U.S. patent application number 13/319851 was filed with the patent office on 2012-05-10 for steatohepatitis-liver cancer model animal.
Invention is credited to Masato Fujii, Hiroyuki Yoneyama.
Application Number | 20120117671 13/319851 |
Document ID | / |
Family ID | 43528923 |
Filed Date | 2012-05-10 |
United States Patent
Application |
20120117671 |
Kind Code |
A1 |
Yoneyama; Hiroyuki ; et
al. |
May 10, 2012 |
STEATOHEPATITIS-LIVER CANCER MODEL ANIMAL
Abstract
Fatty liver was induced by administering agents for inducing
organ inflammation to experimental animals to evoke insulin
resistance and by rearing them with high-fat diets. As a result,
steatohepatitis was successfully induced in the animals. The
animals show pathological findings similar to those of humans. By
using these model animals, substances for treating or preventing
diseases can be efficiently screened and the efficacy of medicinal
substances can be effectively evaluated.
Inventors: |
Yoneyama; Hiroyuki; (Tokyo,
JP) ; Fujii; Masato; (Tokyo, JP) |
Family ID: |
43528923 |
Appl. No.: |
13/319851 |
Filed: |
July 31, 2009 |
PCT Filed: |
July 31, 2009 |
PCT NO: |
PCT/JP2009/063675 |
371 Date: |
January 30, 2012 |
Current U.S.
Class: |
800/9 ; 424/9.2;
435/29; 435/7.21; 514/23 |
Current CPC
Class: |
A01K 2227/10 20130101;
A01K 2207/25 20130101; A01K 67/027 20130101; A01K 2207/20 20130101;
A01K 2267/0331 20130101 |
Class at
Publication: |
800/9 ; 424/9.2;
514/23; 435/29; 435/7.21 |
International
Class: |
A01K 67/027 20060101
A01K067/027; G01N 33/566 20060101 G01N033/566; C12Q 1/02 20060101
C12Q001/02; A61K 49/00 20060101 A61K049/00; A61K 31/7008 20060101
A61K031/7008 |
Claims
1. A non-human animal model for steatohepatitis produced by
administering an agent for inducing organ inflammation.
2. The non-human animal of claim 1, wherein the steatohepatitis is
a non-alcoholic steatohepatitis.
3. A non-human animal model for diabetic disorder produced by
administering an agent for inducing organ inflammation.
4. The non-human animal of claim 1, wherein the agent for inducing
organ inflammation is an N-acetyl-.beta.-D-glucosaminidase
inhibitor.
5. The non-human animal of claim 1, which comprises the step of
inducing fatty liver by administering an agent for inducing organ
inflammation to the animal and rearing the animal with a high-fat
diet.
6. The non-human animal of claim 1, wherein the non-human animal is
a mouse.
7. A method of producing a non-human animal model of
steatohepatitis, which comprises the step of inducing inflammation
in an organ of the non-human animal.
8. A method of screening for a substance for treating or preventing
steatohepatitis, which comprises the steps of: (a) administering a
test substance to the non-human animal model of steatohepatitis of
claim 1; and (b) evaluating an ameliorating effect on
steatohepatitis.
9. A method of evaluating a medicinal substance for efficacy
against steatohepatitis amelioration, which comprises the steps of:
(a) administering a test medicinal substance to the non-human
animal model of steatohepatitis of claim 1; and (b) evaluating an
ameliorating effect on steatohepatitis.
10. A method of screening for a substance for treating or
preventing a diabetic disorder, which comprises the steps of: (a)
administering a test substance to the non-human animal model for a
diabetic disorder of claim 3; and (b) evaluating an ameliorative
effect on diabetic disorder.
11. A method of evaluating the side effects risks of a
pharmaceutical agent for treating or preventing a diabetic
disorder, which comprises the steps of: (a) administering a test
pharmaceutical agent to the non-human animal model for a diabetic
disorder of claim 3; and (b) evaluating the pharmaceutical agent
for treating or preventing diabetic disorder for side effects.
12. A non-human animal model for liver cancer, which is produced by
further rearing the non-human animal of claim 1.
13. The non-human animal of claim 12, which is structurally
characterized by the following pathological morphology: (a) massive
type cord-like liver cell carcinoma; (b) infiltration of
inflammatory cells; or (c) liver cancer caused by cirrhosis
developed such that it displaces normal liver cells.
14. A method of screening for a substance for treating or
preventing liver cancer, which comprises the steps of: (a)
administering a test substance to the non-human animal model for
liver cancer of claim 12; and (b) evaluating a therapeutic effect
on liver cancer.
15. A method of evaluating a medicinal substance for efficacy
against liver cancer treatment, which comprises the steps of: (a)
administering a test medicinal substance to the non-human animal
model of liver cancer of claim 12; and (b) evaluating a therapeutic
effect on liver cancer.
16. The non-human animal of claim 3, wherein the agent for inducing
organ inflammation is an N-acetyl-.beta.-D-glucosaminidase
inhibitor.
17. The non-human animal of claim 3, which comprises the step of
inducing fatty liver by administering an agent for inducing organ
inflammation to the animal and rearing the animal with a high-fat
diet.
18. The non-human animal of claim 3, wherein the non-human animal
is a mouse.
