U.S. patent application number 16/648744 was filed with the patent office on 2020-08-27 for methods and pharmaceutical compositions for modulating autophagy.
The applicant listed for this patent is ASSISTANCE PUBLIQUE-HOPITAUX DE PARIS (APHP), INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE), SORBONNE UNIVERSITE, UNIVERSITE DE PARIS. Invention is credited to Jose Manuel BRAVO-SAN, Guido KROEMER.
Application Number | 20200268837 16/648744 |
Document ID | / |
Family ID | 1000004829914 |
Filed Date | 2020-08-27 |
![](/patent/app/20200268837/US20200268837A1-20200827-D00001.png)
![](/patent/app/20200268837/US20200268837A1-20200827-D00002.png)
![](/patent/app/20200268837/US20200268837A1-20200827-D00003.png)
![](/patent/app/20200268837/US20200268837A1-20200827-D00004.png)
![](/patent/app/20200268837/US20200268837A1-20200827-D00005.png)
![](/patent/app/20200268837/US20200268837A1-20200827-D00006.png)
![](/patent/app/20200268837/US20200268837A1-20200827-D00007.png)
United States Patent
Application |
20200268837 |
Kind Code |
A1 |
KROEMER; Guido ; et
al. |
August 27, 2020 |
METHODS AND PHARMACEUTICAL COMPOSITIONS FOR MODULATING
AUTOPHAGY
Abstract
Autophagy is typically activated by starvation, allowing cells
and organisms to mobilize their energy reserves. It is known that
pharmacological modulation of autophagy represents a therapeutic
potential. Here the inventors report that a protein that is
released from cells in an unconventional, autophagy-dependent
manner, namely, diazepam binding inhibitor (DBI), regulates
autophagy. In particular, the inventors demonstrate that DBI
inhibits autophagy and that the supply of recombinant DBI to mice
enhanced glycolysis, enhanced lipogenesis, and inhibited fatty acid
oxidation. The inventors show that neutralisation of DBI by a
monoclonal antibody and an active immunization by means of an
immunogenic DBI derivative eliciting autoantibodies induce
autophagy and lead to metabolic changes that increase
starvation-induced weight loss, reduce food intake upon refeeding,
and reduce weight gain in response to hypercaloric diets.
Accordingly, the present invention relates to methods and
pharmaceutical compositions for modulating autophagy based on the
modulation of the activity or expression of DBI.
Inventors: |
KROEMER; Guido; (Paris cedex
06, FR) ; BRAVO-SAN; Jose Manuel; (Paris cedex 06,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE
MEDICALE)
ASSISTANCE PUBLIQUE-HOPITAUX DE PARIS (APHP)
SORBONNE UNIVERSITE
UNIVERSITE DE PARIS |
Paris
Paris
Paris
Paris |
|
FR
FR
FR
FR |
|
|
Family ID: |
1000004829914 |
Appl. No.: |
16/648744 |
Filed: |
September 19, 2018 |
PCT Filed: |
September 19, 2018 |
PCT NO: |
PCT/EP2018/075286 |
371 Date: |
March 19, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/24 20130101;
C07K 16/18 20130101; C07K 2317/21 20130101; G01N 33/68 20130101;
A61K 39/0005 20130101; A61K 39/39 20130101; A61K 38/17
20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C07K 16/18 20060101 C07K016/18; A61K 39/00 20060101
A61K039/00; A61K 39/39 20060101 A61K039/39; G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 20, 2017 |
EP |
173062293.0 |
Claims
1. A method of inhibiting autophagy in a subject in need thereof
comprising administering to the subject a therapeutically effective
amount of an agent that promotes the activity or expression of DBI
diazepam binding protein (DBI).
2. The method of claim 1 wherein the subject is underweight.
3. The method of claim 2 wherein the subject suffers from a wasting
disorder.
4. The method of claim 2 wherein the subject suffers from anorexia
cachexia, anorexia of the aged, anorexia nervosa, cachexia
associated with cancer, cachexia associated with AIDS, cachexia
associated with heart failure, cachexia associated with cystic
fibrosis, cachexia associated with rheumatoid arthritis, cachexia
associated with kidney disease, cachexia associated with chronic
obstructive pulmonary disease (COPD), cachexia associated with ALS,
cachexia associated with renal failure, cachexia associated with
aberrant appetite, cachexia associated with fat mass, cachexia
associated with energy balance, and/or cachexia associated with
involuntary weight loss.
5. The method of claim 1 wherein the subject suffers from a disease
selected from the group consisting of cancer diseases,
neurodegenerative diseases, cardiovascular diseases, infectious
diseases, auto-immune diseases and/or inflammatory diseases.
6. The method of claim 1 wherein the agent that promotes the
activity of DBI comprises a polypeptide comprising i) an amino acid
sequence having at least 80% of identity with SEQ ID NO:1, or ii)
an amino acid sequence having at least 80% of identity with the
amino acid sequence ranging from the amino acid residue at position
17 to the amino acid residue at position 50 in SEQ ID NO:1, or iii)
an amino acid sequence having at least 80% of identity with the
amino acid sequence ranging from the amino acid residue at position
33 to the amino acid residue at position 50 in SEQ ID NO:1, or iv)
an amino acid sequence having at least 80% identity with the amino
acid sequence ranging from the amino acid residue at position 43 to
the amino acid residue at position 50 in SEQ ID NO:1.
7. The method of claim 1 wherein the agent that promotes the
expression of DBI is a nucleic acid molecule that encodes the
polypeptide of claim 6.
8. The method of claim 7 wherein the nucleic acid molecule
comprises a nucleic acid sequence having at least 50% identity with
SEQ ID NO: 2.
9. The method of claim 1 wherein the agent that promotes the
activity of DBI is a small organic molecule or peptidomimetic that
mimics the activity of DBI.
10. A method of stimulating autophagy in a subject in need thereof
comprising administering to the subject a therapeutically effective
amount of an agent that inhibits the activity or expression of
DBI.
11. The method of claim 10 wherein the subject is overweight.
12. The method of claim 11 wherein the subject suffers from
obesity.
13. The method of claim 10 wherein the subject suffers from type 2
diabetes or metabolic syndrome.
14. The method of claim 10 wherein the subject suffers from cancer,
neurodegenerative disease, infectious disease, pulmonary disease,
cystic fibrosis, liver disease, pancreatitis, or a
proteinopathy.
15. The method of claim 14 wherein the subject suffers from cancer
and the method further comprises, after the step of administering,
a step of administering to the subject a therapeutically effective
amount of a chemotherapeutic agent.
16. The method of claim 10 wherein the agent that inhibits the
activity of DBI is an antibody or an aptamer directed against
DBI.
17. The method of claim 16 wherein the antibody is directed against
the peptide fragment ranging from the amino acid residue at
position 43 to the amino acid residue at position 50 of DBI.
18. The method of claim 16 wherein the antibody is a monoclonal
chimeric antibody, a monoclonal humanised antibody, or a monoclonal
human antibody.
19. The method of claim 10 wherein the agent that inhibits the
expression of DBI is siRNA, an endonuclease, an antisense
oligonucleotide or a ribozyme.
20. The method of claim 10 wherein the agent that inhibits the
activity of DBI is a vaccine composition that elicits neutralizing
autoantibodies against DBI when administered to the subject.
21. The method of claim 20 wherein the vaccine composition
comprises an antigen comprising a polypeptide comprising i) an
amino acid sequence having at least 80% of identity with SEQ ID
NO:1, or ii) an amino acid sequence having at least 80% of identity
with the amino acid sequence ranging from the amino acid residue at
position 17 to the amino acid residue at position 50 in SEQ ID
NO:1, or iii) an amino acid sequence having at least 80% of
identity with the amino acid sequence ranging from the amino acid
residue at position 33 to the amino acid residue at position 50 in
SEQ ID NO:1, or iv) an amino acid sequence having at least 80% of
identity with the amino acid sequence ranging from the amino acid
residue at position 43 to the amino acid residue at position 50 in
SEQ ID NO:1.
22. The method of claim 21 wherein the polypeptide is conjugated to
a carrier protein.
23. The method of claim 20 wherein the vaccine composition
comprises an adjuvant.
24. A method of screening a compound suitable for modulating
autophagy comprising i) providing a candidate compound ii)
determining whether the candidate compound is capable of modulating
the activity or expression of DBI and iii) positively selecting the
candidate compound which is capable of modulating the activity or
expression of DBI.
25. A method of determining whether a subject is at risk of weight
modulation comprising i) determining the level of DBI in a blood
sample obtained from the subject, ii) comparing the level
determined at step i) with a predetermined reference value and when
a differential between the level determined at step i) and the
predetermined reference value is determined, administering to the
subject a therapeutically effective amount of an agent that
modulates the activity or expression of DBI.
26. A method of treating a non-alcoholic fatty liver disease
(NAFLD) in a subject in need thereof comprising administering to
the subject a therapeutically effective amount of an agent that
inhibits the activity or expression of DBI.
27. The method of claim 26 wherein the NAFLD is nonalcoholic
steatohepatitis (NASH).
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods and pharmaceutical
compositions for modulating autophagy.
BACKGROUND OF THE INVENTION
[0002] Autophagy ("self-eating") constitutes one of the most
spectacular, though subtly regulated phenomena in cell biology and
plays a key role in the maintenance of cellular and organismal
homeostasis by facilitating the turnover of cytoplasmic structures
and allowing cells to adapt to changing and stressful conditions
including nutrient deprivation (1, 2). The cellular secretion of
several leaderless proteins (which can only be released through an
unconventional pathway bypassing Golgi) is strongly associated with
autophagy (3-7). One such protein is a phylogenetically ancient
factor known as diazepam binding protein (DBI) or acyl coenzyme A
(CoA)-binding protein (ACBP) (3, 4). Human or mouse DBI is a small
protein of 87 amino acids (10 kDa) that has two totally distinct
functions, namely as ACBP within cells (where it binds to
long-chain acyl CoA molecules) and as DBI outside cells (where the
entire protein or its cleavage products, triacontatetraneuropeptide
[TTN, residues 17-50] and octadecaneuroptide [ODN, residues 33-50],
can interact with the benzodiazepine binding site of the
gamma-aminobutyric acid type A receptor, GABAAR, and modulate its
activity as a GTP protein-coupled receptor, GPCR) (8-10). DBI and
its proteolytic fragments also bind to the peripheral-type
benzodiazepine receptor (PBR) (11-13), and a still unidentified
GPCR (ODN-GPCR) (14-17). However, the role of DBI secretion in the
feedback regulation of autophagy has never been investigated.
SUMMARY OF THE INVENTION
[0003] The present invention relates to methods and pharmaceutical
compositions for modulating autophagy. In particular, the present
invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION
[0004] Autophagy is typically activated by starvation, allowing
cells and organisms to mobilize their energy reserves. Here the
inventors report that a protein that is released from cells in an
unconventional, autophagy-dependent manner, namely, diazepam
binding inhibitor (DBI), also known as acyl coenzyme A-binding
protein (ACBP), regulates autophagy at three levels. First,
autophagy causes DBI secretion, depleting this pro-autophagic
factor from the cell (autocrine regulation). Second, autophagy
causes DBI accumulation in the extracellular space, allowing DBI to
act on other cells to inhibit autophagy (paracrine regulation).
Third, circulating DBI stimulates feeding behavior, hence removing
the primary cause of autophagy induction (endocrine regulation). In
humans, plasma DBI levels increase in obesity. Extra supply of
recombinant DBI to mice enhanced glycolysis, enhanced lipogenesis,
and inhibited fatty acid oxidation. The inventors also designed
three strategies to neutralize DBI, namely, by inducible whole-body
knockout, passive immunization, and active immunization by means of
an immunogenic DBI derivative eliciting autoantibodies. These
strategies favor metabolic changes that increase starvation-induced
weight loss and reduce food intake upon refeeding.
[0005] General Definitions:
[0006] As used herein, the term "subject", "individual," or
"patient" is used interchangeably and refers to any subject for
whom diagnosis, treatment, or therapy is desired, particularly
humans. Other subjects may include cattle, dogs, cats, guinea pigs,
rabbits, rats, mice, horses, and the like. In some preferred
embodiments the subject is a human.
[0007] The term "autophagy" refers to macroautophagy, unless stated
otherwise, is the catabolic process involving the degradation of a
cell's own components; such as, long lived proteins, protein
aggregates, cellular organelles, cell membranes, organelle
membranes, and other cellular components. The mechanism of
autophagy may include: (i) the formation of a membrane around a
targeted region of the cell, separating the contents from the rest
of the cytoplasm, (ii) the fusion of the resultant vesicle with a
lysosome and the subsequent degradation of the vesicle contents.
The term autophagy may also refer to one of the mechanisms by which
a starving cell re-allocates nutrients from unnecessary processes
to more essential processes. Also, for example, autophagy may
inhibit the progression of some diseases and play a protective role
against infection by intracellular pathogens. Acute, intermittent
or continuous stimulation of autophagy can delay aging and
aging-related diseases including arteriosclerosis, cardiac
insufficiency, cancer and neurodegeneration. Stimulation of
autophagy can also reduce high-fat or high-sugar diet or high-salt
induced weight gain, obesity, metabolic syndrome, hypertension and
diabetes.
[0008] As used herein, the term "body mass index" has its general
meaning in the art and refers to refers to the ratio which is
calculated as body weight per height in meter squared (kg/m.sup.2).
The BMI provides a simple means of assessing how much an
individual's body weight departs from what is normal or desirable
for a person of his or her height. Common definitions of BMI
categories are as follows: starvation: BMI--less than 15
kg/m.sup.2; underweight--BMI less than 18.5 kg/m.sup.2; ideal--BMI
from 18.5 to 25 kg/m.sup.2; overweight--BMI from 25 to 30
kg/m.sup.2; obese--BMI from 30 to 40 kg/m.sup.2; morbidly
obese--BMI greater than 40 kg/m.sup.2. While simple, the BMI method
of characterizing the body weight property of a person is not
always correct. For example, the BMI does not take into account
factors such as frame size, muscularity or varying proportions of
e.g. bone, cartilage, and water weight among individuals. Thus, the
accuracy of BMI in relation to actual levels of body fat mass may
be distorted by such factors as fitness level, muscle mass, bone
structure, gender, and ethnicity. Also, people with short stature
and old people tend to have lower BMI values. It is considered,
however, that the skilled person, e.g. a physician, will be able to
take these factors into account when making the BMI assessment of
any given individual.
[0009] As used herein, the term "cancer" has its general meaning in
the art and includes, but is not limited to, solid tumors and blood
borne tumors. The term cancer includes diseases of the skin,
tissues, organs, bone, cartilage, blood and vessels. The term
"cancer" further encompasses both primary and metastatic cancers.
Examples of cancers that may treated by methods and compositions of
the invention include, but are not limited to, cancer cells from
the bladder, blood, bone, bone marrow, brain, breast, colon,
esophagus, gastrointestine, gum, head, kidney, liver, lung,
nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue,
or uterus. In addition, the cancer may specifically be of the
following histological type, though it is not limited to these:
neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant
and spindle cell carcinoma; small cell carcinoma; papillary
carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma;
basal cell carcinoma; pilomatrix carcinoma; transitional cell
carcinoma; papillary transitional cell carcinoma; adenocarcinoma;
gastrinoma, malignant; cholangiocarcinoma; hepatocellular
carcinoma; combined hepatocellular carcinoma and
cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic
carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,
familial polyposis coli; solid carcinoma; carcinoid tumor,
malignant; branchiolo-alveolar adenocarcinoma; papillary
adenocarcinoma; chromophobe carcinoma; acidophil carcinoma;
oxyphilic adenocarcinoma; basophil carcinoma; clear cell
adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;
papillary and follicular adenocarcinoma; nonencapsulating
sclerosing carcinoma; adrenal cortical carcinoma; endometroid
carcinoma; skin appendage carcinoma; apocrine adenocarcinoma;
sebaceous adenocarcinoma; ceruminous; adenocarcinoma;
mucoepidermoid carcinoma; cystadenocarcinoma; papillary
cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous
cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell
carcinoma; infiltrating duct carcinoma; medullary carcinoma;
lobular carcinoma; inflammatory carcinoma; paget's disease,
mammary; acinar cell carcinoma; adenosquamous carcinoma;
adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian
stromal tumor, malignant; thecoma, malignant; granulosa cell tumor,
malignant; and roblastoma, malignant; Sertoli cell carcinoma;
leydig cell tumor, malignant; lipid cell tumor, malignant;
paraganglioma, malignant; extra-mammary paraganglioma, malignant;
pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic
melanoma; superficial spreading melanoma; malig melanoma in giant
pigmented nevus; epithelioid cell melanoma; blue nevus, malignant;
sarcoma; fibrosarcoma; fibrous histiocytoma, malignant;
myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;
embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal
sarcoma; mixed tumor, malignant; mullerian mixed tumor;
nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma,
malignant; brenner tumor, malignant; phyllodes tumor, malignant;
synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal
carcinoma; teratoma, malignant; struma ovarii, malignant;
choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;
hemangioendothelioma, malignant; kaposi's sarcoma;
hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;
juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma,
malignant; mesenchymal chondrosarcoma; giant cell tumor of bone;
ewing's sarcoma; odontogenic tumor, malignant; ameloblastic
odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma;
pinealoma, malignant; chordoma; glioma, malignant; ependymoma;
astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma;
astroblastoma; glioblastoma; oligodendroglioma;
oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;
ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory
neurogenic tumor; meningioma, malignant; neurofibrosarcoma;
neurilemmoma, malignant; granular cell tumor, malignant; malignant
lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;
malignant lymphoma, small lymphocytic; malignant lymphoma, large
cell, diffuse; malignant lymphoma, follicular; mycosis fungoides;
other specified non-Hodgkin's lymphomas; malignant histiocytosis;
multiple myeloma; mast cell sarcoma; immunoproliferative small
intestinal disease; leukemia; lymphoid leukemia; plasma cell
leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid
leukemia; basophilic leukemia; eosinophilic leukemia; monocytic
leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid
sarcoma; and hairy cell leukemia.
