°C.

Fig. 2 shows that the TNB-instillation resulted in a markedly increased colonic MPO activity after 4 and 7 says compared to colon from sham-instilled intact colon. The MPO activity of colons from the TNB-instilled bhGH-treated rats was 41% less than that of the TNB-instilled control rats at day 4 (p<0.005).

Assessment of microscopical damage score 2 cm distal to the suture two specimens were collected for light microscopy, placed on filter paper, the mucosa oriented upwards and fixed immediately in a 4% formaldehyde solution buffered by a 0.15 M phosphate buffer (pH=7.4). The segments were embedded in paraffine capsules by standarized histologi- cal procedures and 5 μm thick sections were cut perpendicular to the longitudinal axis of the colon and stained with hema- toxylin and eosin (H&E) and van Gieson. Microscopical evalua¬ tion was performed at a magnification of x60 from coded sec¬ tions to avoid observers bias, and the colonic ulceration and inflammation was scored as above.

Criteria for macroscopical and microscopical scoring of colo¬ nic ulceration and inflammation

Score Apperance

Macroscopical

0 Normal

1 Localized hyperemia, no ulcers

2 Ulceration withour hyperemia or bowel wall thickening

3 Ulceration with inflammation at 1 site

4 2 or more sites of ulceration and inflam¬ mation

5 Major sites of damage extending >1 cm along length of colon

6-10 Area of damage extending >2 cm along len¬ gth of colon, score was increased b 1 for each additional cm

Microscopical

0 Normal

1 Damage limited to surface epithelium

2 Focal ulceratin limited to mucosa

3 Focal, transmural inflammation, and ul¬ ceration

4 Extensive transmural ulceration and in¬ flammation bordered by normal mucosa

5 Extensive transmural ulceration and in¬ flammation involving entire section Colonic strength

Biomechanical test was performed on three specimens collected 3 to 4,5 cm distal to the suture. Specimens were transported to the test laboratory i Ringer's solution (pH=7.4, 20°C) and placed between two horizontal clamps in a testing machine (Alwetron TCT 5, Lorenzen & Wettre AB, Stockholm, Sweden) as disclosed in Scand. J. Gastroenterol. (1992), 25, 1137-1143.

The distance between the clamps (jaw-space) was 4 mm. The specimens were kept moistened by Ringer's solution during the test.

Each specimen was stretched at a constant deformation rate of 20 mnrmin^÷1 until rupture, stress and strain values were re¬ corded continuously on an X-Y recorder (Philips PM8272) and stress-strain curves were read into a computer by a digitizer and transformed into stress-strain curves, recording the load value for each strain increment of 1%. Breaking strength was defined as the maximum load required to rupture the speci¬ mens. Extensibility was the deformation at maximun load divi¬ ded by the starting length of the specimen. The starting len- gth of specimen was defined as the sum of jaw-space and the deformation until a preselected load of 0.05 newton was ob¬ tained. Energy absorption of the specimens was determined by integrating the area between^'the curve and the strain axis from the starting point to the point of rupture. Maximum stiffness of the tissue was measured as the maximum slope of the stress-strain curve (= tan β) .

Biomechanical parameters of the colonic specimens at day 4 showed that the breaking strength of the TNB-instilled con¬ trol rats was decreased by 43% compared to intact colon (p<0.005), whereas there was no difference in the breaking strength between bGH treated TNB-rats and intact colon on the same day. The breaking strength of the TNB-instilled control rats was reduced by 50% compared to TNB-instilled bGH rats (p<0.005) . Statistical analysis

Data are expressed as mean values ±SE for the groups. Dif¬ ferences between groups were analyzed by the Kruskal-Wallis test followed by the Mann-Whitney two-sample test. Statisti- cal variance, less than 5% were considered statistically sig¬ nificant.

Claims

1. A method of treating an inflammation in intestinal tissue in a mammal wherein growth hormone is administered to the mammal.

52. A method as claimed in claim 1 wherein the condition to be treated is colitis.

3. A method as claimed in claim 1 or 2 wherein the condition to be treated is Crohn's disease.

4. A method as claimed in any of claims 1 - 3 wherein the

10 mammal to be treated is a human being, and wherein the growth hormone is human growth hormone.

5. Use of a growth hormone for the preparation of a pharma¬ ceutical preparation for treating an inflammation in intesti¬ nal tissue.

156. Use as claimed in claim 5 for the preparation of a phar¬ maceutical preparation for treating colitis.

7. Use as claimed in claim 5 or 6 for the preparation of a pharmaceutical preparation for treating Crohn's disease.

8. Use of a growth hormone as claimed in any of claims 5 - 7 0 wherein human growth hormone is used for the preparation of a pharmaceutical preparation for treating Crohn's disease in a human.

9. A method for preparing a pharmaceutical composition for treating colitis comprising mixing growth hormone, optionally 5 in combination with a pharmaceutical acceptable carrier, with excipients for lyophilizing and lyophilizing the resulting mixture, said growth hormone being pure so as to yield a sin¬ gle major band an a polyacryl amide gel.

10. A method for normalizing the strength of intestinal tis¬ sue of a mammal suffering from colitis or Crohn's disease wherein growth hormone is administered to the mammal.