19. A non-human animal model for liver cancer, which is produced by
further rearing the non-human animal of claim 3.
20. The non-human animal of claim 19, which is structurally
characterized by the following pathological morphology; (a) massive
type cord-like liver cell carcinoma; (b) infiltration of
inflammatory cells; or (c) liver cancer caused by cirrhosis
developed such that it displaces normal liver cells.
21. A method of screening for a substance for treating or
preventing liver cancer, which comprises the steps of: (a)
administering a test substance to the non-human animal model for
liver cancer of claim 19; and (b) evaluating a therapeutic effect
on liver cancer.
22. A method of evaluating a medicinal substance for efficacy
against liver cancer treatment, which comprises the steps of: (a)
administering a test medicinal substance to the non-human model of
liver cancer of claim 19; and (b) evaluating a therapeutic effect
on liver cancer.
Description
TECHNICAL FIELD
[0001] The present invention relates to steatohepatitis/liver
cancer model animals, and uses thereof.
BACKGROUND ART
[0002] Previously, non-alcoholic fatty liver disease was believed
to be a benign disease that does not progress. However, it was
revealed that even non-drinkers develop inflammation similar to
alcoholic hepatitis and show hepatic fibrosis histology, and now
the non-alcoholic fatty liver disease is known as a disease with
poor prognosis. In particular, metabolic syndromes due to obesity,
diabetes, or the like have been drawing attention in recent years.
It is becoming a common view that nonalcoholic steatohepatitis
(NASH) is one of such syndromes. However, the mechanism remains
unclear, and effective methods and/or agents for treating NASH have
not been established. This is partly because NASH is due to human
lifestyle-related diseases, and thus appropriate experimental
animals have not been established.
[0003] For the development of effective methods and agents for
treating NASH, it is essential to elucidate the pathological
condition of NASH, which progresses to lethal diseases such as
liver cirrhosis and liver cancer. However, experimental animals
that are currently used as a NASH model mouse in research include
single-gene modified mice such as leptin receptor-deficient mice
(Non-patent Document 1), hepatocyte-specific Pten-deficient mice
(Non-patent Document 2), and retinoic acid receptor a
dominant-negative transgenic mice (Non-patent Document 3), and mice
induced with a special diet such as a methionine/choline-deficient
diet (Non-patent Document 4). However, the human pathogenesis
differs from that in the genetically-modified mice in which a
single-gene mutation is responsible for the development and
progression of the pathological condition, and is unlikely to be
due to only the intake of a particular nutrient. Furthermore,
insulin resistance and hepatic fibrosis cannot be simultaneously
monitored in these mice. Furthermore, the ALT, an index in
serobiochemical analysis, is only slightly elevated in mice that
develop fibrosis, and therefore several mouse tissue slices are
required to assess their pathological condition. Conversely, the
pathological condition is assumed to be different from that of
human because ALT is elevated to a markedly high level.
Furthermore, the pathological condition recovers spontaneously
after treatment. This makes it difficult to test and assess drugs
for their efficacy. Thus, for developing methods and agents for
treating NASH, it is desirable to establish an experimental animal
model that is compatible with the human clinical condition.
[0004] Although various studies have been conducted, there is no
experimental animal exhibiting pathological conditions similar to
those of human. Thus, under the current circumstances, it is
difficult to conduct detailed screening to elucidate the
pathogenesis or to establish therapeutic methods.
PRIOR ART DOCUMENTS
[0005] [Non-patent Document 1] Sahai A et al., Am J Physiol
Gastroentest Liver Physiol 287: G1035, 2004 [0006] [Non-patent
Document 2] Horie Y et al., J Clin Invest 113: 1774, 2004 [0007]
[Non-patent Document 3] Yanagitani A et al., Hepatology 40: 366,
2004 [0008] [Non-patent Document 4] Rinella M et al., Journal of
Hepatology 40: 47, 2004
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0009] An objective of the present invention is to provide
steatohepatitis model animals and liver cancer model animals that
exhibit pathological findings similar to those of humans, and uses
thereof. More specifically, an objective of the present invention
is to provide techniques for producing model animals that develop
into fatty liver, steatohepatitis, hepatic fibrosis, liver
cirrhosis, and liver cancer from insulin resistance.
Means for Solving the Problems
[0010] The present inventors conducted dedicated studies to achieve
the above-described objectives. The present inventors induced
insulin resistance by administering an agent for inducing organ
inflammation, and induced fatty liver in mice by feeding them with
a high fat diet. As a result, the present inventors successfully
developed steatohepatitis in mice. A detailed observation of the
mice revealed the following pathological findings: [0011] (1)
macrovesicular fat deposition in liver cells and liver cell
ballooning; [0012] (2) infiltration of inflammatory cells; and
[0013] (3) fibrosis around mainly the central vein.
[0014] The above-described pathological findings are characteristic
of human nonalcoholic steatohepatitis (NASH). Specifically, by
using the above-described method, the present inventors for the
first time successfully produced mice that exhibit similar
pathological findings as those of human NASH.