[0010] The terms "polypeptide" and "protein", are used
interchangeably and refer to a polymeric form of amino acids of any
length, which can include coded and non-coded amino acids,
chemically or biochemically modified or derivatized amino acids,
and polypeptides having modified peptide backbones. The term
includes fusion proteins, including, but not limited to, fusion
proteins with a heterologous amino acid sequence, fusions with
heterologous and homologous signal sequences, with or without
N-terminal methionine residues; immunologically tagged proteins;
and the like.
[0011] As used herein, the term "DBI" has its general meaning in
the art and refers to the diazepam binding inhibitor, acyl-CoA
binding protein encoding by the DBI gene (Gene ID: 1622). The term
is also known as EP; ACBP; ACBD1; and CCK-RP. An exemplary human
amino acid sequence is represented by the NCNI reference sequence
NP_001073331.1 (SEQ ID NO:1) (acyl-CoA-binding protein isoform 1).
An exemplary human nucleic acid sequence is represented by the NCNI
reference sequence NM_001079862.2 (SEQ ID NO:2) (acyl-CoA-binding
protein isoform 1).
TABLE-US-00001 SEQ ID NO: 1 MSQAEFEKAA EEVRHLKTKP SDEEMLFIYG
HYKQATVGDI NTERPGMLDF TGKAKWDAWN ELKGTSKEDA MKAYINKVEE LKKKYGI SEQ
ID NO: 2 GCTCGCCCGA GCAGGGTTGG GGCGAGTGGA CCGCGCCTCT AAAGGCGCTT
GCCAGTGCAA TCTGGGCGAT CGCTTCCTGG TCCTCGCCTC CTCCGCTGTC TCCCTGGAGT
TCTTGCAAGT CGGCCAGGAT GTCTCAGGCT GAGTTTGAGA AAGCTGCAGA GGAGGTTAGG
CACCTTAAGA CCAAGCCATC GGATGAGGAG ATGCTGTTCA TCTATGGCCA CTACAAACAA
GCAACTGTGG GCGACATAAA TACAGAACGG CCCGGGATGT TGGACTTCAC GGGCAAGGCC
AAGTGGGATG CCTGGAATGA GCTGAAAGGG ACTTCCAAGG AAGATGCCAT GAAAGCTTAC
ATCAACAAAG TAGAAGAGCT AAAGAAAAAA TACGGGATAT GAGAGACTGG ATTTGGTTAC
TGTGCCATGT GTTTATCCTA AACTGAGACA ATGCCTTGTT TTTTTCTAAT ACCGTGGATG
GTGGGAATTC GGGAAAATAA CCAGTTAAAC CAGCTACTCA AGGCTGCTCA CCATACGGCT
CTAACAGATT AGGGGCTAAA ACGATTACTG ACTTTCCTTG AGTAGTTTTT ATCTGAAATC
AATTAAAAGT GTATTTGTTA CTTTAAATAA CTTTAAAAAA AAAA
[0012] As used herein, the term "DBI activity" refers to any
biological activity of DIB that includes among others: inhibition
of autophagy, induction of hypoglycaemia, stimulation of food
intake, stimulation of weight gain, reduction of fatty acid
oxidation, upregulation of glucose transporter, upregulation of
PPARG, stimulation of glucose uptake, stimulation of glycolysis or
stimulation of lipogenesis.
[0013] As used herein, the term "treatment" or "treat" refer to
both prophylactic or preventive treatment as well as curative or
disease modifying treatment, including treatment of patient at risk
of contracting the disease or suspected to have contracted the
disease as well as patients who are ill or have been diagnosed as
suffering from a disease or medical condition, and includes
suppression of clinical relapse. The treatment may be administered
to a subject having a medical disorder or who ultimately may
acquire the disorder, in order to prevent, cure, delay the onset
of, reduce the severity of, or ameliorate one or more symptoms of a
disorder or recurring disorder, or in order to prolong the survival
of a subject beyond that expected in the absence of such treatment.
By "therapeutic regimen" is meant the pattern of treatment of an
illness, e.g., the pattern of dosing used during therapy. A
therapeutic regimen may include an induction regimen and a
maintenance regimen. The phrase "induction regimen" or "induction
period" refers to a therapeutic regimen (or the portion of a
therapeutic regimen) that is used for the initial treatment of a
disease. The general goal of an induction regimen is to provide a
high level of drug to a patient during the initial period of a
treatment regimen. An induction regimen may employ (in part or in
whole) a "loading regimen", which may include administering a
greater dose of the drug than a physician would employ during a
maintenance regimen, administering a drug more frequently than a
physician would administer the drug during a maintenance regimen,
or both. The phrase "maintenance regimen" or "maintenance period"
refers to a therapeutic regimen (or the portion of a therapeutic
regimen) that is used for the maintenance of a patient during
treatment of an illness, e.g., to keep the patient in remission for
long periods of time (months or years). A maintenance regimen may
employ continuous therapy (e.g., administering a drug at a regular
intervals, e.g., weekly, monthly, yearly, etc.) or intermittent
therapy (e.g., interrupted treatment, intermittent treatment,
treatment at relapse, or treatment upon achievement of a particular
predetermined criteria [e.g., disease manifestation, etc.]).
[0014] By a "therapeutically effective amount" is meant a
sufficient amount of the agent of the present invention for
reaching a therapeutic effect. It will be understood, however, that
the total daily usage of the compounds and compositions of the
present invention will be decided by the attending physician within
the scope of sound medical judgment. The specific therapeutically
effective dose level for any particular subject will depend upon a
variety of factors including the disorder being treated and the
severity of the disorder; activity of the specific compound
employed; the specific composition employed, the age, body weight,
general health, sex and diet of the subject; the time of
administration, route of administration, and rate of excretion of
the specific compound employed; the duration of the treatment;
drugs used in combination or coincidental with the specific
compound employed; and like factors well known in the medical arts.
For example, it is well within the skill of the art to start doses
of the compound at levels lower than those required to achieve the
desired therapeutic effect and to gradually increase the dosage
until the desired effect is achieved. However, the daily dosage of
the products may be varied over a wide range from 0.01 to 4,000 mg
per adult per day. Typically, the compositions contain 0.01, 0.05,
0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, 500 and
1000 mg of the active ingredient for the symptomatic adjustment of
the dosage to the subject to be treated. A medicament typically
contains from about 0.01 mg to about 1000 mg of the active
ingredient. An effective amount of the drug is ordinarily supplied
at a dosage level from 0.0002 mg/kg to about 50 mg/kg of body
weight per day, especially from about 0.001 mg/kg to 10 mg/kg of
body weight per day.
[0015] Methods of Inhibiting Autophagy:
[0016] Accordingly, the first object of the present invention
relates to a method of inhibiting autophagy in a subject in need
thereof comprising administering to the subject a therapeutically
effective amount of an agent that promotes the activity or
expression of DBI.
[0017] In some embodiments, the method of inhibiting autophagy
according to the invention is particularly suitable for stimulating
appetite and consequently weight gain. In particular, the method of
the present invention is also particularly suitable for promoting
glucose uptake and lipogenesis. Accordingly, the method of the
present invention is particularly suitable for the treatment of
various diseases as described herein after.
[0018] Accordingly, in some embodiments, the subject is
underweight. As herein, the term "underweight" refers to a subject
having a body mass index of below 18.5.
[0019] Underweight may be due to several causes, such as rapid
metabolism, poor/inadequate diet or starvation (malnutrition),
malabsorption due to defective intestinal function, endocrine
disturbances e.g. type I diabetes, psychological problems (such as
anorexia nervosa, body dysmorphic disorder, stress and anxiety) and
weight loss, due to chronic illnesses and ageing. While in general
the underlying cause of the underweight will have to be treated per
se, the underweight too may be a health hazard, and as such have to
be treated in itself Indeed, persons suffering from underweight
generally have poor physical stamina, a weakened immune system, as
well as being at higher risk of developing diseases such as
osteoporosis, heart disease and vascular disease. Additionally, in
the female sex, underweight can lead to delayed sexual development,
retarded amenorrhoea or complications during pregnancy.
[0020] In some embodiments, the subject suffers from a wasting
disorder. As used herein, the term "wasting disorder" has its
general meaning in the art and includes but is not limited to
anorexia cachexia, anorexia of the aged, anorexia nervosa, cachexia
associated with cancer, cachexia associated with AIDS, cachexia
associated with heart failure, cachexia associated with cystic
fibrosis, cachexia associated with rheumatoid arthritis, cachexia
associated with kidney disease, cachexia associated with chronic
obstructive pulmonary disease (COPD), cachexia associated with ALS,
cachexia associated with renal failure or cachexia associated, and
other disorders associated with aberrant appetite, fat mass, energy
balance, and/or involuntary weight loss.
[0021] In some embodiments, the subject suffers from "cachexia". As
used herein, the term "cachexia" is used for a condition of
physical wasting with loss of body fat and muscle mass. Generally,
cachexia may be associated with and due to conditions such as
cancer, required immunodeficiency syndrome (AIDS), cardiac
diseases, infectious diseases, shock, burn, endotoxinemia, organ
inflammation, surgery, diabetes, collagen diseases, radiotherapy,
and chemotherapy. In many of these diseases, cachexia may
significantly contribute to morbidity or mortality. Another
particular group of individuals that are susceptible to developing
a cachectic state are those individuals that have undergone a
gastrectomy, such as may be practiced on gastric cancer and ulcer
patients.
[0022] In some embodiments, the subject suffers from anorexia. As
used herein, the term "anorexia" has its general meaning in the art
and refers to any eating disorder characterized by markedly reduced
appetite or total aversion to food. In some embodiments, the
subject suffers from anorexia nervosa. In general, subjects
suffering from anorexia nervosa have a BMI of less than 17.5
kg/m.sup.2.
[0023] Accordingly, the present invention is drawn to methods of
treating a patient exhibiting one or more wasting disorders such as
anorexia cachexia, anorexia of the aged, anorexia nervosa, cachexia
associated with cancer, cachexia associated with AIDS, cachexia
associated with heart failure, cachexia associated with cystic
fibrosis, cachexia associated with rheumatoid arthritis, cachexia
associated with kidney disease, cachexia associated with COPD,
cachexia associated with ALS, cachexia associated with renal
failure or cachexia associated, or hip fracture, and in reducing
the mortality and morbidity of critically ill patients, comprising
administering to said patient in need of such treatment a
therapeutically effective of an agent that promotes the activity or
expression of DBI.
[0024] In some embodiments, the subject suffers from a disease
selected from the group consisting of cancer diseases,
neurodegenerative diseases, cardiovascular diseases, infectious
diseases, auto-immune diseases and/or inflammatory diseases.
[0025] In some embodiments, the subject suffers from a cancer. In
particular, autophagy seems to be indispensable for tumor
progression, providing the tumours with building blocks and energy
for its increased metabolic requirements. The modulation of the
tumours' metabolic environment by the administration of Agent that
promotes the activity or expression of DBI alone or in combination
with chemotherapeutic drugs may lead to a suppression of basal and
starvation-induced autophagy, thus sensitizing tumour cells to the
death. Accordingly, the Agent that promotes the activity or
expression of DBI of the present invention would be suitable for
the treatment of advanced cancer. In some embodiments, the cancer
is an autophagy competent cancer. As used herein the term
"autophagy competent cancer" denotes a cancer wherein autophagy
could occur. In some embodiments, an ATG5 or ATG7 deficiency is not
detected. In the context of the invention, the term "ATG5 or ATG7
deficiency" denotes that the tumor cells of the subject or a part
thereof have an ATG5 or ATG7 dysfunction, a low or a null
expression of ATG5 or ATG7 gene. Said deficiency may typically
result from a mutation in ATG5 or ATG7 gene so that the pre-ARNm is
degraded through the NMD (non sense mediated decay) system. Said
deficiency may also typically result from a mutation so that the
protein is misfolded and degraded through the proteasome. Said
deficiency may also result from a loss of function mutation leading
to a dysfunction of the protein. Said deficiency may also result
from an epigenetic control of gene expression (e.g. methylation) so
that the gene is less expressed in the cells of the subject. Said
deficiency may also result from a repression of the ATG5 or ATG7
gene induce by a particular signalling pathway. Said deficiency may
also result from a mutation in a nucleotide sequence that control
the expression of ATG5 or ATG7 gene.
[0026] In some embodiments, the subject suffers from a
neurodegenerative disease for which inhibition of autophagy would
be suitable. Typically, the subject suffers from amyotrophic
lateral sclerosis. As used herein, the term "amyotrophic lateral
sclerosis (ALS)" includes the spectrum of neurodegenerative
syndromes known under the names of Classical (Charcot's) ALS, Lou
Gehrig's disease, motor neuron disease (MND), progressive bulbar
palsy (PBP), progressive muscular atrophy (PMA), primary lateral
sclerosis (PLS), bulbar onset ALS, spinal onset ALS and ALS with
multi-system involvement (Wijesekera LC and Leigh PN. Amyotrophic
lateral sclerosis. Orphanet).
[0027] In some embodiments, the subject suffers from sarcopenia. As
used herein, the term "sarcopenia" means the gradual decrease in
skeletal muscle mass caused by aging, which can directly cause a
decrease in muscle strength, resulting in a decrease and impairment
in various physical functions.
[0028] Agents that Promote the Activity or Expression of DBI:
[0029] In some embodiments, the agent that promotes the activity of
DBI is a polypeptide having at least having at least 80% of
identity with the sequence of SEQ ID NO:1 or a fragment thereof
[0030] According to the invention a first amino acid sequence
having at least 80% of identity with a second amino acid sequence
means that the first sequence has 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity
with the second amino acid sequence. Sequence identity is
frequently measured in terms of percentage identity (or similarity
or homology); the higher the percentage, the more similar are the
two sequences. Methods of alignment of sequences for comparison are
well known in the art. Various programs and alignment algorithms
are described in: Smith and Waterman, Adv. Appl. Math., 2:482,
1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson
and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins
and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS,
5:151-153, 1989; Corpet et al. Nuc. Acids Res., 16:10881-10890,
1988; Huang et al., Comp. Appls Biosci., 8:155-165, 1992; and
Pearson et al., Meth. Mol. Biol., 24:307-31, 1994). Altschul et
al., Nat. Genet., 6:119-129, 1994, presents a detailed
consideration of sequence alignment methods and homology
calculations. By way of example, the alignment tools ALIGN (Myers
and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman,
1988) may be used to perform sequence comparisons (Internet
Program.RTM. 1996, W. R. Pearson and the University of Virginia,
fasta20u63 version 2.0u63, release date December 1996). ALIGN
compares entire sequences against one another, while LFASTA
compares regions of local similarity. These alignment tools and
their respective tutorials are available on the Internet at the
NCSA Website, for instance. Alternatively, for comparisons of amino
acid sequences of greater than about 30 amino acids, the Blast 2
sequences function can be employed using the default BLOSUM62
matrix set to default parameters, (gap existence cost of 11, and a
per residue gap cost of 1). When aligning short peptides (fewer
than around 30 amino acids), the alignment should be performed
using the Blast 2 sequences function, employing the PAM30 matrix
set to default parameters (open gap 9, extension gap 1 penalties).
The BLAST sequence comparison system is available, for instance,
from the NCBI web site; see also Altschul et al., J. Mol. Biol.,
215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272,
1993; Madden et al. Meth. Enzymol., 266:131-141, 1996; Altschul et
al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang &
Madden, Genome Res., 7:649-656, 1997.
[0031] As used herein, the term "fragment" refers to a physically
contiguous portion of the primary structure of the polypeptide
(i.e. SEQ ID NO:1). In some embodiments, the fragment comprises at
least 8 consecutive amino acids of SEQ ID NO: 1. In some
embodiments, the fragment comprises 8; 9; 10; 11; 12; 13; 14; 15;
16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32;
33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49;
50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66;
67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81: 82; 83;
84; 85; or 86 consecutive amino acids. According to the present
invention the fragment shall retain the activity of DBI.
[0032] In some embodiments, the fragment consist in the amino acid
sequence ranging from the amino acid residue at position 17 to the
amino acid residue at position 50 (i.e. the
triacontatetraneuropeptide or TTN).
