WO1997011721A1

WO1997011721A1 - Method to identify therapeutically active compounds

Info

Publication number: WO1997011721A1
Application number: PCT/SE1996/001183
Authority: WO
Inventors: Wubshet Mamo; Björn MELLGÅRD
Original assignee: Astra AB
Current assignee: AstraZeneca AB
Priority date: 1995-09-25
Filing date: 1996-09-23
Publication date: 1997-04-03
Anticipated expiration: 1998-03-25
Also published as: SE9503309D0; AU7102496A

Abstract

The present invention is related to a method of identifying therapeutic compounds and vaccines active against Helicobacter pylori infections comprising an in vivo animal model. The method is characterized by inoculation of an immunologically competent rodent animal selected from the group of NZB mice, DBA mice, KK mice or a rat with a H. pylori strain associated with gastritis in humans and thereafter to the infected animal administration of a test compound or a vaccine candidate and determination of the effectiveness of said compound or vaccine on the infection. The invention also relates to the use of said animal model in the development of diagnostic tests for such infections, as well as a New Zealand Black (NZB) mouse infected with human Helicobacter pylori strains.

Description

1996-09-16

METHOD TO IDENTIFY THERAPEUTICALLY ACTIVE COMPOUNDS

Field of the invention.

The present invention relates to a method for identification of compounds and vaccine candidates suitable for the therapeutic treatment of gastric disorders associated with Helicobacter infections. More particularly, the present invention relates to providing in vivo animal models useful in the screening and evaluation of prophylactic and therapeutic agents and vaccines for the treatment of gastritis, ulcer and other gastroduodenal diseases associated with Helicobacter pylon infections. The animal models provided by the present invention may also be useful in the development of diagnostic tests for such infections.

Background of the invention.

The relationship between gastroduodenal disorders and infections with Helicobacter pylori (H pylori ) is well established today. Helicobacter pylori was previously named Campylobacter pylori or Campylobacter pyloridis or just Campylobacter like organisms. In the following, the names H pylori , C pylori and C pyloridis are used interchangable. The above-mentioned relationship has been discussed by, for instance, Marchall et al, Microbios Lett. 25: 83 - 88 (1984). Marchall isolated C pyloridis from human gastric mucosa. Goodwin et al, J.Clin. Pathol. 39: 353 - 365 (1986) also suggested that gastroduodenal ulceration is associated with C pyloridis.

The mechanism by which H pylori invades and colonizes the stomach, its mode of action, such as persistence and role in gastroduodenal ulceration are important for the studies concerning the development of new therapies for the treatment of H pylori infections and related gastroduodenal diseases. At present there is no single compound therapy or treatment regimen which consistently provides eradication of said infections. Experimental work for studying H pylori related infections cannot be successfully investigated on human patients, due to ethical regulations.

There has been a long lasting need for animal models which can be used in investigations of H pylori -associated gastroduodenal diseases. So far, only a few animal species such as monkeys and baboons (See e g Euler et al, J. Clin. Microbiol. 28:2285 - 2290 (1990)) and gnotobiotic pigs (See e g Krakowka et al, Reviews of Inf. Diseases 13:681 - 685 (1991)) have been found to be susceptible to infection by H pylori and have been used as models for H pylori associated human gastroduodenal diseases. However, these rather large animals, particularly monkeys are expensive and technically difficult to handle.

Thus, there has been a long standing interest to establish a small animal model such as a rodent model. Rodents, and particularly mice, are easy to handle, inexpensive and easily maintained when used for biomedical research. Moreover, in a pharmaceutical application it is of importance that small animals, such as mice, consume a lesser amount of test substances compared to larger laboratory animals, such as monkeys and pigs.

There are several earlier reports indicating that rodents cannot be infected by H pylori isolated from humans, see for instance Ehler et al Zbl. Bakt. Hyg. A. 268: 341 - 346 (1988). However, the reported investigations have mainly been based on limited inbred strains of mice. A thorough systematic search of a large number of strains of mice in infection trials with H pylori has not been carried out.

Thus, so far, there have been problems to successfully infect conventional, immuno-competent mice with H pylori strains isolated from humans. Prior art discloses only a few examples of small animal models such as immune-deficient nude mice infected with H pylori. (See for instance Karita et al, Am. J. Gastroenterol. 86: 1596 - 1603(1991)). However, nude mice are deficient in T-cell production and investigations on these mouse strains as models for gastritis are inappropriate, and are not relevant for analysis of immune response against potential therapeutic agents or vaccine candidates on test. Vaccines have become of increasing interest in the last several years for possible prophylactic as well as therapeutic treatment. The need for inexpensive small animal models for investigations of H pylori has therefore increased in the recent years. In addition , the nude mice used hithereto are difficult to handle in experimental work, since these animals easily develop to many types of uncontrolled infections owing to their poor iirtmunological status.