[0015] The NASH animal model produced by the present inventors is
different from conventional animal models in the following points:
[0016] (1) similarly to that observed in the histophathological
findings of human, fatty degeneration and fibrosis of liver cells
progress mainly around the central vein instead of the portal vein;
and [0017] (2) the histophathological images of human "burned-out
NASH" are observed, in which only hepatic fibrosis is seen,
although fat deposition and loss of inflammatory cells are observed
as the pathological condition progresses.
[0018] The animal model of the present invention is characteristic
in that it is produced without genetic modification. Furthermore,
the model animals of the present invention unfailingly (100%)
develop a pathological condition similar to the progression and
prognosis of human NASH at a constant time course, and are the
first model animals that exhibit the same course of progression of
pathological condition in human. In addition, the model animals of
the present invention have a remarkably beneficial effect in that
all of insulin resistance, fatty liver, steatohepatitis, hepatic
fibrosis, and liver cirrhosis can be observed at the same time.
[0019] Moreover, the present inventors newly found that the
above-described NASH model animals develop liver cancer following
liver cirrhosis as they continue the rearing. This is the same
change of pathological condition observed in humans. Thus, the
animals prepared by the methods of the present invention are very
useful as model animals of human liver cancer.
[0020] Human liver cancer causes the liver surface to bulge.
However, conventional model animals for liver cancer, which are
prepared by administering a chemical substance, develop liver
cancer that does not cause bulging of the liver surface. Meanwhile,
model animals of the present invention, which are prepared without
genetic modification and drug administration, exhibit bulging of
the liver surface in a fashion similar to the case of human liver
cancer. Thus, the model of the present invention is a model much
closer to human liver cancer. Furthermore, liver cirrhosis is not
developed by administering chemical substances, while model animals
of the present invention develop massive type cord-like liver cell
carcinoma. Furthermore, infiltration of inflammatory cells and
development of liver cancer caused by cirrhosis to displace normal
liver cells are observed in the model animals of the present
invention. In the model, the origin of liver cancer is
macrovesicular fatty liver which shows a pathological condition
very similar to that of human NASH. The liver cancer develops from
hepatic fibrosis and liver cirrhosis. Thus, the animal model of the
present invention is very useful, as it has not been reported
previously.
[0021] As described above, the present inventors successfully
produced model animals for steatohepatitis and liver cancer which
show similar pathological findings to those of human, and thereby
completed the present invention. By using these model animals, it
is possible to efficiently screen for substances for treating or
preventing diseases, and effectively evaluate the efficacy of
medicinal substances.
[0022] The present invention relates to model animals which develop
into fatty liver, steatohepatitis, hepatic fibrosis, cirrhosis, and
liver cancer from insulin resistance, and more specifically, the
present invention provides: [0023] [1] A non-human animal model for
steatohepatitis produced by administering an agent for inducing
organ inflammation; [0024] [2] The non-human animal of [1], wherein
the steatohepatitis is a non-alcoholic steatohepatitis; [0025] [3]
a non-human animal model for diabetes produced by administering an
agent for inducing organ inflammation; [0026] [4] the non-human
animal of any one of [1] to [3], wherein the agent for inducing
organ inflammation is an N-acetyl-.beta.-D-glucosaminidase
inhibitor; [0027] [5] the non-human animal of any one of [1] to
[4], which comprises the step of inducing fatty liver by
administering an agent for inducing organ inflammation to the
animal and rearing the animal with a high-fat diet; [0028] [6] the
non-human animal of any one of [1] to [5], wherein the non-human
animal is a mouse; [0029] [7] a method of producing a non-human
animal model of steatohepatitis, which comprises the step of
inducing inflammation in an organ of the non-human animal; [0030]
[8] a method of screening for a substance for treating or
preventing steatohepatitis, which comprises the steps of: [0031]
(a) administering a test substance to the non-human animal model of
steatohepatitis of [1]; and [0032] (b) evaluating an ameliorating
effect on steatohepatitis; [0033] [9] a method of evaluating a
medicinal substance for efficacy against steatohepatitis
amelioration, which comprises the steps of: [0034] (a)
administering a test medicinal substance to the non-human animal
model of steatohepatitis of [1]; and [0035] (b) evaluating an
ameliorating effect on steatohepatitis; [0036] [10] a method of
screening for a substance for treating or preventing a diabetic
disorder, which comprises the steps of: [0037] (a) administering a
test substance to the non-human animal model for a diabetic
disorder of [3]; and [0038] (b) evaluating an ameliorative effect
on diabetic disorder; [0039] [11] a method of evaluating the side
effects risks of a pharmaceutical agent for treating or preventing
a diabetic disorder, which comprises the steps of: [0040] (a)
administering a test pharmaceutical agent to the non-human animal
model for a diabetic disorder of [3]; and [0041] (b) evaluating the
pharmaceutical agent for treating or preventing diabetic disorder
for side effects; [0042] [12] a non-human animal model for liver
cancer, which is produced by further rearing the non-human animal
of any one of [1] to [6]; [0043] [13] the non-human animal of [12],
which is structurally characterized by the following pathological
morphology: [0044] (a) massive type cord-like liver cell carcinoma;
[0045] (b) infiltration of inflammatory cells; or [0046] (c) liver
cancer caused by cirrhosis developed such that it displaces normal
liver cells; [0047] [14] a method of screening for a substance for
treating or preventing liver cancer, which comprises the steps of:
[0048] (a) administering a test substance to the non-human animal
model for liver cancer of [12] or [13]; and [0049] (b) evaluating a
therapeutic effect on liver cancer; and [0050] [15] a method of
evaluating a medicinal substance for efficacy against liver cancer
treatment, which comprises the steps of: [0051] (a) administering a
test medicinal substance to the non-human animal model of liver
cancer of [12] or [13]; and [0052] (b) evaluating a therapeutic
effect on liver cancer.