[0033] In some embodiments, the fragment consists in the amino acid
sequence ranging from the amino acid residue at position 33 to the
amino acid residue at position 50 (i.e. the octadecaneuropeptide or
ODN).
[0034] In some embodiments, the fragment consists in the amino acid
sequence ranging from the amino acid residue at position 43 to the
amino acid residue at position 50 (i.e. the octapeptide or OP).
[0035] Accordingly, in some embodiments, the agent that promotes
the activity of DBI consists in a polypeptide comprising: [0036] an
amino acid sequence having at least 80% of identity with SEQ ID
NO:1, or [0037] an amino acid sequence having at least 80% of
identity with the amino acid sequence ranging from the amino acid
residue at position 17 to the amino acid residue at position 50 in
SEQ ID NO:1, or [0038] an amino acid sequence having at least 80%
of identity with the amino acid sequence ranging from the amino
acid residue at position 33 to the amino acid residue at position
50 in SEQ ID NO:1, or [0039] an amino acid sequence having at least
80% of identity with the amino acid sequence ranging from the amino
acid residue at position 43 to the amino acid residue at position
50 in SEQ ID NO:l.
[0040] In some embodiments, the polypeptide of the present
invention is fused to at least one heterologous polypeptide to form
a fusion protein. In some embodiments, the polypeptide of the
present invention is fused either directly or via a spacer at its
C-terminal end to the N-terminal end of the heterologous
polypeptide, or at its N-terminal end to the C-terminal end of the
heterologous polypeptide. As used herein, the term "directly" means
that the (first or last) amino acid at the terminal end (N or
C-terminal end) of the polypeptide of the present invention is
fused to the (first or last) amino acid at the terminal end (N or
C-terminal end) of the heterologous polypeptide. In other words, in
this embodiment, the last amino acid of the C-terminal end of said
polypeptide is directly linked by a covalent bond to the first
amino acid of the N-terminal end of said heterologous polypeptide,
or the first amino acid of the N-terminal end of said polypeptide
is directly linked by a covalent bond to the last amino acid of the
C-terminal end of said heterologous polypeptide. As used herein,
the term "spacer" refers to a sequence of at least one amino acid
that links the polypeptide of the invention to the heterologous
polypeptide. Such a spacer may be useful to prevent steric
hindrances. Typically a spacer comprises 2, 3; 4; 5; 6; 7; 8; 9;
10; 11; 12; 13; 14; 15; 16; 17; 18; 19; or 20 amino acids.
[0041] In some embodiments, the polypeptide of the present
invention is fused to a signal sequence. A signal sequence can be
used to facilitate secretion and isolation of the secreted protein
or other proteins of interest. Signal sequences are typically
characterized by a core of hydrophobic amino acids which are
generally cleaved from the mature protein during secretion in one
or more cleavage events. Such signal peptides contain processing
sites that allow cleavage of the signal sequence from the mature
proteins as they pass through the secretory pathway.
[0042] In some embodiments, the fusion protein according to the
invention is an immunoadhesin. As used herein, the term
"immunoadhesin" designates antibody-like molecules which combine
the binding specificity of the polypeptide of the present invention
with the effector functions of immunoglobulin constant domains.
Structurally, the immunoadhesins comprise a fusion of the
polypeptide of the present invention and an immunoglobulin constant
domain sequence. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM. The immunoglobulin sequence typically, but
not necessarily, is an immunoglobulin constant domain (Fc region).
Immunoadhesins can possess many of the valuable chemical and
biological properties of human antibodies. Since immunoadhesins can
be constructed from a human protein sequence with a desired
specificity linked to an appropriate human immunoglobulin hinge and
constant domain (Fc) sequence, the binding specificity of interest
can be achieved using entirely human components. Such
immunoadhesins are minimally immunogenic to the patient, and are
safe for chronic or repeated use. In some embodiments, the Fc
region is a native sequence Fc region. In some embodiments, the Fc
region is a variant Fc region. In still another embodiment, the Fc
region is a functional Fc region. As used herein, the term "Fc
region" is used to define a C-terminal region of an immunoglobulin
heavy chain, including native sequence Fc regions and variant Fc
regions. Although the boundaries of the Fc region of an
immunoglobulin heavy chain might vary, the human IgG heavy chain Fc
region is usually defined to stretch from an amino acid residue at
position Cys226, or from Pro230, to the carboxyl-terminus thereof.
The adhesion portion and the immunoglobulin sequence portion of the
immunoadhesin may be linked by a minimal linker. The immunoglobulin
sequence typically, but not necessarily, is an immunoglobulin
constant domain. The immunoglobulin moiety in the chimeras of the
present invention may be obtained from IgG1, IgG2, IgG3 or IgG4
subtypes, IgA, IgE, IgD or IgM, but typically IgG1 or IgG3. In some
embodiments, the polypeptide of the invention and the
immunoglobulin sequence portion of the immunoadhesin are linked by
a minimal linker. As used herein, the term "linker" refers to a
sequence of at least one amino acid that links the polypeptide of
the invention and the immunoglobulin sequence portion. Such a
linker may be useful to prevent steric hindrances. In some
embodiments, the linker has 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14;
15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30
amino acid residues. However, the upper limit is not critical but
is chosen for reasons of convenience regarding e.g.
biopharmaceutical production of such polypeptides. The linker
sequence may be a naturally occurring sequence or a non-naturally
occurring sequence. If used for therapeutical purposes, the linker
is preferably non-immunogenic in the subject to which the
immunoadhesin is administered. One useful group of linker sequences
are linkers derived from the hinge region of heavy chain antibodies
as described in WO 96/34103 and WO 94/04678. Other examples are
poly-alanine linker sequences.
[0043] The polypeptides of the present invention are produced by
any technique known per se in the art, such as, without limitation,
any chemical, biological, genetic or enzymatic technique, either
alone or in combination. For instance, knowing the amino acid
sequence of the desired sequence, one skilled in the art can
readily produce said polypeptides, by standard techniques for
production of amino acid sequences. For instance, they can be
synthesized using well-known solid phase method, preferably using a
commercially available peptide synthesis apparatus (such as that
made by Applied Biosystems, Foster City, California) and following
the manufacturer's instructions. Alternatively, the polypeptides of
the present invention can be synthesized by recombinant DNA
techniques as is now well-known in the art. For example, these
fragments can be obtained as DNA expression products after
incorporation of DNA sequences encoding the desired (poly)peptide
into expression vectors and introduction of such vectors into
suitable eukaryotic or prokaryotic hosts that will express the
desired polypeptide, from which they can be later isolated using
well-known techniques.
[0044] In some embodiments, it is contemplated that the polypeptide
of the invention used in the therapeutic methods of the present
invention may be modified in order to improve their therapeutic
efficacy. Such modification of therapeutic compounds may be used to
decrease toxicity, increase circulatory time, or modify
biodistribution. For example, the toxicity of potentially important
therapeutic compounds can be decreased significantly by combination
with a variety of drug carrier vehicles that modify
biodistribution. A strategy for improving drug viability is the
utilization of water-soluble polymers. Various water-soluble
polymers have been shown to modify biodistribution, improve the
mode of cellular uptake, change the permeability through
physiological barriers; and modify the rate of clearance from the
body. To achieve either a targeting or sustained-release effect,
water-soluble polymers have been synthesized that contain drug
moieties as terminal groups, as part of the backbone, or as pendent
groups on the polymer chain. Polyethylene glycol (PEG) has been
widely used as a drug carrier, given its high degree of
biocompatibility and ease of modification. Attachment to various
drugs, proteins, and liposomes has been shown to improve residence
time and decrease toxicity. PEG can be coupled to active agents
through the hydroxyl groups at the ends of the chain and via other
chemical methods; however, PEG itself is limited to at most two
active agents per molecule. In a different approach, copolymers of
PEG and amino acids were explored as novel biomaterials which would
retain the biocompatibility properties of PEG, but which would have
the added advantage of numerous attachment points per molecule
(providing greater drug loading), and which could be synthetically
designed to suit a variety of applications.
[0045] In some embodiments, the agent that promotes the expression
of DBI is a nucleic acid molecule that encodes for the polypeptide
as described above. As used herein, the term "nucleic acid
molecule" has its general meaning in the art and refers to a DNA or
RNA molecule. However, the term captures sequences that include any
of the known base analogues of DNA and RNA such as, but not limited
to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-fiuorouracil, 5-bromouracil, 5-
carboxymethylaminomethyl-2-thiouracil,
5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1- methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-
methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil,
beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-
5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
-uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0046] In some embodiments, the nucleic acid molecule comprises a
nucleic acid sequence having at least 50% with SEQ ID NO: 2.
According to the invention a first nucleic acid sequence having at
least 50% of identity with a second nucleic acid sequence means
that the first sequence has 50; 51; 52; 53; 54; 55; 56; 57; 58; 59;
60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76;
77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93;
94; 95; 96; 97; 98; 99; or 100% of identity with the second nucleic
acid sequence.
[0047] In some embodiments, the nucleic acid molecule of the
present invention is included in a suitable vector, such as a
plasmid, cosmid, episome, artificial chromosome, phage or a viral
vector. Typically, the vector is a viral vector which is an
adeno-associated virus (AAV), a retrovirus, bovine papilloma virus,
an adenovirus vector, a lentiviral vector, a vaccinia virus, a
polyoma virus, or an infective virus. In some embodiments, the
vector is an AAV vector. As used herein, the term "AAV vector"
means a vector derived from an adeno- associated virus serotype,
including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, and mutated forms thereof AAV vectors can have
one or more of the AAV wild-type genes deleted in whole or part,
preferably the rep and/or cap genes, but retain functional flanking
ITR sequences. Retroviruses may be chosen as gene delivery vectors
due to their ability to integrate their genes into the host genome,
transferring a large amount of foreign genetic material, infecting
a broad spectrum of species and cell types and for being packaged
in special cell- lines. In order to construct a retroviral vector,
a nucleic acid encoding a gene of interest is inserted into the
viral genome in the place of certain viral sequences to produce a
virus that is replication-defective. In order to produce virions, a
packaging cell line is constructed containing the gag, pol, and/or
env genes but without the LTR and/or packaging components. When a
recombinant plasmid containing a cDNA, together with the retroviral
LTR and packaging sequences is introduced into this cell line (by
calcium phosphate precipitation for example), the packaging
sequence allows the RNA transcript of the recombinant plasmid to be
packaged into viral particles, which are then secreted into the
culture media. The media containing the recombinant retroviruses is
then collected, optionally concentrated, and used for gene
transfer. Retroviral vectors are able to infect a broad variety of
cell types. Lentiviruses are complex retroviruses, which, in
addition to the common retroviral genes gag, pol, and env, contain
other genes with regulatory or structural function. The higher
complexity enables the virus to modulate its life cycle, as in the
course of latent infection. Some examples of lentivirus include the
Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian
Immunodeficiency Virus (SIV). Lentiviral vectors have been
generated by multiply attenuating the HIV virulence genes, for
example, the genes env, vif, vpr, vpu and nef are deleted making
the vector biologically safe. Lentiviral vectors are known in the
art, see, e.g.. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of
which are incorporated herein by reference. In general, the vectors
are plasmid-based or virus-based, and are configured to carry the
essential sequences for incorporating foreign nucleic acid, for
selection and for transfer of the nucleic acid into a host cell.
The gag, pol and env genes of the vectors of interest also are
known in the art. Thus, the relevant genes are cloned into the
selected vector and then used to transform the target cell of
interest. Recombinant lentivirus capable of infecting a
non-dividing cell wherein a suitable host cell is transfected with
two or more vectors carrying the packaging functions, namely gag,
pol and env, as well as rev and that is described in U.S. Pat. No.
5,994,136, incorporated herein by reference. This describes a first
vector that can provide a nucleic acid encoding a viral gag and a
pol gene and another vector that can provide a nucleic acid
encoding a viral env to produce a packaging cell. Introducing a
vector providing a heterologous gene into that packaging cell
yields a producer cell which releases infectious viral particles
carrying the foreign gene of interest. The env preferably is an
amphotropic envelope protein which allows transduction of cells of
human and other species. Typically, the nucleic acid molecule or
the vector of the present invention include "control sequences",
which refers collectively to promoter sequences, polyadenylation
signals, transcription termination sequences, upstream regulatory
domains, origins of replication, internal ribosome entry sites
("IRES"), enhancers, and the like, which collectively provide for
the replication, transcription and translation of a coding sequence
in a recipient cell. Not all of these control sequences need always
be present so long as the selected coding sequence is capable of
being replicated, transcribed and translated in an appropriate host
cell. Another nucleic acid sequence, is a "promoter" sequence,
which is used herein in its ordinary sense to refer to a nucleotide
region comprising a DNA regulatory sequence, wherein the regulatory
sequence is derived from a gene which is capable of binding RNA
polymerase and initiating transcription of a downstream
(3'-direction) coding sequence. Transcription promoters can include
"inducible promoters" (where expression of a polynucleotide
sequence operably linked to the promoter is induced by an analyte,
cofactor, regulatory protein, etc.), "repressible promoters" (where
expression of a polynucleotide sequence operably linked to the
promoter is induced by an analyte, cofactor, regulatory protein,
etc.), and "constitutive promoters".
[0048] In some embodiments, the agent that promotes the activity of
DBI is a small organic molecule or peptidomimetics that mimics the
activity of DBI. As used herein, the term "small organic molecule"
refers to a molecule of a size comparable to those organic
molecules generally used in pharmaceuticals. The term excludes
biological macromolecules (e. g., proteins, nucleic acids, etc.).
Preferred small organic molecules range in size up to about 5000
Da, more preferably up to 2000 Da, and most preferably up to about
1000 Da. As used herein, the term "peptidomimetics" is used to
refer to any molecule whose essential elements (pharmacophore)
mimic a natural peptide or protein in 3D space and which retain the
ability to interact with the biological target and produce the same
biological effect. Peptidomimetics include small protein-like chain
designed to mimic a peptide which may typically be obtained either
by modifying an existing peptide, or by designing similar systems
that mimic peptides, such as, e.g., peptoids and .beta.-peptides.
Irrespective of the approach, the altered chemical structure is
designed to adjust the molecular properties advantageously in that,
e.g., the stability or biological activity is increased or
decreased. According modifications involve changes to the peptide
that will not occur naturally including but not limited to altered
backbones and the incorporation of non- natural amino acids. The
term "amino acid mimetics," as used herein, refers to chemical
compounds that have a structure that is different from the general
chemical structure of an amino acid, but functions in a manner
similar to a naturally occurring amino acid.
[0049] Methods of Stimulating Autophagy:
[0050] Accordingly, the first object of the present invention
relates to a method of stimulating autophagy in a subject in need
thereof comprising administering to the subject a therapeutically
effective amount of an agent that inhibits the activity or
expression of DBI.
[0051] In some embodiments, the method of stimulating autophagy
according to the invention is particularly suitable for inhibiting
appetite and consequently weight loss. The method is also
particularly for reducing glycaemia and lipogenesis. Accordingly,
the method of the present invention is particularly suitable for
the treatment of various diseases as described herein after.
[0052] In some embodiments, the subject is overweight. In
particular, the subject is obese. Obesity refers to a condition
whereby an otherwise healthy subject has a BMI greater than or
equal to 30 kg/m.sup.2, or a condition whereby a subject with at
least one co-morbidity has a BMI greater than or equal to 27
kg/m.sup.2. An "obese subject" is an otherwise healthy subject with
a BMI greater than or equal to 30 kg/m.sup.2 or a subject with at
least one co-morbidity with a BMI greater than or equal 27
kg/m.sup.2. A "subject at risk of obesity" is an otherwise healthy
subject with a BMI of 25 kg/m.sup.2 to less than 30 kg/m.sup.2 or a
subject with at least one co-morbidity with a BMI of 25 kg/m.sup.2
to less than 27 kg/m.sup.2. The increased risks associated with
obesity may occur at a lower BMI in people of Asian descent. In
Asian and Asian-Pacific countries, including Japan, "obesity"
refers to a condition whereby a subject has a BMI greater than or
equal to 25 kg/m.sup.2. An "obese subject" in these countries
refers to a subject with at least one obesity-induced or
obesity-related co-morbidity that requires weight reduction or that
would be improved by weight reduction, with a BMI greater than or
equal to 25 kg/m.sup.2. In these countries, a "subject at risk of
obesity" is a person with a BMI of greater than 23 kg/m2 to less
than 25 kg/m.sup.2.
[0053] In some embodiments, the subject suffers from type 2
diabetes. As used herein, the term "type 2 diabetes" or
"non-insulin dependent diabetes mellitus (NIDDM)" has its general
meaning in the art. Type 2 diabetes often occurs when levels of
insulin are normal or even elevated and appears to result from the
inability of tissues to respond appropriately to insulin. Most of
the Type 2 diabetics are obese.
[0054] In some embodiments, the subject suffers from metabolic
syndrome. As used herein, the term "Metabolic Syndrome" refers to a
subject characterized by having three or more of the following
symptoms: abdominal obesity, hyperglyceridemia, low HDL
cholesterol, high blood pressure, and high fasting plasma glucose.