Recently, Ghiara et al. (Science Vol. 267, 1655 - 1658 (1995)) have described a mouse model for studying the pathogenesis of H pylori infections, especially a model for testing candidate vaccines against H pylori. Specific pathogen-free CDI mice as well as conventional BALB/c and CDI mice were inoculated orally with different H pylori strains. Strains which had been isolated after a 2 weeks passage in mice were used in the subsequent studies, i e the isolated human H pylori strains were adapted to the animal model by repeated passages in mice animal. During these several passages in the mice the organism developed a capacity to infect the stomach of the mice. The reason for these repeated passages in the mice could be that strains of H pylori freshly isolated from humans hardly infect the mice.

It has now surprisingly been shown that the novel animal models described in the present application could be infected by freshly isolated H pylori strains which had not been passaged in the animal, i e had not been adapted to the specific animal. Outline of the invention.

Immunologically compentent rodents, and especially some specific mouse strains, have now surprisingly been found to be capable of developing infections by H pylori . Fresh isolates obtained directly from human gastric mucosa are used to establish H pylori infection. By the present invention the above mentioned disadvantages with the previously known animal models for H pylori infections are therefore avoided and there is provided new in vivo animal models which can be used in a method for identifying therapeutic drugs/ agents against H pylori infections. The new animal models can also be used in the development of vaccines against such infections and related diseases. Further, the animal models may be useful in the development of diagnostic tests for such infections.

In the animal models according to the present invention, especially in the inbred mouse strains, the H pylori infection is detectable 8 - 10 days following inoculation. Fresh isolates obtained from H pylori strains are used for inoculation. These fresh isolates are isolated from human patients. The isolates have not been passaged in animals, but they have in most instances been stored in a -70°C to minirnize laboratory passage (in vitro passage).

One specific immunocompetent rodent of interest for the present invention is New Zealand Black inbred mouse strain, shortly named NZB mice. Other inbred mouse strains which can be used in the method according to the present invention are the mouse strains named KK and DBA-1. KK and DBA-1 mouse strains have been previously used in biomedical research concerned with autoimmune diseases, see for instance, the International Index of Laboratory Animals, 6th edition, Michael F.W. Festing (1993).

Another rodent of interest for the present invention is an immunocompetent rat, such as Sprague-Dawley rats. The latter have been inoculated by bacteria reisolated from colonized mice, i.e. human isolates which have been passaged in mice.

In order to be useful as a screening model for H pylori, animals must be susceptible to infection with H pylori and especially with fresh isolates of H pylori obtained from humans. Moreover, to establish adequate H pylori infection in the animals, the immunological status of the specific animal and the virulence of the infecting organism are important factors.

As mentioned above, one preferred immunocompetent mouse strain which it is possible to infect with H pylori is the above mentioned NZB mouse strain. Such NZB mice were developed by Dr. Bielschowsky in 1970 as black-coated inbred mouse strains (Bieleshowsky et al. Cancer Res. 30: 834 ). It has to be noted that the NZB mice used according to the present invention are not genetically or immunologically transformed. The mice can be either of male or female sex. NZB mice are not previously known to have been infected by H pylori. They are hithereto used as standard animal for studies of the ethiology and pathogenisis of autoimmune diseases and therapeutic effect of immunosuppressive agents.

Several earlier studies have indicated that infection by H pylori , for instance, is dependent on the bacterial strains as well as the species of the animals. The results obtained in animal models suitable for the present invention, especially the mouse strains used in the experiment as well as the parameters used, confirmed colonization and infection of H pylori in the stomach of inoculated mice. Gastritis was evaluated according to the clinical condition of the infected animal and histopathological changes in the stomach tissues as well as induced antibody response were correlated with bacterial colonization. According to histopathological findings, infiltration of the lamina propia with polymorphonuclear leucocytes, monocytes, lymphocytes and plasma cells, alteration in the structure and integrity of the mucosal epithelial glands and gastric erosion were the main characteristics of infection.