Effects of the Invention
[0053] To produce experimental animals that develop pathological
conditions similar to those of humans, insulin resistance was
induced in mice, and fatty liver was induced by feeding them with a
high fat diet.
[0054] Mice of different ages were sacrificed and each organ,
mainly liver, was analyzed histopathologically (HE staining, fat
staining, immunostaining for macrophages and fibroblasts). NAFLD
Activity Score (NAS; reference: "Kleiner D E et al., Hepatology.
2005 June; 41(6): 1313-21") was calculated to assess the
pathological features in detail. Model animals of the present
invention can also be assessed for NASH by using the same NAFLD
Activity Score as for human. Thus, the model animals of the present
invention are very useful as NASH model animals.
[0055] Furthermore, serobiochemical tests were carried out using
FUJIFILM DRI-CHEM. Gene expression analysis was performed using
Real-Time RT-PCR (Takara).
[0056] As described above, the present inventors successfully
produced steatohepatitis model animals (for example, NASH model
animals) and liver cancer model animals that show pathological
findings similar to those of humans.
[0057] The present invention provides simple techniques for stably
producing animals that develop at an early stage a pathological
condition similar to human NASH, which leads to fatty liver,
steatohepatitis, hepatic fibrosis, and liver cirrhosis, followed by
spontaneous development of liver cancer as a result of progression
of the pathological condition, by inducing insulin resistance in
experimental animals such as mice, and loading them with a high fat
diet.
[0058] Furthermore, diabetic disorders (diabetic nephritis,
retinopathy, hyperlipidemia, and arteriosclerosis) can also be
simultaneously observed in the animals. Thus, the present invention
also provides techniques for producing experimental animals that
enable the pathological conditions of metabolic syndrome to be
observed at the same time.
[0059] Meanwhile, in ob/ob mice and db/db mice which are commonly
used as an NASH model, the pathological condition does not develop
uniformly with aging. It is necessary to monitor the pathological
condition of the mice to accurately assess the disease state of
NASH, which makes the experiments cumbersome and complicated.
Furthermore, the pathological lesions are not always irreversible.
This has made the efficacy assessment difficult (Horie Y et al., J
din Invest 113: 1774-1783, 2004; Yanagitani A et al., Hepatology
40: 366-375, 2004; Anstee Q M et al., Int J Exp Path 87: 1-16,
2006). Meanwhile, in the model animals of the present invention,
the period leading to the mature pathological condition is constant
and its progression is irreversible. Thus, the model animals of the
present invention can be used to solve the above-described
problems.
[0060] The model animals of the present invention can be used in
preclinical tests for various therapeutic agents, and are very
useful in developing agents and searching for therapeutic
targets.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] FIG. 1 shows a result of serobiochemical test in mice of the
present invention.
[0062] FIG. 2 shows in photographs a result of fat staining of
liver from a mouse of the present invention.
[0063] FIG. 3 shows in photographs a result of immunostaining of
liver from a mouse of the present invention.
[0064] FIG. 4 shows in photographs a result of HE staining of liver
from a 20-week-old mouse of the present invention.
[0065] FIG. 5 shows a result of HE staining of liver from C57BL/6J
in photographs and the NAFLD Activity Score in a graph.
[0066] FIG. 6 shows in photographs a result of F4/80 immunostaining
of pancreas and adipose tissue from C57BL/6J.
[0067] FIG. 7 shows in photographs a result of immunostaining of
F4/80 and ER-TR7 of 8-week-old BALB/c and C3H/HeN mice.
[0068] FIG. 8 shows photographs of liver cancer in 20-week-old
C3H/HeN.
[0069] FIG. 9 shows in photographs reproducible results of NASH
model mice reared with other high fat diets.
[0070] FIG. 10 shows in photographs and graphs results of
pharmacological tests using the NASH model mice. 1 shows the result
of histological analysis in photographs. 2 shows the result of gene
expression analysis in photographs.
[0071] FIG. 11 shows the onset of diabetic complications in
photographs. 1 shows diabetic nephropathy in photographs, while 2
shows diabetic retinopathy (immunostaining images of new blood
vessels; CD31) in photographs.
MODE FOR CARRYING OUT THE INVENTION
[0072] The present invention relates to steatohepatitis model
animals produced by inducing insulin resistance.
[0073] In a preferred embodiment, the present invention provides
steatohepatitis model animals (herein sometimes referred to as
"model animals of the present invention") prepared by administering
agents for inducing organ inflammation.
[0074] Model animals of the present invention are prepared by
administering to experimental animals agents for inducing organ
inflammation and then preferably feeding them with high fat
diets.
[0075] Animals to be used in the present invention are not
particularly limited, as long as they are commonly used as
experimental animals. Such animals typically include non-human
animals, preferably non-human vertebrates, more preferably
non-human mammals, and still more preferably rodents. Examples of
animals that can be used to prepare model animals of the present
invention specifically include mice, rats, rabbits, dogs, chickens,
and monkeys (such animals are sometimes also referred to simply as
"experimental animals").