The criteria for these symptoms are defined in the third Report of
the National Cholesterol Education Program Expert Panel in
Detection, Evaluation and Treatment of High blood Cholesterol in
Adults (Ford, E S. et al. 2002).
[0055] In some embodiments, the subject suffers from a cancer.
Although the underlying mechanism has not been characterized yet,
it has been shown that pre-chemotherapy starvation (the most potent
autophagy-inducing physiological stimulus able to systemically
induce autophagy) significantly increased treatment efficiency and
limits the tumour growth. Furthermore, it has been demonstrated
that tumours with PI3K over-activation are resistant to dietary
restriction, suggesting an important role for autophagy in the
chemiosensitization process. This invention might lead to a less
aggressive and equivalently effective treatment based on the
punctual administration of an agent of the present invention.
Accordingly, a further object of the present invention relates to a
method for treating a cancer in a subject in need thereof
comprising administering the subject with a therapeutically
effective amount of agent of the present invention and a
therapeutically effective amount of a chemotherapeutic agent
wherein the agent of the present invention is administered prior to
the chemotherapeutic agent. In some embodiments, the agent of the
present invention is administered 12; 13; 14; 15; 16; 17; 18; 19;
20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36;
37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53;
54; 55; 56h before the administration of the chemotherapeutic
agent. Chemotherapeutic agents include, but are not limited to
alkylating agents such as thiotepa and cyclosphosphamide; alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(especially bullatacin and bullatacinone); a camptothecin
(including the synthetic analogue topotecan); bryostatin;
callystatin; CC-1065 (including its adozelesin, carzelesin and
bizelesin synthetic analogues); cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g. , calicheamicin, especially
calicheamicin gammall and calicheamicin omegall; dynemicin,
including dynemicin A; bisphosphonates, such as clodronate; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores, aclacinomysins,
actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and
doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;
mercaptopurine; methotrexate; platinum coordination complexes such
as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum;
etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine;
vinorelbine; novantrone; teniposide; edatrexate; daunomycin;
aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1);
topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);
retinoids such as retinoic acid; capecitabine; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0056] In some embodiments, the subject suffers from a
neurodegenerative disease. Examples of neurodegenerative diseases
include but are not limited to Adrenoleukodystrophy (ALD),
Alexander's disease, Alper's disease, Alzheimer's disease,
Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia
telangiectasia, Batten disease (also known as
Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform
encephalopathy (B SE), Canavan disease, Cockayne syndrome,
Corticobasal degeneration, Creutzfeldt-Jakob disease,
Frontotemporal lobar degeneration, Huntington's disease,
HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy
body dementia, Neuroborreliosis, Machado-Joseph disease
(Spinocerebellar ataxia type 3), MELAS--Mitochondrial
Encephalopathy, Lactic Acidosis and Stroke, Multiple System
Atrophy, Multiple sclerosis, Niemann Pick disease, Parkinson's
disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary
lateral sclerosis, Prion diseases, Progressive Supranuclear Palsy,
Refsum's disease, Sandhoff disease, Schilder's disease,
Spinocerebellar ataxia (multiple types with varying
characteristics), Spinal muscular atrophy,
Steele-Richardson-Olszewski disease, Tabes dorsalis, Tay-Sachs
Disease, and Toxic encephalopathy. Preferred neurodegenerative
diseases include Alzheimer's disease. Neurodegenerative diseases
(i.e Alzheimer disease, Parkinson disease, Huntington disease) are
a series of different age-dependent or genetic-dependent
pathologies, characterized by progressive neuronal death as
consequence of accumulation of aggregates of misfolded proteins,
damaged organelles, impaired function of cellular clearence
mechanisms. Being autophagy a physiological mechanism dedicated to
the degradation of potentially harmful and aggregation-prone
long-lived proteins, as well as of the recycle of damaged
organelles, it is considered as a protective factor against
neuronal cell death. In the context of this invention, the
treatment of patients with agent of the present invention may
results in an improvement of the cellular clearance functions and
in an amelioration of the symptomatology of different diseases. For
example, Huntington disease is a pathology characterized by the
progressive expansion of poly-glutamine tail of the protein
huntingtin, resulting in its intra-neuronal aggregation. Huntingtin
has been demonstrated to be a specific target of the autophagic
pathway, and the increase in basal autophagy by administration of
the agent of the present invention can reduce the rate of neuronal
death. In two forms of familiar Parkinson disease, recessive
mutations in two genes encoding for PINK1 and PARK2, involved in
mitophagy, partially account for the pathogenesis of this disease
and may render the patients suitable for treatment with agent of
the present invention. In the same way, autophagy induction may
contribute to the removal of alpha-synuclein aggregates (Lewi
bodies), responsible for the pathogenesis of sporadic forms of
Parkinson disease, most likely due to a saturation of the
autophagic system.
[0057] In some embodiments, the subject suffers from an infectious
disease. Autophagic process actively participates in a multipronged
defense against microorganisms, contributing to their elimination
either via the selective delivery of microorganisms to degradative
lysosomes (a process referred to as xenophagy) or via the delivery
of microbial nucleic acids to endolysosomal compartment (with
subsequent activation of innate and adaptive immunity). Clinically
relevant pathogens are degraded in vitro by xenophagy; among these,
there are bacteria such as group A Streptococcus pyogenes,
Mycobacterium tuberculosis, Shigella flexneri, Salmonella enterica,
Listeria monocytogenes; viruses such as herpes simplex virus type 1
(HSV 1) and parasites such as Toxoplasma gondii. Moreover in vivo
evidences showed that autophagy genes have a protective role
against numerous pathogens, including L. monocytogenes, M.
tuberculosis, S. enterica, T. gondii, HSV 1. It has been recently
shown that the infection mediated by pathogens like Shigella and
Salmonella triggers an aminoacids starvation response eventually
leading to the elimination of these pathogens via autophagy. Here
use of agent of the present invention for triggering a
pro-autophagic and anti microbial response against bacterial and
virus infection may be suitable.
[0058] In some embodiments, the subject suffers from pulmonary
emphysema. Mutations in the protein .alpha.1-antitrypsin causes
pulmonary emphysema, a disease characterized by the accumulation of
the aggregated form of the mutant proteins. As for others
proteinopathy, autophagy induction by the administration of agent
of the present invention (e.g. HC, UK-5099) might ameliorate the
symptoms.
[0059] In some embodiments, the subject suffers from cystic
fibrosis. A recent pre-clinical study has found as a consequence of
a dysfunctional aggrephagy the pathogenicity of cystic fibrosis,
due to an impaired clearance of aggregates of the mutant CTFR.
Induction of autophagy mediated by administration of an agent of
the present invention may represent a suitable strategy.
[0060] In some embodiments, the subject suffers from a liver
disease. The potential impact of autophagy in vivo was discovered
from liver studies and this underlines the important role played by
autophagy in the physiology of liver.
[0061] Accordingly, a further object of the present invention
relates to a method of treating a non-alcoholic fatty liver disease
in a subject in thereof comprising administering to the subject a
therapeutically effective amount of an agent that inhibits the
activity or expression of DBI.
[0062] As used herein, the term "non-alcoholic fatty liver disease"
or "NAFLD" has its general meaning in the art and refers to one
cause of a fatty liver, occurring when fat is deposited in the
liver not due to excessive alcohol use. Non-alcoholic fatty liver
disease (NAFLD) represents one of the most recurrent and severe
pathologies, especially among obese and diabetic patients, yet a
specific therapy is far from being available. NAFLD is defined as
the accumulation of fat in the liver, but not as secondary
consequence of alcohol consumption. The pro-autophagic potential of
agent of the present invention can be used as therapy for NAFLD for
different causes: selective degradation of TG droplets (lipophagy),
suppression of lipogenetic pathways (i.e. inhibition of Citrate
export from the mitochondria by BTC). Conversely, autophagy inducer
Perhexiline can play an adaptive and protective role in ALD,
conferring to hepatocyte protection after ethanol intoxication and
inhibiting adipocytes differentiation.
[0063] NAFLD can be sub-classified as non-alcoholic steatohepatitis
(NASH) and nonalcoholic fatty liver (NAFL). Nonalcoholic fatty
liver (NAFL) is a type of NAFLD and is a condition in which fat
accumulates in the liver cells. NAFL has minimal risk of
progressing to cirrhosis. Nonalcoholic steatohepatitis (NASH) is
the more extreme form of NAFLD, and is regarded as a major cause of
fibrosis and cirrhosis of the liver of unknown cause. The major
feature in NASH is fat in the liver, along with inflammation and
damage. NASH can be severe and can lead to fibrosis and cirrhosis,
in which the liver is permanently damaged and scarred and no longer
able to work properly. Most patients with NAFLD have few or no
symptoms.
[0064] Patients may complain of fatigue, malaise, and dull
right-upper-quadrant abdominal discomfort. Mild jaundice may be
noticed although this is rare. Accordingly the complications of
NAFLD typically include liver fibrosis and subsequently cirrhosis.
Liver fibrosis is characterized by the accumulation of
extracellular matrix that can be distinguished qualitatively from
that in normal liver. Left unchecked, hepatic fibrosis progresses
to cirrhosis (defined by the presence of encapsulated nodules),
liver and organ failure, and death.
[0065] In some embodiments, the agent that inhibits the activity or
expression of DBI is particularly suitable for the treatment of
NASH.
[0066] In some embodiments, the subject suffers from pancreatitis,
which is an inflammatory disease of the exocrine pancreas,
culminating in a massive necrotic cell death of acinar cells.
Although the mechanisms promoting this pathology are still unclear,
there is a consensus on the notion that autophagy is impaired in
this pathological process. Acinar cells are characterized by large
autophagosomes unable to become autophagolysosomes, mainly due to
the depletion of lysosomal proteins (i.e. LAMP2). Furthermore, it
has been recently shown that loss of Ikka inhibits autophagy flux
and promotes the formation of p62-positive protein aggregates, thus
contributing to the initiation of the disease. In addition, during
the acute phase of the disease, a selective autophagy process
called `zymophagy` prevents acinar cells death through degradation
of harmful activated zymogen granules. agent of the present
invention such as the hydroxycitric acid can be tested for their
capacity to trigger zimophagy. Moreover, these agents, alone or in
combination with a lysosomal-targeted therapy, can be suitable for
ameliorating the symptomatology of the disease by restoring a
normal autophagic flux.
[0067] In some embodiments, the subject suffers from a
proteinopathy. Inducing autophagy by using agent of the present
invention may be particularly suitable for the treatment of
proteionpathies. Examples of proteinopathies include, but are not
limited to Alzheimer's disease, cerebral .beta.-amyloid angiopathy,
retinal ganglion cell degeneration, prion diseases (e.g. bovine
spongiform encephalopathy, kuru, Creutzfeldt- Jakob disease,
variant Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker
syndrome, fatal familial insomnia) tauopathies (e.g. frontotemporal
dementia, Alzheimer's disease, progressive supranuclear palsy,
corticobasal degeration, frontotemporal lobar degeneration),
frontemporal lobar degeneration, amyotrophic lateral sclerosis,
Huntington's disease, familial British dementia, Familial Danish
dementia, hereditary cerebral hemorrhage with amyloidosis
(Iclandic), CADASIL, Alexander disease, Seipinopathies, familial
amyloidotic neuropathy, senile systemic amyloidosis,
serpinopathies, AL amyloidosis, AA amyloidosis, type II diabetes,
aortic medial amyloidosis, ApoAI amyloidosis, ApoII amyloidosis,
ApoAIV amyloidosis, familial amyloidosis of the Finish type,
lysozyme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis,
inclusion body myositis/myopathy, cataracts, medullary thyroid
carcinoma, cardiac atrial amyloidosis, pituitary prolactinoma,
hereditary lattice corneal dystrophy, cutaneous lichen amyloidosis,
corneal lactoferrin amyloidosis, corneal lactoferrin amyloidosis,
pulmonary alveolar proteinosis, odontogenic tumor amylois, seminal
vesical amyloid, cystic fibrosis, sickle cell disease and critical
illness myopathy.
[0068] Agents that Inhibit the Activity or Expression of DBI:
[0069] In some embodiments, the agent that inhibits the activity of
DBI is an antibody directed against DBI.
[0070] As used herein, the term "antibody" is thus used to refer to
any antibody-like molecule that has an antigen binding region, and
this term includes antibody fragments that comprise an antigen
binding domain such as Fab', Fab, F(ab')2, single domain antibodies
(DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv,
Fd, linear antibodies, minibodies, diabodies, bispecific antibody
fragments, bibody, tribody (scFv-Fab fusions, bispecific or
trispecific, respectively); sc-diabody; kappa(lamda) bodies
(scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv
tandems to attract T cells); DVD-Ig (dual variable domain antibody,
bispecific format); SIP (small immunoprotein, a kind of minibody);
SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART
(ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody
mimetics comprising one or more CDRs and the like. The techniques
for preparing and using various antibody-based constructs and
fragments are well known in the art (see Kabat et al., 1991,
specifically incorporated herein by reference). Diabodies, in
particular, are further described in EP 404, 097 and WO 93/1 1 161;
whereas linear antibodies are further described in Zapata et al.
(1995). Antibodies can be fragmented using conventional techniques.
For example, F(ab')2 fragments can be generated by treating the
antibody with pepsin. The resulting F(ab')2 fragment can be treated
to reduce disulfide bridges to produce Fab' fragments. Papain
digestion can lead to the formation of Fab fragments. Fab, Fab' and
F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers,
minibodies, diabodies, bispecific antibody fragments and other
fragments can also be synthesized by recombinant techniques or can
be chemically synthesized. Techniques for producing antibody
fragments are well known and described in the art. For example,
each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall
et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and
Young et al., 1995 further describe and enable the production of
effective antibody fragments. In some embodiments, the antibody of
the present invention is a single chain antibody. As used herein
the term "single domain antibody" has its general meaning in the
art and refers to the single heavy chain variable domain of
antibodies of the type that can be found in Camelid mammals which
are naturally devoid of light chains. Such single domain antibody
is also called "nanobody.RTM.". For a general description of
(single) domain antibodies, reference is also made to the prior art
cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989
Oct. 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003,
21(11):484-490; and WO 06/030220, WO 06/003388. In natural
antibodies, two heavy chains are linked to each other by disulfide
bonds and each heavy chain is linked to a light chain by a
disulfide bond. There are two types of light chain, lambda (1) and
kappa (k). There are five main heavy chain classes (or isotypes)
which determine the functional activity of an antibody molecule:
IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence
domains. The light chain includes two domains, a variable domain
(VL) and a constant domain (CL). The heavy chain includes four
domains, a variable domain (VH) and three constant domains (CHI,
CH2 and CH3, collectively referred to as CH). The variable regions
of both light (VL) and heavy (VH) chains determine binding
recognition and specificity to the antigen. The constant region
domains of the light (CL) and heavy (CH) chains confer important
biological properties such as antibody chain association,
secretion, trans-placental mobility, complement binding, and
binding to Fc receptors (FcR). The Fv fragment is the N-terminal
part of the Fab fragment of an immunoglobulin and consists of the
variable portions of one light chain and one heavy chain. The
specificity of the antibody resides in the structural
complementarity between the antibody combining site and the
antigenic determinant. Antibody combining sites are made up of
residues that are primarily from the hypervariable or
complementarity determining regions (CDRs). Occasionally, residues
from nonhypervariable or framework regions (FR) can participate to
the antibody binding site or influence the overall domain structure
and hence the combining site. Complementarity Determining Regions
or CDRs refer to amino acid sequences which together define the
binding affinity and specificity of the natural Fv region of a
native immunoglobulin binding site. The light and heavy chains of
an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2,
L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An
antigen-binding site, therefore, typically includes six CDRs,
comprising the CDR set from each of a heavy and a light chain V
region. Framework Regions (FRs) refer to amino acid sequences
interposed between CDRs. The residues in antibody variable domains
are conventionally numbered according to a system devised by Kabat
et al. This system is set forth in Kabat et al., 1987, in Sequences
of Proteins of Immunological Interest, US Department of Health and
Human Services, NIH, USA (hereafter "Kabat et al."). This numbering
system is used in the present specification. The Kabat residue
designations do not always correspond directly with the linear
numbering of the amino acid residues in SEQ ID sequences. The
actual linear amino acid sequence may contain fewer or additional
amino acids than in the strict Kabat numbering corresponding to a
shortening of, or insertion into, a structural component, whether
framework or complementarity determining region (CDR), of the basic
variable domain structure. The correct Kabat numbering of residues
may be determined for a given antibody by alignment of residues of
homology in the sequence of the antibody with a "standard" Kabat
numbered sequence. The CDRs of the heavy chain variable domain are
located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2) and
residues 95-102 (H-CDR3) according to the Kabat numbering system.
The CDRs of the light chain variable domain are located at residues
24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3)
according to the Kabat numbering system.
[0071] In some embodiments, the antibody is directed against the
fragment consisting in the amino acid sequence ranging from the
amino acid residue at position 43 to the amino acid residue at
position 50 (i.e. the octapeptide or OP).
[0072] In some embodiments, the antibody of the present invention
is a chimeric antibody, typically a chimeric mouse/human antibody.