Thus, the new animal models can be used in a method for identifying therapeutically active compounds for treatment of H pylori infections in mammals and man, wherein the method includes the following steps

a) inoculation of an immunologically compentent rodent animal with a H pylori strain associated with gastritis in humans,

b) establishing said infection in the animal,

c) administering to the animal a dosage form of a test compound suspected of having effect against H pylori infections, and

d) determining the effect of said test compound on the infection.

Immunologically compentent rodent of interest are inbred mouse strains, such as New Zealand Black mouse, DBA-1 mouse and KK mouse as well as rats.

Further the invention discloses a method for identifying biological probes clinically useful for detecting H pylori infections in mammals including humans. Such methods include for instance a biological indicator suspected of interacting selectively with H pylori or a substance interacting with a product released by the bacteria and identifying a selective positive interaction with such a released product for detecting H pylori infections in mammals.

The accompanied Figures and the following Examples support and illustrate the claimed invention. Brief description of the Figures.

Figure 1 shows the number of H pylori colony forming units, i e bacteria, recovered from the stomach biopsies of NZB mice 1 to 10 weeks post inoculation.

Figures 2 (a) and 2 (b) show the number of H pylori colony forming units, i e bacteria, recovered from the stomach biopsies of DBA-1 mice and KK mice respectively 1 to 5 weeks post inoculation.

Figure 3 shows that H pylori colonization occurs around the crypts in the lamina propia of infected mouse stomach.

Figure 4 shows that H pylori- specific antibodies bind to the H pylori colonizing the stomach, i e are present on the thin-sectioned samples of infected NZB mouse stomach, confirr ing the colonizing bacteria of H pylori, by fluorescence staining.

Figure 5 shows in 5(a) a scanning electromicroscope (SEM) picture showing the presence of H pylori in the mucus blanket of the gastric epithelium. 5(b) shows H pylori cultivated in vitro and sedimented on a filter paper, used for comparison.

Figure 6 shows the results from therapeutic immunisation in H pylori infected mice. Mice infected were NZB and DBA, respectively.

Examples.

1. Material and methods.

1.1 Animals

6 - 7 weeks old mice of the inbred mouse strain NZB were used. The mice were of male or female sex. The mice were bred and maintained in specific pathogen-free conditions and were examined to ensure the absence of specific bacteria and common murine diseases. They were housed in conventional Mak III cages and kept in room temperature, 50 - 60% relative humidity and fresh air exchange in accordance with Swedish regulations on Laboratory Animal Care. The mice were fed with autoclaved commercial rodent diet and sterile drinking water ad libitum. In some instances the animals did not receive any food, i e only water, 24 hours before the inoculation of bacteria.

Prior to inoculation with bacteria some of the mice were given antibiotics per os for 3 days. Such optional antibiotic treatment of the animals was made to eliminate interference of normal flora competing with the experimentally inoculated H pylori. Doses of antibiotics were 40mg/ml Ampicillin; 200 mg/ml Nalidixan; 40 mg/ml metronidazole; 160 mg/ml Vancomycin; 200 mg/ml Trimethoprin and each mouse received 0.3 ml of antibiotics twice a day up to 4 hours before adrninistration of bacteria. Some of the mice were not pretreated with antibiotics before the inoculation with H pylori. Also, some of the mice were pretreated with an inhibitor of intragastric acid secretion to increase the gastric pH in the animal before inoculation with H pylori. For instance, such an inhibitor of intragastric acid secretion used in the experimental studies was omeprazole.

Mice from the inbred mouse strains DBA-1 and KK were also tested as animals for the in vivo model useful in the screening method according to the present invention. These mice from the mouse strains DBA-1 and KK were treated in the same way as the NZB mice. Also immunocompetent Sprague-Dawley rats were inoculated with H pylori.

1.2 Inoculation of animals.

In one study two different H pylori strains A-9 and A-18 obtained from the stomach of human patients with active gastritis were used for inoculation of mice,

9 male sex. The mice were inoculated per os with 0.2 ml of H pylori (6 x 10 bact/ml) in peptone water. As a control mice of the different species were inoculated with 0.2 ml peptone water. Bacterial colonization and infection were followed up to 5 weeks post inoculation for DBA-1 and KK mice, and up to 10 weeks post inoculation for NZB mice.

In another study the H pylori strain A-9 and another strain AH 69 isolated from the stomach of a human patient with duodenal ulcer were used for inoculation of NZB mice, female sex. The strain AH 69 was also used for infection of NZB and DBA mice. The therapeutic effect was evaluated in oral immunization experiment four weeks post infection.