[0076] The genetic background of the animals to be used to produce
model animals of the present invention is not particularly limited;
and it is possible to use animals with any genetic background. In
general, wild-type animals can be preferably used.
[0077] In the present invention, the agents for inducing organ
inflammation (herein sometimes also referred to simply as "agents")
are not particularly limited, as long as they have an activity of
directly or indirectly inducing inflammation in various organs.
Organs for inducing inflammation include, for example, pancreas,
adipose tissues, and muscle tissues. The agents for inducing organ
inflammation of the present invention also include agents that
directly or indirectly induce inflammation in the peripheral
tissues.
[0078] The agents for inducing organ inflammation of the present
invention preferably include N-acetyl-.beta.-D-glucosaminidase
inhibitors.
[0079] In a preferred embodiment, model animals of the present
invention are produced by administering
N-acetyl-.beta.-D-glucosaminidase inhibitors to the above-described
experimental animals. Such inhibitors include, for example,
streptozotocin and Pugnac.
[0080] Alternatively, nucleic acids having an activity of
inhibiting N-acetyl-.beta.-D-glucosaminidase can also be used as an
agent of the present invention. Specifically, such nucleic acids
include, for example, siRNAs that suppress the expression of the
O-G1cNAcase gene (GenBank accession No. NM.sub.--023799.3),
antisenses of the gene, and ribozymes that target the gene.
[0081] In producing the model animals of the present invention, the
dosage form of the agents for inducing organ inflammation is not
particularly limited. For example, the dosage form includes, for
example, subcutaneous administration (subcutaneous injection,
etc.), intravenous administration, oral administration, and
intraperitoneal administration.
[0082] The dose of the agent to be administered is not limited, and
is typically 50 to 500 .mu.g, preferably 100 to 300 .mu.g, and more
preferably 200 .mu.g when the agent is streptozotocin.
[0083] The timing for administering the agent is as follows. The
agent is typically administered one to five days after birth
(neonatal period, preferably one to five days old, and more
preferably two days old), and preferably two days after birth.
[0084] As described above, insulin resistance can be induced by
administering the agents. After administration of the agents as
described above, the animals are reared to produce model animals of
the present invention. In general, the animals are preferably fed
with a high fat diet. Such high fat diets include various general
animal diets that are commercially available.
[0085] The major ingredients of the above-described high fat diets
include, for example, crude fat, crude protein, crude fiber, crude
ash, nitrogen-free extract, and water. The high fat diets of the
present invention are not particularly limited; however, the
content of crude fat is 20% or more, and preferably 30% or more;
and the ratio of fat-derived calories to total calories is
typically 50% or more, and preferably 60% or more. Ingredients to
be formulated into the high fat diets include, for example,
powdered beef tallow, milk casein, powdered egg white, L-cystine,
safflower oil, crystalline cellulose, maltodextrin, lactose, and
sucrose. These substances are shown as an example of ingredients of
the high fat diets, and are not necessarily contained in the
diets.
[0086] The above-described high fat diets include, but are not
limited to, for example, those that have a higher content of crude
fat (for example, higher by about 30% or more) than normal diets.
An example of such high fat diets include those commercially
available as diets for laboratory animals, such as High Fat Diet32
(CLEA Japan Inc.) and D12492 (Research Diets).
[0087] For example, when the animals are mice, the above-described
high fat diet feeding begins typically at the age of 2 to 6 weeks,
preferably 3 to 5 weeks, and more preferably 4 weeks. In addition,
for example, in the case of mice, the amount of high fat diet given
each time is about 3 to 6 g. In general, mice are preferably fed
with the high fat diet for one week or more. Those skilled in the
art can appropriately regulate (adjust) the amount of high fat diet
depending on the type, size, weight, or such of the experimental
animals to be used. Fatty liver can be induced by feeding them with
a high fat diet. Thus, in a preferred embodiment, the present
invention provides steatohepatitis model animals, which comprise
the step of inducing fatty liver by administering to the animals
agents for inducing organ inflammation and by feeding them with a
high fat diet.
[0088] Animals produced by the above-described method develop
symptoms of steatohepatitis and are useful as steatohepatitis model
animals. The model animals of the present invention have the
characteristic of simultaneously developing pathological conditions
that are observed in animals prepared by the methods of the present
invention. It is preferred that the model animals simultaneously
develop insulin resistance and/or hepatic fibrosis. However, such
pathological conditions are not limited to these examples.
[0089] In a preferred embodiment, the model animals of the present
invention are characteristic in that the pathological conditions of
metabolic syndrome can be observed at the same time, since diabetic
disorders (diabetic nephritis, retinopathy, hyperlipidemia, and
arteriosclerosis) can be observed simultaneously in model animals
of the present invention.
[0090] Furthermore, since the model animals of the present
invention have the characteristic that pathological condition does
not recover spontaneously, the animals can be suitably used in
testing and assessing drug efficacy.
[0091] In a preferred embodiment of the model animals of the
present invention, the above-described steatohepatitis is
nonalcoholic hepatitis (NASH). Specifically, the present invention
provides nonalcoholic hepatitis (NASH) model animals, which are
prepared by administering agents for inducing organ inflammation.