The term "chimeric antibody" refers to a monoclonal antibody which
comprises a VH domain and a VL domain of an antibody derived from a
non-human animal, a CH domain and a CL domain of a human antibody.
As the non-human animal, any animal such as mouse, rat, hamster,
rabbit or the like can be used. In particular, said mouse/human
chimeric antibody may comprise the heavy chain and the light chain
of the antibody of the present invention.
[0073] In some embodiments, the antibody is a humanized antibody.
As used herein, "humanized" describes antibodies wherein some, most
or all of the amino acids outside the CDR regions are replaced with
corresponding amino acids derived from human immunoglobulin
molecules. Methods of humanization include, but are not limited to,
those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089,
5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated
by reference.
[0074] In some embodiments, the antibody is a fully human antibody.
Fully human monoclonal antibodies also can be prepared by
immunizing mice transgenic for large portions of human
immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat.
Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and
references cited therein, the contents of which are incorporated
herein by reference.
[0075] In some embodiments, the antibody is a neutralizing
antibody. As used herein, the term "neutralizing antibody" is an
antibody that that inhibits, reduces or completely the activity of
DBI. Whether an antibody is a neutralizing antibody can be
determined by in vitro assays described in the EXAMPLE. Typically,
the neutralizing antibody of the present invention inhibits the
activity of DBI by at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or
100%.
[0076] In some embodiments, the neutralizing antibody of the
present invention does not mediate antibody-dependent cell-mediated
cytotoxicity and thus does not comprise an Fc portion that induces
antibody dependent cellular cytotoxicity (ADCC). In some
embodiments, the neutralizing antibody does not comprise an Fc
domain capable of substantially binding to a FcgRIIIA (CD16)
polypeptide. In some embodiments, the neutralizing antibody lacks
an Fc domain (e.g. lacks a CH2 and/or CH3 domain) or comprises an
Fc domain of IgG2 or IgG4 isotype. In some embodiments, the
neutralizing antibody consists of or comprises a Fab, Fab',
Fab'-SH, F (ab') 2, Fv, a diabody, single-chain antibody fragment,
or a multispecific antibody comprising multiple different antibody
fragments. In some embodiments, the neutralizing antibody is not
linked to a toxic moiety. In some embodiments, one or more amino
acids selected from amino acid residues can be replaced with a
different amino acid residue such that the antibody has altered C2q
binding and/or reduced or abolished complement dependent
cytotoxicity (CDC). This approach is described in further detail in
U.S. Pat. Nos. 6,194,551 by ldusogie et al.
[0077] In some embodiments, the agents that inhibits the activity
of DBI is an aptamer directed against DBI. Aptamers are a class of
molecule that represents an alternative to antibodies in term of
molecular recognition. Aptamers are oligonucleotide sequences with
the capacity to recognize virtually any class of target molecules
with high affinity and specificity. Such ligands may be isolated
through Systematic Evolution of Ligands by EXponential enrichment
(SELEX) of a random sequence library. The random sequence library
is obtainable by combinatorial chemical synthesis of DNA. In this
library, each member is a linear oligomer, eventually chemically
modified, of a unique sequence. Peptide aptamers consists of a
conformationally constrained antibody variable region displayed by
a platform protein, such as E. coli Thioredoxin A that are selected
from combinatorial libraries by two hybrid methods (Colas et al.,
1996).
[0078] In some embodiments, the agent that inhibits the expression
of DBI is an inhibitor of expression. An "inhibitor of expression"
refers to a natural or synthetic compound that has a biological
effect to inhibit the expression of a gene. In a preferred
embodiment of the invention, said inhibitor of gene expression is a
siRNA, an endonuclease, an antisense oligonucleotide or a
ribozyme.
[0079] In some embodiments, the inhibitor of expression is a siRNA.
Small inhibitory RNAs (siRNAs) can also function as inhibitors of
expression for use in the present invention. DBI gene expression
can be reduced by contacting a patient or cell with a small double
stranded RNA (dsRNA), or a vector or construct causing the
production of a small double stranded RNA, such that DBI gene
expression is specifically inhibited (i.e. RNA interference or
RNAi).
[0080] In some embodiments, the inhibitor of expression is an
endonuclease. The term "endonuclease" refers to enzymes that cleave
the phosphodiester bond within a polynucleotide chain. Some, such
as Deoxyribonuclease I, cut DNA relatively nonspecifically (without
regard to sequence), while many, typically called restriction
endonucleases or restriction enzymes, cleave only at very specific
nucleotide sequences. The mechanism behind endonuclease-based
genome inactivating generally requires a first step of DNA single
or double strand break, which can then trigger two distinct
cellular mechanisms for DNA repair, which can be exploited for DNA
inactivating: the error prone non-homologous end joining (NHEJ) and
the high-fidelity homology-directed repair (HDR). In a particular
embodiment, the endonuclease is CRISPR-Cas. As used herein, the
term "CRISPR-Cas" has its general meaning in the art and refers to
clustered regularly interspaced short palindromic repeats
associated which are the segments of prokaryotic DNA containing
short repetitions of base sequences. In some embodiment, the
endonuclease is CRISPR-cas9, which is from Streptococcus pyogenes.
The CRISPR/Cas9 system has been described in U.S. Pat. No.
8,697,359 B1 and US 2014/0068797. In some embodiment, the
endonuclease is CRISPR-Cpf1 which is the more recently
characterized CRISPR from Provotella and Francisella 1 (Cpf1) in
Zetsche et al. ("Cpf1 is a Single RNA-guided Endonuclease of a
Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
[0081] In some embodiments, the inhibitor of expression is an
antisense oligonucleotide. The term "antisense oligonucleotide"
refers to an oligonucleotide sequence that is inverted relative to
its normal orientation for transcription and so expresses an RNA
transcript that is complementary to a target gene mRNA molecule
expressed within the host cell (e.g., it can hybridize to the
target gene mRNA molecule through Watson-Crick base pairing). An
antisense strand may be constructed in a number of different ways,
provided that it is capable of interfering with the expression of a
target gene. For example, the antisense strand can be constructed
by inverting the coding region (or a portion thereof) of the target
gene relative to its normal orientation for transcription to allow
the transcription of its complement, (e.g., RNAs encoded by the
antisense and sense gene may be complementary). Furthermore, the
antisense oligonucleotide strand need not have the same intron or
exon pattern as the target gene, and noncoding segments of the
target gene may be equally effective in achieving antisense
suppression of target gene expression as coding segments. As used
herein, the term "oligonucleotide" refers to a nucleic acid
sequence, 3'-5' or 5'-3' oriented, which may be single-or
double-stranded. The antisense oligonucleotide used in the context
of the invention may in particular be DNA or RNA. According to the
invention, the antisense oligonucleotide of the present invention
targets an mRNA encoding DBI (e.g. SEQ ID NO:2), and is capable of
reducing the amount of DBI in cells. As used herein, an
oligonucleotide that "targets" an mRNA refers to an oligonucleotide
that is capable of specifically binding to said mRNA. That is to
say, the antisense oligonucleotide comprises a sequence that is at
least partially complementary, preferably perfectly complementary,
to a region of the sequence of said mRNA, said complementarity
being sufficient to yield specific binding under intra-cellular
conditions. As immediately apparent to the skilled in the art, by a
sequence that is "perfectly complementary to" a second sequence is
meant the reverse complement counterpart of the second sequence,
either under the form of a DNA molecule or under the form of a RNA
molecule. A sequence is "partially complementary to" a second
sequence if there are one or more mismatches. The antisense
oligonucleotide of the present invention that target an mRNA
encoding DBI may be designed by using the sequence of said mRNA as
a basis, e.g. using bioinformatic tools. For example, the sequence
of SEQ ID NO: 2 can be used as a basis for designing nucleic acids
that target an mRNA encoding DBI. Preferably, the antisense
oligonucleotide according to the invention is capable of reducing
the amount of DBI in cells, e.g. in cancerous cells. Methods for
determining whether an oligonucleotide is capable of reducing the
amount of DBI in cells are known to the skilled in the art. This
may for example be done by analyzing DBI protein expression by
Western blot, and by comparing DBI protein expression in the
presence and in the absence of the antisense oligonucleotide to be
tested. In some embodiments, the antisense oligonucleotide of the
present invention has a length of from 12 to 50 nucleotides, e.g.
12 to 35 nucleotides, from 12 to 30, from 12 to 25, from 12 to 22,
from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 22, from 18
to 22, or about 19, 20 or 21 nucleotides. The antisense
oligonucleotide according to the invention may for example comprise
or consist of 12 to 50 consecutive nucleotides, e.g. 12 to 35, from
12 to 30, from 12 to 25, from 12 to 22, from 15 to 35, from 15 to
30, from 15 to 25, from 15 to 22, from 18 to 22, or about 19, 20 or
21 consecutive nucleotides of a sequence complementary to the mRNA
of SEQ ID NO: 2. In some embodiments, the antisense oligonucleotide
of the present invention is further modified, preferably chemically
modified, in order to increase the stability and/or therapeutic
efficiency of the antisense oligonucleotide in vivo. In particular,
the antisense oligonucleotide used in the context of the invention
may comprise modified nucleotides. Chemical modifications may occur
at three different sites: (i) at phosphate groups, (ii) on the
sugar moiety, and/or (iii) on the entire backbone structure of the
antisense oligonucleotide. For example, the antisense
oligonucleotide may be employed as phosphorothioate derivatives
(replacement of a non-bridging phosphoryl oxygen atom with a sulfur
atom) which have increased resistance to nuclease digestion.
2'-methoxyethyl (MOE) modification (such as the modified backbone
commercialized by ISIS Pharmaceuticals) is also effective.
Additionally or alternatively, the antisense oligonucleotide of the
present invention may comprise completely, partially or in
combination, modified nucleotides which are derivatives with
substitutions at the 2' position of the sugar, in particular with
the following chemical modifications: O-methyl group (2'-O-Me)
substitution, 2-methoxyethyl group (2'-O-MOE) substitution, fluoro
group (2'-fluoro) substitution, chloro group (2'-C1) substitution,
bromo group (2'-Br) substitution, cyanide group (2'-CN)
substitution, trifluoromethyl group (2'-CF3) substitution, OCF3
group (2'-OCF3) substitution, OCN group (2'-OCN) substitution,
O-alkyl group (2'-O-alkyl) substitution, S-alkyl group (2'-S-alkyl)
substitution, N-alkyl group (2'-N-akyl) substitution, O-alkenyl
group (2'-O-alkenyl) substitution, S-alkenyl group (2'-S-alkenyl)
substitution, N-alkenyl group (2'-N-alkenyl) substitution, SOCH3
group (2'-SOCH3) substitution, SO2CH3 group (2'-SO2CH3)
substitution, ONO2 group (2'-ONO2) substitution, NO2 group (2'-NO2)
substitution, N3 group (2'-N3) substitution and/or NH2 group
(2'-NH2) substitution. Additionally or alternatively, the antisense
oligonucleotide of the present invention may comprise completely or
partially modified nucleotides wherein the ribose moiety is used to
produce locked nucleic acid (LNA), in which a covalent bridge is
formed between the 2' oxygen and the 4' carbon of the ribose,
fixing it in the 3'-endo configuration. These constructs are
extremely stable in biological medium, able to activate RNase H and
form tight hybrids with complementary RNA and DNA.
[0082] Accordingly, in a preferred embodiment, the antisense
oligonucleotide used in the context of the invention comprises
modified nucleotides selected from the group consisting of LNA,
2'-OMe analogs, 2'-phosphorothioate analogs, 2'-fluoro analogs,
2'-Cl analogs, 2'-Br analogs, 2'-CN analogs, 2'-CF3 analogs,
2'-OCF3 analogs, 2'-OCN analogs, 2'-O-alkyl analogs, 2'-S-alkyl
analogs, 2'-N-alkyl analogs, 2'-O-alkenyl analogs, 2'-S-alkenyl
analogs, 2'-N-alkenyl analogs, 2'-SOCH3 analogs, 2'-SO2CH3 analogs,
2'-ONO2 analogs, 2'-NO2 analogs, 2'-N3 analogs, 2'-NH2 analogs and
combinations thereof. More preferably, the modified nucleotides are
selected from the group consisting of LNA, 2'-OMe analogs,
2'-phosphorothioate analogs and 2'-fluoro analogs. In some
embodiments, the antisense is a Tricyclo-DNA antisense. The term
"tricyclo-DNA (tc-DNA)" refers to a class of constrained
oligodeoxyribonucleotide analogs in which each nucleotide is
modified by the introduction of a cyclopropane ring to restrict
conformational flexibility of the backbone and to optimize the
backbone geometry of the torsion angle y as (Ittig D, et al.,
Nucleic Acids Res, 2004, 32:346-353; Ittig D, et al., Prague,
Academy of Sciences of the Czech Republic. 1:21-26 (Coll. Symp.
Series, Hocec, M., 2005); Ivanova et al., Oligonucleotides 2007,
17:54-65; Renneberg D, et al., Nucleic Acids Res, 2002, 15
30:2751-2757; Renneberg D, et al., Chembiochem, 2004, 5:1114-1118;
and Renneberg D, et al., JACS, 2002, 124:5993-6002). In detail, the
tc-DNA differs structurally from DNA by an additional ethylene
bridge between the centers C(3') and C(5') of the nucleosides, to
which a cyclopropane unit is fused for further enhancement of
structural rigidity. See e.g. WO2010115993 for examples of
tricyclo- DNA (tc-DNA) antisense oligonucleotides. The advantage of
the tricyclo-DNA chemistry is that the structural properties of its
backbone allow a reduction in the length of an AON while retaining
high affinity and highly specific hybridization with a
complementary nucleotide sequence. Unexpectedly, tc-DNA AON may be
advantageously used in microgram dosages in the in vivo setting
using intramuscular application, which are at least 10-fold less
than the dosages required for conventional antisense
oligonucleotide technologies. In addition, tc-DNA retains full
activity with reduced antisense lengths. Thus, for example, tc-DNA
AON of 13 to 15 nucleotides are highly effective in the ex vivo and
in vivo applications exemplified by the present disclosure.
[0083] In some embodiments, the agent that inhibits the activity of
DBI consists in a vaccine composition suitable for eliciting
neutralizing autoantibodies against DBI when administered to the
subject. For the purpose of the present invention, the term
"vaccine composition" is intended to mean a composition which can
be administered to humans or to animals in order to induce an
immune system response; this immune system response can result in
the production of antibodies against DBI. Typically, the vaccine
composition comprises at least one antigen derived from DBI. As
used herein the term "antigen" refers to a molecule capable of
being specifically bound by an antibody or by a T cell receptor
(TCR) if processed and presented by MHC molecules. The term
"antigen", as used herein, also encompasses T-cell epitopes. An
antigen is additionally capable of being recognized by the immune
system and/or being capable of inducing a humoral immune response
and/or cellular immune response leading to the activation of B-
and/or T-lymphocytes. An antigen can have one or more epitopes or
antigenic sites (B- and T- epitopes). In some embodiments, the
antigen consists in a polypeptide comprising an amino acid sequence
having at least 80% of identity with the sequence of SEQ ID NO:1 or
a fragment thereof (e.g. an epitope). In some embodiments, the
antigen consists in a polypeptide comprising i) an amino acid
sequence having at least 80% of identity with SEQ ID NO:1, or ii)
an amino acid sequence having at least 80% of identity with the
amino acid sequence ranging from the amino acid residue at position
17 to the amino acid residue at position 50 in SEQ ID NO:1, or iii)
an amino acid sequence having at least 80% of identity with the
amino acid sequence ranging from the amino acid residue at position
33 to the amino acid residue at position 50 in SEQ ID NO:1, or iv)
an amino acid sequence having at least 80% of identity with the
amino acid sequence ranging from the amino acid residue at position
43 to the amino acid residue at position 50 in SEQ ID NO:l. In some
embodiments, the polypeptide is conjugated to a carrier protein
which is generally sufficiently foreign to elicit a strong immune
response to the vaccine. Illustrative carrier proteins are
inherently highly immunogenic. Both bovine serum albumin (BSA) and
keyhole limpet hemocyanin (KLH) have commonly been used as carriers
in the development of conjugate vaccines when experimenting with
animals and are contemplated herein as carrier proteins. Proteins
which have been used in the preparation of therapeutic conjugate
vaccines include, but are not limited to, a number of toxins of
pathogenic bacteria and their toxoids. Suitable carrier molecules
are numerous and include, but are not limited to: Bacterial toxins
or products, for example, cholera toxin B-(CTB), diphtheria toxin,
tetanus toxoid, and pertussis toxin and filamentous hemagglutinin,
shiga toxin, pseudomonas exotoxin; Lectins, for example, ricin-B
subunit, abrin and sweet pea lectin; Sub virals, for example,
retrovirus nucleoprotein (retro NP), rabies ribonucleoprotein
(rabies RNP), plant viruses (e.g. TMV, cow pea and cauliflower
mosaic viruses), vesicular stomatitis virus-nucleocapsid protein
(VSV-N), poxvirus vectors and Semliki forest virus vectors;
Artificial vehicles, for example, multiantigenic peptides (MAP),
microspheres; Yeast virus-like particles (VLPs); Malarial protein
antigen; and others such as proteins and peptides as well as any
modifications, derivatives or analogs of the above. Other useful
carriers include those with the ability to enhance a mucosal
response, more particularly, LTB family of bacterial toxins,
retrovirus nucleoprotein (retro NP), rabies ribonucleoprotein
(rabies RNP), vesicular stomatitis virus-nucleocapsid protein
(VSV-N), and recombinant .pox virus subunits.