In still another study Sprague-Dawley rats were inoculated by bacteria reisolated from mice colonized with H pylori. The colonized mice had been inoculated with the H pylori strain AH 69 isolated from a human patient with duodenal ulcer. Optionally the rats were pretreated with omeprazole to increase the intragastric pH before inoculation with the bacteria. 2. Results

2.1 Recovery and identification of H pylori from infected animals

Bacterial colonization and infection up to 10 weeks post inoculation for the group of male NZB mice are summarised in Figure 1 and up to 5 weeks post inoculation for KK and DBA-1 mice in Figures 2 (a) and 2 (b).

Further results show that H pylori strain A-9 was recovered from the pyloric- antrum region of the stomach of 8 out of 9 mice, two weeks post inoculation. Four weeks later H pylori could be recovered from all mice inoculated. An average number of 1 000 bacteria/ 25 mm ² stomach biopsies could be recovered. The biopsies were homogenized and were analysed for urease, catalase and oxidase production. All analysed biopsy homogenates showed a rapid positive urease, catalase and oxidase reaction. None of the biopsy homogenates from the control group showed positive reactions.

Bacterial colonies grown on agar plates after incubation of the biopsy homogenates for 36-96 hours and suspected to be H pylori were also assayed for urease, catalase and oxidase production. These colonies also showed positive reactions.

According to microscopic examination of wet mounts from the NZB mice, strain A-9 produced large proportions of spreading colonies and all were motile. The motility was rapid and directional.

In the study of female NZB mice the results are summarized in Table 1, below. Table 1. Infection of H pylori in female NZB mice.

Group Mice Bact.^» Pre *^•- Inocul Infect Infect CFU CFU

No Strain treatm weeks rate % antr corpus antr/co

1 9 AH69 - 10⁹x3d 4,5 33/66 50 1800

2 10 AH69 fasted 10⁹x2/d 4,5 0/40 0 500

3 10 AH69 antib 10⁹x3d 4,5 40/70 280 800

4 10 AH69 fasted 10⁹x2/d 4,5 90/100 1500 3800

5 10 A-9 fasted 10⁹x2/d 4,5 70/60 700 1000

6 20 A-9 antib 10⁹x2/d 4,5 55/75 900 1500

Remarks: * Groups 1, 2 and 3, the bacterial strains were grown on blood agar plates. Groups 4, 5 and 6, the strains were grown in liquid media.

** Pretreatment was by fasting, i.e. the animals did not receive food or the mice were pretreated with antibiotics. Inoculation was made during 3 days alternatively 1 day and 2 times. The infection rate was measured in antrum and corpus respectively. CFU stands for colony forming units/25 mm². The figures in the drawings are mean values.

The study of rats gave the following result. Eight to nine weeks after inoculation with AH 69, H pylori was found to be colonizing the gastric antrum of all inoculated rats. An average of 15000 colony forming units/ 25 mm ² of mucosa were obtained. Some of the rats were also colonized by H pylori in the gastric corpus with an average of 100 colony forming units/ 25 mm ². 2.2 Confirmation of infection

Bacteria isolated from the stomach of inoculated mice were assayed for DNA dependent discriminary ribotyping and quantitative PCR tests. The DNA pattern as well as PCR confirmed that those bacteria isolated from the stomach of the infected mice were identical to the inoculum H pylori strain A-9 or strain A-l 8 originally isolated from human gastritis patients.

H pylori adhesion to epithelial tissue is partly mediated via Lewis b (Le ) receptors.

Stomach tissues from NZB mice were screened for the presence of Le^b receptors and the results show the presence of such receptors. Adhesion is an important prerequisite for many bacteria that colonize mucosal surfaces and may be important for the induction of inflammation by H pylori. The detection of Le ⁵ receptors suggest that NZB mice are appropriate models for studying adhesion of H pylori to gastric mucosa which is considered as one of the pathogenic mechanisms of H pylori associated gastritis.

Results from imrnunohistochemistry studies show that H pylori - specific antibodies were bound to the tissue surface along the length of the gastric crypts in the stomach, see Figure 4. The white dots in the picture of Figure 4 indicate fluorescent antibodies.

These results were also confirmed by serum antibody response. Serum samples pooled from the blood of infected NZB mice showed a well detectable serum titer of antibodies against H pylori.

The infection of the animals (NZB mice) was further confirmed by electron microscopy, see Figures 5 (a) and 5 (b). Further Warthin-Starry Silver stained biopsy preparations showed that animals (NZB mice) inoculated with H pylon had been infected in the pyloric-antrum region, see Figure 3.