The animals steadily develop in a constant time course pathological
conditions with similar progression and prognosis to those of human
NASH.
[0092] In a preferred embodiment, nonalcoholic hepatitis model
animals of the present invention have at least one (preferably,
all) of the following pathological findings: [0093] (1)
macrovesicular fat deposition in liver cells and liver cell
ballooning; [0094] (2) infiltration of inflammatory cells; and
[0095] (3) fibrosis around mainly the central vein.
[0096] Accordingly, in a preferred embodiment, model animals of the
present invention are structurally characterized by the
above-described pathological morphologies.
[0097] As the rearing continued, the above-described
steatohepatitis model animals of the present invention developed
liver cirrhosis which led to liver cancer. Thus, steatohepatitis
model animals of the present invention are also useful, for
example, as animals (starting materials) for producing liver
cirrhosis model animals or liver cancer model animals Specifically,
the present invention provides materials for preparing liver
cirrhosis or liver cancer model animals, which comprise
steatohepatitis model animals of the present invention.
[0098] Furthermore, methods for producing model animals of the
present invention as described above are also included in the
present invention. In a preferred embodiment, the present invention
provides methods for producing steatohepatitis model animals, which
comprise the step of administering agents for inducing organ
inflammation to non-human animals
[0099] Furthermore, substances for treating or preventing
steatohepatitis can be screened by using model animals of the
present invention. Specifically, the present invention provides
methods of screening for substances for treating or preventing
steatohepatitis, which comprise the steps of: [0100] (a)
administering a test substance to a steatohepatitis model animal of
the present invention; and [0101] (b) evaluating its ameliorating
effect on steatohepatitis.
[0102] Test substances to be used in these methods are not
particularly limited. For example, such substances include single
compounds such as natural compounds, organic compounds, inorganic
compounds, proteins, and peptides, as well as compound libraries,
expression products of gene libraries, cell extracts, cell culture
supernatants, products of fermenting microorganisms, extracts of
marine organisms, and plant extracts, but are not limited
thereto.
[0103] Methods for administering test substances or medicinal
substances of the present invention are not particularly limited;
however, they can be administered, for example, orally or by
injection. When such a test substance is a protein, for example, a
viral vector carrying a gene encoding the protein may be
constructed and can be introduced into model animals of the present
invention using their infectability.
[0104] In the step of (b), the ameliorating effect on
steatohepatitis can be evaluated by determining whether
steatohepatitis is ameliorated by assessing the pathological
findings of the model animals.
[0105] The pathological findings of steatohepatitis include, for
example, the above-described pathological findings (pathological
morphologies). Herein, "amelioration" means that the symptoms of
steatohepatitis are alleviated or restored to normal. By using as
an indicator the pathological findings described herein, those
skilled in the art can appropriately evaluate whether the symptoms
of steatohepatitis are ameliorated in the model animals.
[0106] In the present invention, substances that produce the
ameliorating effect in the step of (b) above can be selected as
substances for treating or preventing steatohepatitis.
[0107] Furthermore, medicinal substances can be assessed for their
efficacy in ameliorating steatohepatitis by using model animals of
the present invention. Specifically, the present invention provides
methods for evaluating the efficacy of medicinal substances in
ameliorating steatohepatitis, which comprise the steps of: [0108]
(a) administering a test medicinal substance to a steatohepatitis
model animal of the present invention; and [0109] (b) evaluating
its ameliorating effect on steatohepatitis.
[0110] The type of medicinal substances that can be evaluated for
efficacy by the above-described methods is not particularly
limited; and such medicinal substances include, for example,
various known pharmaceutical agents (low-molecular-weight
compounds, proteins, nucleic acids, or such).
[0111] When a test medicinal substance exerts an ameliorating
effect on steatohepatitis, the medicinal substance is judged to
have therapeutic effect on steatohepatitis.
[0112] Furthermore, model animals of the present invention are
characterized in showing diabetic disorders (diabetic nephritis,
retinopathy, or such) which develop as a complication of diabetes,
simultaneously in conjunction with steatohepatitis. Thus, model
animals of the present invention are useful as diabetes model
animals.
[0113] Specifically, the present invention provides diabetes model
non-human animals prepared by administering agents for inducing
organ inflammation. Agents for treating or preventing diabetic
disorders (diabetic nephritis, retinopathy, or such) can be
developed by using diabetes model animals of the present invention.
For example, candidate compounds for treating or preventing
diabetic disorders can be screened by administering test substances
to diabetes model animals of the present invention, and evaluating
their ameliorating effect on diabetic disorders.
[0114] In a preferred embodiment, the present invention provides
methods of screening for substance for treating or preventing
diabetic disorders, which comprise the steps of: [0115] (a)
administering a test substance to a non-human animal model of
diabetic disorder of the present invention; and [0116] (b)
evaluating its ameliorating effect on diabetic disorder.
[0117] Many NASH patients are also diabetes patients, and they are
thought to develop various complications. Model animals of the
present invention develop diabetic complications and are thus very
useful as model animals, because risks such as side effects
discovered at clinical trials can be evaluated at earlier stages by
using the model animals.