[0084] In some embodiments, the vaccine composition of the present
invention comprises an adjuvant. The term "adjuvant" can be a
compound that lacks significant activity administered alone but can
potentiate the activity of another therapeutic agent. In some
embodiments, the adjuvant is Incomplete Freund's adjuvant (IFA) or
other oil based adjuvant is present between 30-70%, preferably
between 40-60%, more preferably between 45-55% proportion weight by
weight (w/w). In some embodiments, the vaccine composition of the
present invention comprises at least one Toll-Like Receptor (TLR)
agonist which is selected from the group consisting of TLR1, TLR2,
TLR3, TLR4, TLRS, TLR6, TLR7, and TLR8 agonists. Other adjuvants
include cytokines, such as interleukins (IL-1, IL-2, and IL-12),
macrophage colony stimulating factor (M-CSF), and tumor necrosis
factor (TNF). In one particular embodiment, the adjuvant is an
emulsion having adjuvanting properties. Such emulsions include
oil-in-water emulsions. Freund's incomplete adjuvant (IFA) is one
such adjuvant. Another suitable oil-in-water emulsion is MF59.TM.
adjuvant, which contains squalene, polyoxyethylene sorbitan
monooleate (also known as Tween.TM. 80 surfactant), and sorbitan
trioleate. Squalene is a natural organic compound originally
obtained from shark liver oil, although also available from plant
sources (primarily vegetable oils), including amaranth seed, rice
bran, wheat germ, and olives. Other suitable adjuvants are
MontanideTM adjuvants (Seppic Inc., Fairfield N.J.) including
Montanide.TM. ISA 50V, which is a mineral oil-based adjuvant;
Montanide.TM. ISA 206; and Montanide.TM. IMS 1312. While mineral
oil may be present in the co-adjuvant, in some embodiments the oil
component(s) of the compositions described herein are all
metabolizable oils.
[0085] Pharmaceutical Compositions:
[0086] The agent that modulates (i.e. promotes or inhibits) the
activity or expression of DBI is administered to the subject in a
form of a pharmaceutical composition. Typically, the agent of the
present invention can be combined with pharmaceutically acceptable
excipients, and optionally sustained-release matrices, such as
biodegradable polymers, to form therapeutic compositions.
"Pharmaceutically" or "pharmaceutically acceptable" refer to
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to a mammal,
especially a human, as appropriate. A pharmaceutically acceptable
carrier or excipient refers to a non-toxic solid, semi-solid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any type. In the pharmaceutical compositions of the
present invention for oral, sublingual, subcutaneous,
intramuscular, intravenous, transdermal, local or rectal
administration, the active principle, alone or in combination with
another active principle, can be administered in a unit
administration form, as a mixture with conventional pharmaceutical
supports, to the subjects. Suitable unit administration forms
comprise oral-route forms such as tablets, gel capsules, powders,
granules and oral suspensions or solutions, sublingual and buccal
administration forms, aerosols, implants, subcutaneous,
transdermal, topical, intraperitoneal, intramuscular, intravenous,
subdermal, transdermal, intrathecal and intranasal administration
forms and rectal administration forms. Typically, the
pharmaceutical compositions contain vehicles, which are
pharmaceutically acceptable for a formulation capable of being
injected. These may be in particular isotonic, sterile, saline
solutions (monosodium or disodium phosphate, sodium, potassium,
calcium or magnesium chloride and the like or mixtures of such
salts), or dry, especially freeze-dried compositions which upon
addition, depending on the case, of sterilized water or
physiological saline, permit the constitution of injectable
solutions. The pharmaceutical forms suitable for injectable use
include sterile aqueous solutions or dispersions; formulations
including sesame oil, peanut oil or aqueous propylene glycol; and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases, the form must be
sterile and must be fluid to the extent that easy syringability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi. Solutions comprising
compounds of the present invention as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms. The agent of
the present invention can be formulated into a composition in a
neutral or salt form. Pharmaceutically acceptable salts include the
acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. The carrier can
also be a solvent or dispersion medium containing, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol,
and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetables oils. The proper fluidity can be
maintained, for example, by the use of a coating, such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminium
monostearate and gelatin. Sterile injectable solutions are prepared
by incorporating the active compounds in the required amount in the
appropriate solvent with several of the other ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the various
sterilized active ingredients into a sterile vehicle which contains
the basic dispersion medium and the required other ingredients from
those enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
preparation of more, or highly concentrated solutions for direct
injection is also contemplated, where the use of DMSO as solvent is
envisioned to result in extremely rapid penetration, delivering
high concentrations of the active agents to a small tumor area.
Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed. For parenteral administration in an aqueous
solution, for example, the solution should be suitably buffered if
necessary and the liquid diluent first rendered isotonic with
sufficient saline or glucose. These particular aqueous solutions
are especially suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. In this
connection, sterile aqueous media which can be employed will be
known to those of skill in the art in light of the present
disclosure. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject.
[0087] Methods of Screening:
[0088] A further object of the present invention relates to a
method of screening a compound suitable for modulating autophagy
comprising i) providing a candidate compound ii) determining
whether the candidate compound is capable of modulating the
activity or expression of DBI and iii) positively selecting the
candidate compound which is capable of modulating the activity or
expression of DBI.
[0089] According to one embodiment of the invention, the candidate
compound of may be selected from a library of compounds previously
synthesised, or a library of compounds for which the structure is
determined in a database, or from a library of compounds that have
been synthesised de novo or natural compounds. The candidate
compound may be selected from the group of (a) proteins or
peptides, (b) nucleic acids and (c) organic or chemical compounds
(natural or not). Illustratively, libraries of pre-selected
candidate nucleic acids may be obtained by performing the SELEX
method as described in documents U.S. Pat. No. 5,475,096 and U.S.
Pat. No. 5,270,163. In some embodiments, the candidate compound is
a peptide derived from DBI or a peptidomimetic of DBI.
[0090] Testing whether a candidate compound can modulate the
activity or expression of DBI can be determined using or routinely
modifying assays known in the art. For example, the method may
involve contacting cells expressing DBI with the candidate
compound, and measuring the DBI mediated activity, and comparing
the cellular response to a standard cellular response. Typically,
the standard cellular response is measured in absence of the
candidate compound. A decreased cellular response over the standard
indicates that the candidate compound is capable of inhibiting the
activity of DBI. On contrary in increased cellular response over
the standard indicates that the candidate compound is capable of
promoting the activity of DBI. In some embodiments, the invention
provides a method for identifying a ligand which binds specifically
to the receptor of DBI. For example, a cellular compartment, such
as a membrane or a preparation thereof, may be prepared from a cell
that expresses a molecule that binds to the receptor of DBI.
Methods of determining the expression of a gene are also well known
in the art and typically reporter assays (e.g. a cell which express
a nucleic acid molecule under the promoter of DBI gene, or cell
which express of form of DBI labelled with a detectable moiety) or
any assays for determining the expression at nucleic acid level
(e.g. RT-PCR).
[0091] In some embodiments, the candidate compounds that have been
positively selected may be subjected to further selection steps in
view of further assaying its properties on autophagy (e.g. by means
of endogenous fluorescent biosensors or exogenous fluorescent
probes such as described in the EXAMPLE). In some embodiments, tthe
candidate compounds that have been positively selected may be
subjected to further selection steps in view of further assaying
its properties on different in vitro or in vivo assays. For
instance, the selected compound is assayed for its ability to
modulate glycaemia, modulate food intake, modulate of weight gain
or loss, modulate fatty acid oxidation, modulate the membrane
expression of glucose transporter (e.g. GLUT-1 or GLUT-4), modulate
the expression of PPARG, modulate glucose uptake (e.g. uptake of
non-radioactive or radioactive glucose isotopes), modulate
glycolysis or modulate lipogenesis. Such assays are typically
described in the EXAMPLE.
[0092] Biomarkers:
[0093] A further object of the present invention relates to a
method of determining whether a subject is at risk of weight
modulation comprising i) determining the level of DBI in a blood
sample obtained from the subject, ii) comparing the level
determined at step i) with a predetermined reference value and ii)
concluding that the subject is at risk of weight modulation when a
differential between the level determined at step i) and the
predetermined reference value is determined.
[0094] In some embodiments, the method of the present invention is
particularly suitable for determining whether the subject is at
risk of weight gain when the level determined at step i) is higher
than the predetermine reference value. In some embodiments, the
method of the present invention is particularly suitable for
determining whether the subject is at risk of weight loss when the
level determined at step i) is lower than the predetermine
reference value.
[0095] The method of the present invention is particularly suitable
for determining whether the subject achieves a response with a diet
or drug suitable for modulating the weight gain or loss. In some
embodiments, the method of the present invention is particularly
suitable for determining the risk of recurrence.
[0096] As used herein, the term "blood sample" means any blood
sample derived from the patient that contains DBI s. In some
embodiments, the blood sample is a serum or plasma sample liable to
contain DBI.
[0097] For instance when the level is determined by any
conventional method for determining the level of a protein in a
sample can be used. In some embodiments, the methods of the
invention comprise contacting the blood sample with a binding
partner capable of selectively interacting with the protein liable
to be present in the blood sample. The binding partner may be an
antibody that may be polyclonal or monoclonal, preferably
monoclonal. In another embodiment, the binding partner may be an
aptamer. Polyclonal antibodies of the invention or a fragment
thereof can be raised according to known methods by administering
the appropriate antigen or epitope to a host animal selected, e.g.,
from pigs, cows, horses, rabbits, goats, sheep, and mice, among
others. Various adjuvants known in the art can be used to enhance
antibody production. Although antibodies useful in practicing the
invention can be polyclonal, monoclonal antibodies are preferred.
Monoclonal antibodies of the invention or a fragment thereof can be
prepared and isolated using any technique that provides for the
production of antibody molecules by continuous cell lines in
culture. Techniques for production and isolation include but are
not limited to the hybridoma technique. In some embodiments, the
binding partner may be an aptamer. Aptamers are a class of molecule
that represents an alternative to antibodies in term of molecular
recognition. Aptamers are oligonucleotide sequences with the
capacity to recognize virtually any class of target molecules with
high affinity and specificity. The binding partners of the
invention such as antibodies or aptamers, may be labelled with a
detectable molecule or substance, such as a fluorescent molecule, a
radioactive molecule or any others labels known in the art. Labels
are known in the art that generally provide (either directly or
indirectly) a signal. As used herein, the term "labelled", with
regard to the antibody, is intended to encompass direct labelling
of the antibody or aptamer by coupling (i.e., physically linking) a
detectable substance, such as a radioactive agent or a fluorophore
(e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or
Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect
labelling of the probe or antibody by reactivity with a detectable
substance. An antibody or aptamer of the invention may be labelled
with a radioactive molecule by any method known in the art. The
afore mentioned assays generally involve the binding of the binding
partner (ie. antibody or aptamer) to a solid support. Solid
supports which can be used in the practice of the invention include
substrates such as nitrocellulose (e. g., in membrane or microtiter
well form); polyvinylchloride (e. g., sheets or microtiter wells);
polystyrene latex (e.g., beads or microtiter plates);
polyvinylidine fluoride; diazotized paper; nylon membranes;
activated beads, magnetically responsive beads, and the like. The
level of biomarker protein may be measured by using standard immuno
diagnostic techniques, including immunoassays such as competition,
direct reaction, or sandwich type assays. Such assays include, but
are not limited to, agglutination tests; enzyme-labelled and
mediated immunoassays, such as ELISAs; biotin/avidin type assays;
radioimmunoassays; Immunoelectrophoresis; immunoprecipitation. More
particularly, an ELISA method can be used, wherein e.g. the wells
of a microtiter plate are coated with a set of antibodies which
recognize said biomarker protein. A blood sample containing or
suspected of containing said biomarker protein is then added to the
coated wells. After a period of incubation sufficient to allow the
formation of antibody- antigen complexes, the plate(s) can be
washed to remove unbound moieties and a detectably labelled
secondary binding molecule added. The secondary binding molecule is
allowed to react with any captured sample marker protein, the plate
washed and the presence of the secondary binding molecule detected
using methods well known in the art. In some embodiments, the
immunoassay may involve the use of 2 antibodies having specificity
for the protein. Typically, a first antibody is used as to "detect"
the protein and the second antibody is used to "capture" the
protein. In some embodiments, the method is achieved by i)
providing a solid support coating with an amount of first
antibodies specific for the protein, ii) bringing the sample into
contact with the solid support, iii) and adding an amount of the
second antibodies conjugated to a label. Measuring the amount of
bound binding partner which is specific for the label reveals the
amount of the protein present in the sample. Typically, the first
antibody is directed to an epitope which does not prevent the
interaction with the second antibody. Typically washing steps (with
any appropriate buffer such as PBS with or without a non-ionic
detergent) are performed after steps ii) and iii). Typically, a
blocking step is performed with a buffer containing BSA or milk
and/or serum (goat or bovine) to block non-specific binding of the
proteins. Measuring the level of the biomarker protein (with or
without immunoassay-based methods) may also include separation of
the compounds: centrifugation based on the compound's molecular
weight; electrophoresis based on mass and charge; HPLC based on
hydrophobicity; size exclusion chromatography based on size; and
solid-phase affinity based on the compound's affinity for the
particular solid-phase that is used. Once separated, said biomarker
protein may be identified based on the known "separation profile"
e. g., retention time, for that compound and measured using
standard techniques. Alternatively, the protein of interest (e.g.
DBI) may be detected and measured by, for example, a mass
spectrometer.
[0098] In some embodiments, the predetermined reference value is a
threshold value or a cut-off value. Typically, a "threshold value"
or "cut-off value" can be determined experimentally, empirically,
or theoretically. A threshold value can also be arbitrarily
selected based upon the existing experimental and/or clinical
conditions, as would be recognized by a person of ordinary skilled
in the art. For example, retrospective measurement of expression
level of DBI in properly banked historical patient samples may be
used in establishing the predetermined reference value. In some
embodiments, the predetermined reference value is derived from the
level of DBI in a control sample derived from one or more subjects
who are substantially healthy (i.e. a normal BMI as above defined).
The predetermined reference value has to be determined in order to
obtain the optimal sensitivity and specificity according to the
function of the test and the benefit/risk balance (clinical
consequences of false positive and false negative). Typically, the
optimal sensitivity and specificity (and so the threshold value)
can be determined using a Receiver Operating Characteristic (ROC)
curve based on experimental data. For example, after determining
the level of the marker in a group of reference, one can use
algorithmic analysis for the statistic treatment of the measured
levels of the marker in samples to be tested, and thus obtain a
classification standard having significance for sample
classification. The full name of ROC curve is receiver operator
characteristic curve, which is also known as receiver operation
characteristic curve. It is mainly used for clinical biochemical
diagnostic tests. ROC curve is a comprehensive indicator that
reflects the continuous variables of true positive rate
(sensitivity) and false positive rate (1-specificity). It reveals
the relationship between sensitivity and specificity with the image
composition method. A series of different cut-off values
(thresholds or critical values, boundary values between normal and
abnormal results of diagnostic test) are set as continuous
variables to calculate a series of sensitivity and specificity
values. Then sensitivity is used as the vertical coordinate and
specificity is used as the horizontal coordinate to draw a curve.
The higher the area under the curve (AUC), the higher the accuracy
of diagnosis. On the ROC curve, the point closest to the far upper
left of the coordinate diagram is a critical point having both high
sensitivity and high specificity values. The AUC value of the ROC
curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic
result gets better and better as AUC approaches 1. When AUC is
between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7
and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the
accuracy is quite high. This algorithmic method is preferably done
with a computer. Existing software or systems in the art may be
used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1
medical statistical software, SPSS 9.0, ROCPOWER.SAS,
DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0
(Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.
[0099] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
FIGURES
[0100] FIG. 1. Effects of DBI and DBI its inhibition on autophagy
in human and mouse. (A-D). Effects of extracellular DBI on
autophagy in cultured cells. H4-GFP-LC3 cells were cultured for 6 h
in the presence of neutralizing DBI antibody (that was optionally
heat-inactivated) in the absence or presence of BAFA1 during the
final 2 h (A). Alternatively, WT H4 cells were cultured in similar
conditions followed by the detection of autophagy-associated LC3-II
(B). H4-GFP-LC3 cells (C) or WT H4 cells (D) were cultured with
recombinant (rec.) DBI protein, with or without BAFA1 and autophagy
was measured. *P<0.05, **P<0.01, ***P<0.001 (Student
t-test) as compared to isotype or untreated controls. (E,F).
Effects of extracellular DBI on autophagy in mice. Mice were
intraperitoneally injected with a neutralizing DBI-specific
antibody (E) or intravenously with recDBI (F). After 4h of
treatment the mice were treated with leupeptine and livers were
recovered 2 hours later and autophagy-related parameters (LC3-II
increase, SQSTM1 reduction) were monitored by immunoblot (n=3 mice
per group).