The results presented above show that colonization of H pylon in the stomach of the animals is strongly correlated with the development of gastritis. Therefore the new animal models are suitable for studying new therapies and vaccine candidates for treatment of gastric disorders associated with H pylon infections as discussed below.

2.3 Therapeutic immunization in H pylon infected mice.

NZB or DBA mice were infected with mouse passaged fresh isolates H pylon, strain AH69. Oral immunizations were started four weeks post infection and from the first immunization day (day 1), mice were subsequently immunized on day 15, 25 and 35. One group mice was dosed with vehicle including Cholera toxin (CT) 10 μg/ mouse/ dose, and one group with the combination of CT + Membrane proteins (Mp). There were ten mice in each group.

Four weeks after the final immunization, mice were sacrificed. Colony forming units (CFU) of H pylon was determined in specimens from antrum and corpus, and serum antibody titer was determined in ELISA against membrane proteins. The results are shown in Figure 6, where the values are expressed as geometric means for CFU and serum titer.

The effect of immunization, expressed as a reduction of CFU, is seen clear in the Mp+CT group compared to vehicle. Thus, this model is shown to be useful and of great value in the screening and development of a therapeutic vaccine against H pylon. Further, the antibody response present in the animals makes the new animal models useful for identifying biological probes clinically useful for detecting H pylori infections in mammals.

Claims

Claims.

1. A method of identifying therapeutic compounds, such as drugs and agents, useful for treatment of Helicobacter pylori infections in mammals, wherein the method includes the following steps:

- inoculation of an immunologically competent rodent animal selected from the group of mouse strains: New Zealand Black (NZB), DBA and KK mice, and rats with a H pylori strain associated with gastritis in humans,

- establishing said infection in the animal,

- administration to the animal a dosage form of a test compound suspected of having effect against H pylori infections, and

- determining the effect of said test compound on the infection.

2. A method according to claim 1, wherein the mouse strain is an inbred strain of New Zealand Black (NZB) mouse.

3. A method according to claim 1, wherein the mouse strain is an inbred strain of DBA mouse.

4. A method according to claim 1, wherein the mouse strain is an inbred strain of KK mouse.

5. A method according to claim 1, wherein the rodent animal is a rat.

6. A method for infecting an immunologically competent rodent animal selected from the group of mouse strains New Zealand Black, DBA and KK mice, and rats, with an isolate of H pylori of human origin, wherein the method includes the following steps:

(i) isolating H pylori from an human patient, and

(ii) administering a dose of the fresh isolate of H pylori to an immunologically competent rodent animal.

7. A method according to claim 6, wherein the method includes a further step:

(iii) isolating H pylori from the rodent animal and adminstering the isolate to a new immunologically competent rodent animal.

8. A method according to any of claims 1 or 6, wherein prior to administration of a H pylori strain to the animal, the animal is pretreated with an inhibitor of intragastric acid secretion to increase the intragastric pH of the animal stomach.

9. A method according to claim 8, wherein the inhibitor of intragastric acid secretion is a proton pump inhibitor.

10. A method according to claim 9, wherein proton pump inhibitor is omeprazole or a pharmaceutically acceptable salt thereof.

11. A method according to any of claims 1 or 6, wherein the H pylori strain is administred orally to the animal and the infection is assessed by a suitable test.

12. A method according to claim 11, wherein the test for assessment of infection in the animal is an immunohistological test.

13. A method according to any of claims 1 or 6, wherein the test compound to be tested is administred to the animal in an intravenous, subcutaneous or oral dosage form.

14. A method of identifying biological probes clinically useful for detecting

Helicobacter pylori infections in mammals wherein the method includes inoculation of an immunologically competent rodent model with a H pylori strain associated with human gastritis, allow the infection to develop, probing the infected mammal with a biological indicator suspected of interacting selectively with H pylori and identifying a selective positive interaction with H pylori with a clinically useful probe for detecting H pylori infections in mammals.

15. A method of identifying biological probes clinically useful for detecting Helicobacter pylori infections in mammals wherein the method includes inoculation of an iinmunologically competent rodent model with a H pylori strain associated with human gastritis, allow the infection to develop, probing the infected mammal with a substance suspected of interacting selectively with a product released during a H pylori infection and identifying a selective positive interaction with such a released product for detecting H pylori infections in mammals.

16. An inbred strain of NZB mouse infected with a Helicobacter pylori strain of human origin.