[0118] Specifically, the present invention provides methods for
evaluating the side effects risks of pharmaceutical agents by using
the diabetes model animals of the present invention.
[0119] In a preferred embodiment, the present invention relates to
methods for evaluating the risk of side effects of pharmaceutical
agents for treating or preventing diabetic disorders, which
comprise the steps of: [0120] (a) administering a test
pharmaceutical agent to the non-human animal model of diabetic
disorder of the present invention; and [0121] (b) evaluating the
side effects of the pharmaceutical agent for treating or preventing
diabetic disorder.
[0122] Furthermore, the present inventors for the first time
discovered that as the rearing continues, the above-described
steatohepatitis model animals of the present invention develop
liver cirrhosis which leads to liver cancer. Thus, the present
invention provides liver cirrhosis model animals and liver cancer
model animals which are prepared by continuing to rear the
above-described steatohepatitis model animals.
[0123] In a preferred embodiment, the present invention relates to
liver cancer model animals which are prepared by continuing the
rearing of steatohepatitis model animals prepared by administering
agents for inducing organ inflammation.
[0124] The above-described steatohepatitis model animals of the
present invention subsequently develop liver cirrhosis. Liver
cancer model animals can be produced by further rearing of the
animals. In producing such model animals, the period of rearing
after liver cirrhosis has developed is typically, for example, 2 to
20 weeks or more, and preferably 10 weeks or more, when the
experimental animal is mouse.
[0125] Liver cancer model animals of the present invention are
structurally characterized, for example, by at least one
(preferably, all) selected from the following pathological findings
(pathological morphologies): [0126] (a) massive type cord-like
liver cell carcinoma; [0127] (b) infiltration of inflammatory
cells; and [0128] (c) liver cancer caused by cirrhosis developed
such that it displaces normal liver cells.
[0129] Liver cancer model animals having the above-described
characteristics exhibit the pathological morphologies described
above, and thus are structurally different from conventional liver
cancer model animals prepared by administering chemical substances
(carcinogenic substances).
[0130] Substances for treating or preventing liver cancer can be
selected by using the above-described liver cancer model animals of
the present invention.
[0131] In a preferred embodiment, the above-described methods of
the present invention includes methods of screening for substances
for treating or preventing liver cancer, which comprise the steps
of: [0132] (a) administering a test substance to a liver cancer
model animal of the present invention; and [0133] (b) evaluating
its therapeutic effect on liver cancer.
[0134] In the above-described methods, the therapeutic effect can
be appropriately evaluated, for example, using as an indicator the
above-described pathological findings of liver cancer. For example,
when massive type cord-like liver cell carcinoma is eliminated in a
model animal of the present invention administered with a test
substance, the test substance is judged to have therapeutic effect
on liver cancer.
[0135] Furthermore, according to the present invention, medicinal
substances can be evaluated for their efficacy in liver cancer
treatment by using liver cancer model animals of the present
invention. In a preferred embodiment, the methods include, for
example, those comprising the steps of: [0136] (a) administering a
test medicinal substance to a liver cancer model animal of the
present invention; and [0137] (b) evaluating its therapeutic effect
on liver cancer.
[0138] All prior art documents cited herein are incorporated herein
by reference.
EXAMPLES
[0139] Hereinbelow, the present invention will be described more
specifically with reference to the Examples, but it is not limited
thereto.
Example 1
Preparation of NASH Model Animals and Liver Cancer Model
Animals
(a) Preparation of NASH Model Mice
[0140] Gestational C57BL6J/JJcl, C3H/HeNJcl, and BALB/cByJJcl (CLEA
Japan Inc.) and C57BL6J/NCrlCrlj (Charles River Japan, Inc.) were
reared and allowed to deliver. Pancreatic inflammation was induced
in male mice of C57BL6J/JcL, BALB/cByJJcl, and C3H/HeNJcl (CLEA
Japan Inc.) two days after birth with cytotoxicity specific to
N-Acetyl-beta-D-glucosaminidase (O-GlcNAcase) in pancreatic .beta.
cells (for example, by subcutaneously administering 10 mg/ml
streptozotocin (SIGMA) at 20 .mu.l/head). Thus, insulin resistance
was induced by directly or indirectly eliciting inflammation in
peripheral tissues. The mice were reared with a CE-2 diet (CLEA
Japan Inc.) and sterile water until four weeks old, and ablactated
when they reached the age of four weeks. Then, the mice were reared
until 20 weeks old with sterile water and High Fat Diet (CLEA Japan
Inc.) or D12492 (Research Diets), which have a higher crude fat
content (or by about 30% or more) than normal diet.
(b) Histological Assessment
[0141] After mice of different ages were fasted for 24 hours, they
were sacrificed under ether anesthesia, and blood was collected.
Each organ was frozen in OCT compound (Sakura Fine Technical), and
then sliced into sections for pathological analysis. A
serobiochemical test showed that the levels of fasting blood
glucose, alanine aminotransferase (ALT), and neutral fat were all
higher in this model as compared to the group of normal animals.