[0101] FIG. 2. Plasma DBI concentrations in patients with anorexia
or obesity. Plasma DBI was measured in cohorts of patients with
anorexia nervosa (A), obesity (B) or obesity before or one year
after bariatric surgery (C), as compared to age- and sex-matched
normal weight controls (A, B). Results are means .+-.SEM.
***P<0.001 (Student t-test). Results are means of 5 mice per
group. *P<0.05, **P<0.01, ***P<0.001. Complete data are
shown in Table S1.
[0102] FIG. 3. Glycolytic and orexogenic effects of DBI. (A-C).
Hydrodynamic injection of a DBI-encoding vector. Mice (n=5 per
group) received i.v. injection of vector only or a construct
expressing mouse Dbi cDNA, and glycemia (A), food intake (B) or
weight gain (C) were monitored over time. Results are means
.+-.SEM. *P<0.05, **P<0.01, ***P<0.001 for comparisons to
vector-only controls. (D-H). Effects of recombinant (rec.) DBI
protein on whole-body metabolism. Mice (n=5 per group) were
injected intravenously with vehicle only or rec. DBI or
alternatively the DBI-derived peptides TTN or ODN and glycemia (D),
while food intake (E, F) and weight gain (G) were measured at the
indicated time points. Alternatively, rec. DBI protein was
administered as indicated by arrows, and fatty acid oxidation was
measured by respirometry over 24 hours (H). *P<0.05,
**P<0.01, ***P<0.001 for comparisons to vehicle controls.
[0103] FIG. 4. Anorexigenic effects of extracellular DBI
neutralization with specific antibodies. (A-D). Effects of DBI
neutralization on glucose as well as on feeding behavior. Plasma
glucose levels were measured in fed or unfed (24 hours) mice (n=5
per group), 30 min after i.p. injection of a monoclonal anti-DBI
(anti-DBI mAb 7A) (A) or a policlonal anti-DBI (anti-DBI, ab16871)
(C) and an isotype control antibody. Food intake after refeeding
was monitored over time (B, D). Results are expressed as
means.+-.SEM (n=5). *P<0.05, ***P<0.001, indicate anti-DBI
effects as compared to isotype controls and $ P<0.05, $$$
P<0.001, denote anti-DBI effects in unfed mice as compared to
control unfed mice.
[0104] FIG. 5. DBI neutralization induces a global suppression of
lipogenesis in liver. The effects of DBI neutralization on the
expression of various hepatic proteins was measured by
immunoblotting, each lane representing one mouse (A). Quantitative
results (B) are means.+-.SEM (N=5) *p<0.05 as compared to fed
mice receiving isotype.
[0105] FIG. 6. Anorexigenic effects of auto-immunization against
DBI. Mice were injected with keyhole limpet hemocyanin (KLH) alone
or KLH conjugated to rec. DBI protein (KLH-DBI). Mice developing
IgG autoantibodies against DBI were compared to KLH-only immunized
controls to monitor weight loss (5 animals per group) in starvation
conditions (A), food intake after 24h starvation (B), and to
measure cumulative weight gain of the mice (7-8 per group) on a
normal (C) or a high-fat diet (D). *P<0.05, **P<0.01,
***P<0.001 for the effects of DBI-specific auto antibodies.
EXAMPLE
[0106] Materials and Methods
[0107] Chemicals, cell lines and culture conditions. Unless
otherwise indicated, media and supplements for cell culture were
purchased from Gibco-Invitrogen (Carlsbad, Calif., USA), plastic
ware from Corning B.V. Life Sciences (Schiphol-Rijk, The
Netherlands), and chemicals from Sigma-Aldrich (St Louis, Mo.,
USA). All cell lines were cultured at 37.degree. C. under 5%
CO.sub.2, in a medium containing 10% fetal bovine serum, 100 mg/L
sodium pyruvate, 10 mM HEPES buffer, 100 units/mL penicillin G
sodium and 100 .mu.g/mL streptomycin sulfate. In addition, cell
type-specific culture conditions include Dulbecco's modified
Eagle's medium (DMEM) for human cervical carcinoma HeLa cells and
human brain neuroglioma H4 cells as well as their
GFP-LC3-expressing derivatives. Minimum Essential Medium Eagle
(EMEM) supplemented as above plus 2mM Glutamine and 1%
non-essential amino acids (NEAA) for human hepatocyte carcinoma Hep
G2 cells. Cells were seeded in 6-, 94-well plates and grown for 24
h before treatment, for the indicated period alone and /or in
combination, with 50 nM bafilomycin Al (BafAl, Tocris), 100 nM
Rapamycin (Rapa), antibody against DBI (antiDBl), recombinant
protein DBI (recDBI). For serum and nutrient deprivation (NF),
cells were cultured in serum-free Hank's balanced salt solution
(HBSS).
[0108] Plasmid transfection and RNA interference in human cell
cultures. Plasmids encoding DBI cDNAs were obtained from OriGene
(Rockville, MD, USA). Transient plasmid transfections were
performed with the AttracteneR reagent (Qiagen, Hilden, Germany),
and, unless otherwise indicated, cells were analyzed 24 h after
transfection. Cells were cultured in 6-wells or 96-wells plates and
transfected at 50% confluence. siRNAs were reverse-transfected with
the help of the RNAi MaxTM transfection reagent (Invitrogen,
Eugene, USA) in the presence of 100 nM of siRNAs specific for DBI
and TSPO (Qiagen), a scrambled siRNA was used as control.
siRNA-mediated protein downregulation was controlled by
immunoblots.
[0109] Immunofluorescence. Cells were fixed with 4% PFA for 15 min
at room temperature, and permeabilized with 0.1% Triton X-100 for
10 min. Non-specific binding sites were blocked with 5% bovine
serum in PBS, followed by overnight staining with primary
antibodies at 4 .degree. C. Cells were stained for the detection of
DBI (Santa Cruz). Primary antibodies were developed with the
appropriate AlexaFluor.sup.TM conjugates (Molecular
Probes-Invitrogen). Nuclei were labeled with Hoechst 33342 (10
.mu.g/m1). Standard and confocal fluorescence microscopy
assessments (40x) were performed on an IRE2 microscope (Leica
Microsystems) equipped with a DC300F camera and with an LSM 510
microscope (Carl Zeiss, Jena, Germany) or a Leica SPE confocal
microscope, respectively. Concerning the quantification of dots
mean area, the images were captured with confocal microscope, using
a 40X objective. The acquired images were converted to 8-bit binary
files, and the area of individual GFP-LC3 puncta with an area
greater than four pixels on each image were calculated by ImageJ
software (NIH). Each experiment was done at least three times, and
40-50 cells per condition were quantified.
[0110] Automated microscopy. H4, Hep G2 or HeLa cells stably
expressing GFP-LC3 were seeded in 96-well imaging plates (BD
Falcon, Sparks, USA) 24 h before stimulation. Cells were treated
with the indicated agents for 4-6h. Subsequently, cells were fixed
with 4% PFA and counterstained with 10 .mu.M Hoechst 33342. Images
were acquired using a BD pathway 855 automated microscope (BD
Imaging Systems, San Jose, USA) equipped with a 40.times. objective
(Olympus, Center Valley, USA) coupled to a robotized Twister II
plate handler (Caliper Life Sciences, Hopkinton, USA). Images were
analyzed for the presence of GFP-LC3 puncta in the cytoplasm by
means of the BD Attovision software (BD Imaging Systems). Cell
surfaces were segmented and divided into cytoplasmic and nuclear
regions according to manufacturer standard proceedings. RB 2x2 and
Marr-Hildreth algorithms were used to recognize cytoplasmic
GFP-GALT, GFP-LC3, RFP-FYVE, GFP-GALT-RFP-LC3, GFP-RFP-LC3 positive
dots. Statistical analyses were implemented on the R software
(http://www.r-project.org/).
[0111] Immunoblotting. For immunoblotting, 25 .mu.g of proteins
were separated on 4-12% Bis-Tris acrylamide (Invitrogen) or 12%
Tris-Glycine SDS-PAGE precast gels (Biorad, Hercules, USA) and
electrotransferred to ImmobilonTM membranes (Millipore Corporation,
Billerica, USA). Membranes were then sliced into different parts
according to the molecular weight of the protein of interest to
allow simultaneous detection of different antigens within the same
experiment. Unspecific binding sites were saturated by incubating
membranes for 1 h in 0.05% Tween 20 (v:v in TBS) supplemented with
5% non-fat powdered milk (w:v in TBS), followed by the overnight
incubation with primary antibodies specific for DBI (XXX),
SQSTM1/p62, (Santa Cruz Biotechnology, Calif., USA), LC3, FASN,
p-p70s6k, p70s6k, p-SREBP, SREBP, GLUT1, GLUT4, TSPO, PPARG (Cell
Signalling, Danvers, Mass., USA). Development was performed with
appropriate horseradish peroxidase (HRP)-labeled secondary
antibodies (Southern Biotech, Birmingham, USA) plus SuperSignal
West Pico chemoluminescent substrate (Thermo Scientific-Pierce). An
anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH;
Chemicon International, Temecula, USA) or anti-actin (Abcam,
Cambridge, Mass., USA) were used to control equal loading of
lanes.
[0112] Mouse experiments and tissue processing. C57BL/6 mice that
were wild type (WT) (Charles River Laboratory, Lentilly, France),
GFP-LC3-transgenic (gift of Prof. N. Mizushima),
Beclin.sup.+/-C57BL/6 (gift of Dr. B. Levine), Ambra.sup.gt/gt
(gift of Dr. P. Boya), Atg4b.sup.-/- (gift of Dr. C. Lopez-Otin)
were bred and maintained according to both the FELASA and the
Animal Experimental Ethics Committee Guidelines (CE n. 26: 2012-65,
2012-67; Val de Marne, France). Mice were housed in a
temperature-controlled environment with 12 h light/dark cycles and
received food and water ad libitum or high fat diet (HFD). Mice
were subjected to 24-48h h starvation or were injected
intraperitoneally or intravenously with DBI,
[0113] DBI-derived peptides or DBI-specific antibodies, and were
sacrificed lh to 6 h later. The tissues were immediately frozen in
liquid nitrogen after extraction and homogenized two cycles for 20
s at 5,500 rpm using Precellys 24 tissue homogenator (Bertin
Technologies, Montigny-lc-Bretonneux, France) in a 20 mM Tris
buffer (pH 7.4) containing 150 mM NaCl, 1% Triton X-100, 10 mM EDTA
and Complete.RTM. protease inhibitor cocktail (Roche Applied
Science). Tissue extracts were then centrifuged at 12,000 g at
4.degree. C. and supernatants were collected. Protein concentration
in the supernatants was evaluated by the bicinchoninic acid
technique (BCA protein assay kit, Pierce Biotechnology, Rockford,
Ill.).
[0114] DBI detection: After in vivo treatments, the blood plasma
from the blood collection tubes was harvested by centrifugation at
15000 rpm for 30 minutes, and the amount of DBI in the plasma was
determined using the DBI ELISA (Mybiosource MBS2025156) as
instructed by the manufacturer. For in vitro experiments, H4, Hep
G2 or HeLa cells were seeded in 96-well imaging plates (BD Falcon,
Sparks, USA) 24 h before stimulation. Cells were treated with the
indicated agents for time indicated and the supernatant was
collected, the amount of DBI in the supernatant was determined
using the DBI ELISA (Abnova KA0532 DBI (Human) ELISA).
[0115] Immunization. Male 6-8-week-old C57BL/6 mice obtained from
Harlan France (Gannat, France) were immunized subcutaneously at the
base of the tail with 100 .mu.g alum-precipitated KLH (Calbiochem,
La Jolla, Calif.) in 100 .mu.l balanced salt solution. DBI-KLH was
manufactured by crosslinking DBI to keyhole limpet haemocyanin
(KLH). Transgenic mice expressing DBI autoantibodies received by
intramuscular injection either saline KLH-DBI, emulsified in
Montanide ISA51vg adjuvant (30 g, 30 g, 30 g, 10 g once per week
during 4 weeks). For the generation of the KLH-DBI conjugate,
murine DBI were mixed at a 1:20 molar ratio and adjusted gradually
to 0.25% final (v/v) glutaraldehyde. The reaction was stopped by
addition of a glycine solution. After ultrafiltration (Millipore;
Billerica, Mass., USA), a formaldehyde solution was added to 0.2%
final (v/v). The reaction was quenched by addition of a glycine
solution followed by an ultrafiltration using a 100 kDa membrane
with 70 mM phosphate buffer (pH 7.8). DBI-KLH was stored at
4.degree. C. IFNgf, which served as a control antigen, was
manufactured in the same way, except that the crosslinking reaction
was carried out in the absence of KLH and the molecular weight
cut-off of the final membrane was 10 kDa. Protein concentrations
were determined by Bradford assay.
[0116] Nematode strains: We followed standard procedures for
maintaining C. elegans strains. Rearing temperature was set at
20.degree. C. for all our experiments. We used the DA2123:
WT;Is[p.sub.lgg-1GFP::LGG-1+rol-6(su1006)], MAH14:
daf-2(e1370);[p.sub.lgg-1GFP::LGG-1+rol-6(su1006)] and MAH28:
aak-2(ok524);[p.sub.lgg-1GFP::LGG-1+rol-6(su1006)] for assessment
of autophagy (40, 41). The first strain was crossed with the
SV62:acbp-1(sv62)I and the quadruple
FE0017:acbp-1(sv62)I;acbp-6(tm2995)II;acbp-4(t5m2896)III;acbp-3(sv73)X
strains (42) to monitor autophagy upon depletion of the acbp family
genes. For pharyngeal pumping measurements, the SV62 and FE0017
strains were used in combination with the DA465: eat-2(ad465)II, a
genetic model for reduced pharyngeal pumping.
[0117] Autophagy measurementin C. elegans: Autophagy was measured
as described (43). Briefly, ten well-fed adult worms of the
respective genetic backgrounds were allowed to lay eggs on NGM or
RNAi plates. Four hours later the parents were removed and the
plates were placed at 20.degree. C. Two days later synchronized
animals were collected, anaesthetized at 10 mM levamisole and
mounted on slides for microscopic examination. The number of
GFP::LGG-1 positive autophagic puncta was measured in hypodermal
seam cells at the L3-L4 larval stages (44). Pharyngeal pumping
Pharyngeal pumping was measured as described (45). Grinder
movements of free-moving animals were measured under the
stereomicroscope. Three independent measurements were performed for
each individual and the average number of pumps per animal was
recorded. Starvation was achieved by placing the animals in NGM
plates devoid of bacteria for 24 hours. The animals were left to
recover on OP50-seeded plates for half an hour prior to
observation.
[0118] Immunohistochemistry of mouse brains. Mice were deeply
anesthetized with pentobarbital (Nembutal, Abbott Laboratories,
Chicago, Ill.; 80 mg/kg ip) and perfused transcardially with
phosphate buffer (PB; 0.1 M), followed by 4% paraformaldehyde (in
0.1 M PB). The brains were removed, postfixed for 2 h in the same
fixative, cryoprotected in 20% sucrose solution (in 0.1 M PB) for
48 h, and snap frozen in CO.sub.2. Coronal sections (20 .mu.m) were
cut in a cryostat (CM 3050 Leica, Nussloch, Germany). The
hypothalamic sections were collected in three separate series and
were thaw mounted on microscopic glass slides (SuperFrost Plus,
Faust, Schaffhausen, Switzerland). After air-drying at room
temperature and rehydrating in PBS, sections were incubated in
blocking solution for 2 h (1.5% rabbit normal serum+avidin; Vector
Laboratories, Burlingame, Calif.). The primary antibody (polyclonal
goat anti-c-Fos, Santa Cruz; 1:10,000+biotin, Vector Laboratories)
was applied for 48 h at 4.degree. C. The unbound antibody was
removed by washing in PBS before the sections were incubated with
the secondary antibody (biotinylated rabbit-anti-goat,
Vectastain-Elite ABC Kit, Vector Laboratories; 1:200) for 2 h at
room temperature. After incubation in ABC solution
(Vectastain-Elite ABC Kit, Vector Laboratories), diaminobenzidene
(DAB) was used as a chromogen [0.04% in PBS with 0.02%
H.sub.2O.sub.2 and for color enhancement 0.08% NiCl.sub.2 (.times.6
H.sub.2O), 0.01% CoCl.sub.2 (.times.6 H.sub.2O)]. Finally, the
sections were dehydrated in graded alcohols, cleared in xylenes,
and coverslipped with Entellan (Merck, Darmstadt, Germany).
[0119] Yeast autophagy measurements: Autophagy was monitored either
by vacuolar localization of Atg8p using fluorescence microscopy of
cells expressing an EGFP--Atg8 fusion protein or by alkaline
phosphatase (ALP) activity according to published methods using
BY4741 wild-type or dbi1 transformed cells.