Thus, the model animals developed insulin resistance and
hyperlipemia (FIG. 1). Histologically, severe fatty liver with
liver cell ballooning was observed at the age of five weeks. Fat
was almost completely eliminated from the liver at the age of eight
weeks, and thus the progression of histopathological condition was
very similar to that of human burned-out NASH (FIG. 2). At the age
of six weeks, accumulation and infiltration of inflammatory cells
including macrophages was observed in their liver, and fibrosis
progressed around the central vein of liver. Result obtained by
further observation of changes over time showed that at the age of
eight weeks, central veins become connected as fibrosis progressed,
and liver cirrhosis with formation of regenerating nodules was
observed at the age of ten weeks (FIG. 3). Furthermore, NAS was
calculated based on the histopathological data. The result showed
that the score was 5 in average at the NASH stage. Thus, it was
demonstrated that the pharmacological effect could be evaluated by
monitoring changes of this score (FIG. 5).
[0142] The pathogenesis of NASH in this mouse model is that
pancreatic inflammation triggers chronic inflammation in peripheral
tissues such as liver and adipose tissues; insulin resistance
develops; and persistent systemic inflammation leads to fatty liver
(FIG. 6). Then, the regenerating nodules enlarged as the mice age.
Infiltration of inflammatory cells, increase of atypical
hepatocytes, and development of cancer to displace normal liver
cells were observed at the age of 20 weeks (FIG. 4). Moreover,
lesions that are histologically consistent with NASH lesion which
leads to liver cancer can also be developed in mice of other lines
(FIGS. 7 and 8). NASH lesions were reproducible even if the type of
high fat diet was changed (FIG. 9).
[0143] Furthermore, as a trial of NASH therapy, angiotensin
receptor antagonist (ARB), which is an antihypertensive agent, was
orally administered to the model of the present invention for two
weeks. Consistent with a clinical report (Georgescu E. F. et al.,
15: 942), comparison of the ARB-administered group and the
non-treated group showed histological improvement in the liver and
ameliorating effects on inflammation and fibrosis as detected by
genetic test, which suggested, the result is highly similar to
clinical results (FIG. 10).
[0144] Furthermore, in the mouse model of the present invention,
adipose tissue inflammation is also induced at the same time and
this potentiates insulin resistance. The resulting persistent
chronic hyperglycemia causes microangiopathy and such, leading to
diabetic complications (diabetic nephropathy, retinopathy, and
neuropathy). Then, pathological lesions in other organs were
studied in detail. As a result, glomerular and interstitial
fibrosis which are characterized by accumulation of inflammatory
macrophages and fibroblasts within and around renal glomeruli were
observed at the age of ten weeks, and thus the animals developed
diabetic nephropathy (FIG. 11). Furthermore, at the age of 20
weeks, neovascularization in the eyes was assessed using a CD31
antibody. In the model mice of the present invention, neovascular
blood hyperplasia was observed in the retina, suggesting that the
mice developed diabetic retinopathy (FIG. 11).
[0145] Thus, the present invention provides NASH model animals
which develop fatty liver leading to liver cancer. The use of such
model animals facilitates the analysis of pathogenesis and
pathological condition of human NASH, and development of techniques
and agents for treating human NASH.
INDUSTRIAL APPLICABILITY
[0146] The causes of fat accumulation in the liver include alcohol,
obesity, diabetes, lipid metabolism abnormality, pharmaceutical
agents, and severe malnutrition. However, the causes are roughly
categorized as alcoholic and nonalcoholic. Alcoholic fatty liver
leads to hepatitis, liver fibrosis, and liver cirrhosis. Meanwhile,
nonalcoholic fatty liver has been believed to be a pathological
condition that does not progress. However, in the late 1990s, as
the obese population increased and the disease concept became
known, it was revealed that nonalcoholic fatty liver is a
high-incidence disease next to type C hepatitis and alcoholic
hepatitis in Europe and the United States. The pathological
condition was reported to progress into liver cirrhosis and finally
liver cancer, which drew attention to the disease. In Japan, the
obese population with obesity is also steadily increasing due to
genetically predisposed low insulin secretion, and westernized
diets and lack of physical activity. Under such circumstances, the
number of patients diagnosed as NASH is increasing, and thus there
is an urgent demand for developing and establishing methods and
agents for treating NASH.
[0147] The model of the present invention is highly similar to the
pathological condition of human NASH in terms of disease
progression, and can be used to determine the stage for analyzing
the phases of insulin resistance, fatty liver, steatohepatitis,
liver fibrosis, and liver cirrhosis, depending on the subject to be
treated. Furthermore, by searching for pathogenic factors involved
in the progression of each phase, the present invention can also
contribute to the development of completely new methods or agents
for treating human NASH, hepatic fibrosis, and liver cirrhosis, as
well as biomarkers for the diseases. In addition, the present
invention is applicable to evaluate pharmacokinetics in NASH
lesions. Moreover, the model of the present invention finally leads
to liver cancer. Thus, the present invention enables the screening
for cancer-suppressing agents or such, investigation of onset
mechanism for liver cancer, and pharmaceuticals that target
molecules in development of liver cancer.
[0148] In addition to the diseases described above, diabetic
disorders can also be analyzed at the same time. Thus, the present
invention is expected to greatly contribute to the development of
methods and agents for liver diseases, elucidation of the
relationships among systemic pathological conditions in a subject
animal, as well as development of therapeutic methods/agents and
biomarkers.
* * * * *