[0120] Results and Discussion:
[0121] Autophagy ("self-eating") constitutes one of the most
spectacular, though subtly regulated phenomena in cell biology and
plays a key role in the maintenance of cellular and organismal
homeostasis by facilitating the turnover of cytoplasmic structures
and allowing cells to adapt to changing and stressful conditions
including nutrient deprivation (1, 2). The cellular secretion of
several leaderless proteins (which can only be released through an
unconventional pathway bypassing Golgi) is strongly associated with
autophagy (3-7). One such protein is a phylogenetically ancient
factor known as diazepam binding protein (DBI) or acyl coenzyme A
(CoA)-binding protein (ACBP) (3, 4). Human or mouse DBI is a small
protein of 87 amino acids (10 kDa) that has two totally distinct
functions, namely as ACBP within cells (where it binds to
long-chain acyl CoA molecules) and as DBI outside cells (where the
entire protein or its cleavage products, triacontatetraneuropeptide
[TTN, residues 17-50] and octadecaneuroptide [ODN, residues 33-50],
can interact with the benzodiazepine binding site of the
gamma-aminobutyric acid type A receptor, GABAAR, and modulate its
activity as a GTP protein-coupled receptor, GPCR) (8-10). DBI and
its proteolytic fragments also bind to the peripheral-type
benzodiazepine receptor (PBR) (11-13), and a still unidentified
GPCR (ODN-GPCR) (14-17). Here, we addressed the question as to
whether DBI secretion might participate in the feedback regulation
of autophagy.
[0122] Human cell lines cultured in nutrient-free (NF) or treated
with rapamycin (RAPA), autophagy-stimulatory conditions manifest a
reduction of intracellular DBI expression that can be suppressed by
addition of lysosomal inhibitors such as bafilomycin A1 (BAFA1),
chloroquine and hydroxychloroquine, as well as by deletion of the
essential autophagy gene/protein ATG5 (data not shown). Soluble DBI
could be detected in culture supernatants in baseline conditions,
yet increased upon NF culture, unless BAFA1 was added or ATGS was
removed (data not shown). Similarly, the intracellular content of
DBI declined in several organs from autophagy competent wild-type
(WT) (but not from autophagy deficient Becn1.sup.+/-) mice
subjected to 24h of starvation (data not shown), a condition that
is known to induce autophagy in most cells of the body (18). In
parallel, DBI levels increased after starvation in the plasma from
WT but not from partially autophagy deficient Becn1.sup.+/-,
Atg4b.sup.-/-
[0123] and Ambra1.sup.gt/gt mice (data not shown). These results
confirm that autophagy induction in vivo causes the release of
intracellular DBI into extracellular compartments.
[0124] Depletion of DBI by small interfering RNAs (siRNAs) reduced
NF-stimulated autophagy in cultured human cells (data not shown),
while its overexpression stimulated autophagic flux (data not
shown). This result was obtained when autophagy was monitored by
following the redistribution of microtubule-associated proteins
1A/1B light chain 3B (LC3) coupled to green fluorescent protein
(GFP) to autophagosomes, as well by measuring LC3 lipidation,
causing an increase in its electrophoretic mobility (data not
shown). In parallel, silencing of DBI increased the kinase activity
of mechanistic target of rapamycin (MTOR), a negative regulator of
autophagy, as indicated by the increased phosphorylation of the
MTOR substrate p70.sup.S6K ((data not shown). Thus, intracellular
DBI, which may intersect with the MTOR pathway through a direct
molecular interaction with Late Endosomal/Lysosomal Adaptor, MAPK
And MTOR Activator 5 (LAMTORS) (19), negatively regulates mTOR and
positively regulates autophagy. The autophagy-dependent depletion
of DBI from cells may activate an autocrine feedback loop that
results in the self-limitation of the autophagic process.
[0125] Knockout of the yeast (Saccharomyces cerevisiae) acbl gene,
which codes for the DBI orthologue, inhibited NF-induced autophagy
(data not shown), while knockout of the nematode (Caenorhabditis
elegans) acbp-1 gene, alone or together with several of its
homologues (which exist in this species but not in mammals),
stimulated autophagy (data not shown). This discrepancy suggests
that this phylogenetically ancient protein might have distinct
autophagy-regulatory functions in uni- versus multicellular
contexts. Indeed, when DBI was siRNA-depleted in a majority of
cultured human cells (which inhibits autophagy in these cells, data
not shown) that were mixed with a minority of still DBI-expressing
cells, this maneuver enhanced autophagy in the latter (data not
shown). Similarly, addition of an antibody that neutralizes
extracellular DBI in the culture medium stimulated autophagic flux
(FIG. 1.A,B), while addition of recombinant (rec) DBI protein (or
that of its fragments TTN and ODN) inhibited NF-induced autophagy
in cultured human cells (FIG. 1C-D). Similarly, neutralization of
extracellular DBI by intraperitoneal (i.p.) injection of a specific
antibody into mice induced autophagy in various organs (FIG. 1E),
while the systemic intravenous (i.v.) or i.p. administration of
rec. DBI protein inhibited starvation-induced autophagy (FIG. 1F).
These results indicate that extracellular DBI suppresses autophagy
(contrasting with the fact that intracellular DBI stimulates
autophagy), meaning that autophagy-induced DBI release from cells
may engage in a paracrine feedback loop.
[0126] In a cohort of 52 patients with anorexia nervosa, plasma DBI
concentrations were abnormally low as compared to age- and
sex-matched controls with a normal body mass index (BMI) (FIG. 2A)
confirming a prior report on 24 anorexic patients (20). More
importantly, DBI concentrations were abnormally high in obese
individuals (and reduced after bariatric surgery), correlating with
high circulating insulin levels (FIG. 2B, C). Similarly,
genetically obese Ob/Ob mice (that have a defect in the leptin
receptor) exhibited enhanced circulating DBI levels (data not
shown). Driven by these findings, we investigated whether DBI might
regulate general metabolism. For this, rec. DBI protein and
anti-DBI antibody were injected into fed and starved mice,
respectively, and two hours later their organs were subjected to
mass spectrometric metabolomics analyses. Rec. DBI protein caused
hypoglycemia. Conversely, DBI neutralization reversed the
starvation-induced hypoglycemia and further exacerbated the
starvation-induced increase in the plasma levels of the ketone body
2-hydroxybutyric acid (data not shown). We therefore decided to
investigate the effects of DBI on weight control in the context of
glucose and fatty acid metabolism.
[0127] Hydrodynamic injection of the cDNA coding for DBI, a
procedure that increases hepatic expression of DBI, led to
hypoglycemia, increased food intake and caused weight gain (FIG.
3A-C). Similarly, systemic (i.p. or i.v.) injection of rec. DBI
protein (or that of its peptide fragments TTN or ODN) stimulated
the triad of hypoglycemia, increased food intake and weight gain
(FIG. 3D-G). In parallel, rec. DBI protein reduced fatty acid
oxidation at the whole-body level, as determined by respirometry
(FIG. 1H). The finding that rec. DBI protein has orexigenic effects
contrasts with prior reports showing that administration of DBI
fragments into the brain is anorexigenic (21, 22). Hence, rec. DBI
protein injected via the i.p. or i.v. routes is likely to act via
peripheral rather than central nervous effects. Indeed, systemic
administration of rec. DBI rapidly (30 min) caused the hepatic
upregulation of glucose transporters (GLUT1) and peroxisome
proliferator-activated receptor gamma-y (PPARG), which upregulates
lipogenesis via fatty acid synthase (FASN) (data not shown).
Accordingly, rec. DBI enhanced the incorporation of .sup.14C atoms
from glucose into visceral fat (data not shown). Moreover, when
added to human Hep G2 liver cells, rec. DBI stimulated both basic
and maximum glycolysis (data not shown). Reversal of DBI-induced
hypoglycemia by i.p. injection of glucose prevented hyperphagy
(data not shown). Thus, DBI drives glucose uptake, glycolysis and
lipogenesis, ultimately causing hypoglycemia that triggers a
feeding response.
[0128] Given the orexigenic effects of rec. DBI protein, we
investigated whether depletion or neutralization of endogenous DBI
would be anorexigenic. Mice bearing a constitutive Dbi knockout
(Dbi.sup.-/-) either die (23) or are affected by multiple defects
including in their epidermal barrier function (24-33) and obviously
cannot be used to differentiate the intra- and extracellular
functions of DBI. We generated mice in which Dbi could be
conditionally knocked out by tamoxifen injection (using tamoxifen
(Tam)-inducible Cre recombinase-mediated excision of the floxed
Dbi) (data not shown). The Tam-inducible whole-body knockout of DBI
killed a fraction of adult C57B1/6 mice fed normal chow (data not
shown), failed to compromise the survival of mice on a high fat
diet (HFD) (data not shown), yet sensitized mice to
starvation-induced death (data not shown). Weight loss induced by
starvation was increased in DBI-depleted mice (data not shown),
although glucose levels were maintained in the normoglycemic range
(data not shown). To neutralize extracellular DBI only, we
generated a monoclonal antibody (mAb 7A, an IgG). Systemic (i.p.)
injection of different anti-DBI antibodies increased plasma glucose
levels in fed as well as in starved mice (FIG. 4A, C) and reduced
food intake post-starvation (FIG. 4B, D). Very similar results were
obtained with several commercial polyclonal antibodies neutralizing
DBI (data not shown). In contrast to the whole-body DBI knockout,
neither mAb 7A nor the polyclonal antibodies caused fatalities,
even in starved mice. Blockade of DBI inhibited whole-body fatty
acid oxidation both in baseline (data not shown) and in starved
conditions (data not shown). In spite of the hyperglycemia induced
by DBI neutralization, starved mice exhibited a decrease in plasma
insulin levels, C-peptide and gastric inhibitory peptide (GIP)
(data not shown).
[0129] In the liver, neutralization of DBI reduced PPARG and FASN
expression, as it provoked the inhibitory phosphorylation of sterol
regulatory element-binding transcription factor 1 (SREBF1),
commensurate with the suppression of lipogenesis (FIG. 5).
Accordingly, neutralization of DBI reduces hepatosteatosis in the
context of an obesogenic high-fat diet.
[0130] Next, we investigated the possibility to break
self-tolerance to DBI and to induce the production of neutralizing
autoantibodies by immunizing mice with DBI coupled to keyhole
limpet hemocyanine (KLH) together with a potent adjuvant (34). The
surge of auto-antibodies that durably neutralized DBI in the
circulation (data not shown) had a major impact on metabolism,
though without lethal effects (as recorded for the whole-body
knockout), leading to enhanced weight loss during starvation (FIG.
6A) and reduced re-feeding post-starvation (FIG. 6B). Moreover, the
weight gain that is usually found in mice fed normal chow or HFD
was reduced upon autoimmunization against DBI (FIG. 6C, D) In
HFD-fed mice, the immunization against DBI downregulated hepatic
lipogenesis-stimulatory factor (FASN), increased hepatic carnitine
palmitoyl transferase-1 (CPT1, which is required for fatty acid
uptake), augmented carnitine fatty acid ethers in the liver,
suppressed hepatosteatosis, reduced hyperlipidemia of multiple free
fatty acids, and upregulated uncoupling protein 1 (UCP1) in brown
fat, as it diminished the amount of white adipose tissue (data not
shown).
[0131] Metabolomic comparisons of distinct tissues from starved
mice and mice subjected to DBI neutralization revealed strong
similarities for brown adipose tissue (BAT) (data not shown) and
plasma (data not shown), more so than in liver and muscle (not
shown). Although the effects of DBI neutralization on metabolism
must be due to peripheral effects (outside of the central nervous
system), the antibody-mediated DBI blockade inhibited neurons of
the orixogenic lateral hypothamalic area (LH) and activated neurons
in the anorexigenic ventromedial nucleus (VMN), as determined by
assessing the phosphorylation of the transcription factor c-Fos
(data not shown). Altogether, these results indicate that both
passive and active immunization against DBI exerts potent
anti-obesity effects.
[0132] Our data point to the model that starvation-induced
autophagy is subjected to three levels of DBI mediated feedback
regulation. Autophagy causes DBI secretion, depleting this
pro-autophagic factor from the cell (autocrine regulation), and DBI
accumulating in the extracellular space then acts on other cells to
inhibit autophagy (paracrine regulation). In addition, circulating
DBI stimulates feeding behavior, hence increasing nutrient uptake
and removing the primary cause of autophagy induction (endocrine
regulation). This latter effect appears to be phylogenetically
conserved because C. elegans subjected to the depletion of one or
several DBI orthologs manifested a reduction in pharyngeal pumping
(data not shown). Thus, DBI may participate in a primitive reflex
in which nutrient depletion stimulates eating behavior via the
induction of autophagy.
[0133] Beyond its autophagy-inhibitory effects, extracellular DBI
has potent modulatory effects on whole body metabolism. In
adolescents with anorexia nervosa, circulating DBI levels are low.
This contrasts with the short-term starvation-induced increase in
DBI levels observed in mice. The reasons for this discrepancy
remain elusive. However, it is tempting to speculate that the
anorexia-associated reduction in DBI levels (perhaps resulting from
a long-term readjustment of the setpoint determining DBI expression
at the transcriptional level) (35) might be responsible for the
phenotype, because deletion of the DBI-encoding gene or
neutralization of the DBI protein had anorexigenic effects on mice,
reducing food intake after starvation. In sharp contrast, provision
of extracellular DBI by systemic injection of the recombinant
protein (or its active peptide fragments) stimulated food intake by
favoring hypoglycemia, secondary to the upregulation of glucose
uptake into hepatocytes and increased glycolysis as well as
lipogenesis. Indeed, patients or mice with morbid obesity exhibited
an increased plasma level of DBI. The reasons for this increase in
DBI expression remain obscure. Obesity is linked to autophagy
inhibition (36, 37), meaning that altered autophagic flux may not
explain the augmentation in circulating DBI. Conversely, the
obesity-associated rise in DBI may contribute to autophagy
inhibition, which in turn counteracts weight loss and predisposes
to weight gain (38, 39). Moreover, elevated levels of extracellular
DBI favor orexigenic and adipogenic reactions, as indicated by the
observation that deletion or neutralization of DBI can dampen
appetite, reduce weight gain, and blunt HFD-induced adiposity and
hepatosteatosis. Neutralization of DBI can be achieved by injecting
monoclonal or polyclonal antibodies, as well as by the induction of
autoantibodies. If the long-term DBI blockade was exempt of
detrimental side effects and constituted a desirable therapeutic
goal, this latter strategy might be particularly useful for the
prevention or treatment of morbid obesity.
REFERENCES:
[0134] Throughout this application, various references describe the
state of the art to which this invention pertains. The disclosures
of these references are hereby incorporated by reference into the
present disclosure.
[0135] 1. B. Levine, N. Mizushima, H. W. Virgin, Autophagy in
immunity and inflammation. Nature 469, 323-335 (2011).
[0136] 2. N. Mizushima, B. Levine, A. M. Cuervo, D. J. Klionsky,
Autophagy fights disease through cellular self-digestion. Nature
451, 1069-1075 (2008).
[0137] 3. J. M. Duran, C. Anjard, C. Stefan, W. F. Loomis, V.
Malhotra, Unconventional secretion of Acbl is mediated by
autophagosomes. J Cell Biol 188, 527-536 (2010).
[0138] 4. R. Manjithaya, C. Anjard, W. F. Loomis, S. Subramani,
Unconventional secretion of Pichia pastoris Acbl is dependent on
GRASP protein, peroxisomal functions, and autophagosome formation.
J Cell Bio1 188, 537-546 (2010).
[0139] 5. N. Dupont et al., Autophagy-based unconventional
secretory pathway for extracellular delivery of IL-1beta. EMBO J
30, 4701-4711 (2011).
[0140] 6. M. Zhang, R. Schekman, Cell biology. Unconventional
secretion, unconventional solutions. Science 340, 559-561
(2013).
[0141] 7. M. Ponpuak et al., Secretory autophagy. Curr Opin Cell
Bio135, 106-116 (2015).
[0142] 8. J. Bormann, Electrophysiological characterization of
diazepam binding inhibitor (DBI) on GABAA receptors.
Neuropharmacology 30, 1387-1389 (1991).
[0143] 9. P. W. Gray, D. Glaister, P. H. Seeburg, A. Guidotti, E.
Costa, Cloning and expression of cDNA for human diazepam binding
inhibitor, a natural ligand of an allosteric regulatory site of the
gamma-aminobutyric acid type A receptor. Proc Natl Acad Sci USA 83,
7547-7551 (1986).
[0144] 10. C. A. Christian et al., Endogenous positive allosteric
modulation of GABA(A) receptors by diazepam binding inhibitor.
Neuron 78, 1063-1074 (2013).
[0145] 11. A. Berkovich, P. McPhie, M. Campagnone, A. Guidotti, P.
Hensley, A natural processing product of rat diazepam binding
inhibitor, triakontatetraneuropeptide (diazepam binding inhibitor
17-50) contains an alpha-helix, which allows discrimination between
benzodiazepine binding site subtypes. Mol Pharmaco137, 164-172
(1990).
[0146] 12. V. Papadopoulos, A. Berkovich, K. E. Krueger, E. Costa,
A. Guidotti, Diazepam binding inhibitor and its processing products
stimulate mitochondrial steroid biosynthesis via an interaction
with mitochondrial benzodiazepine receptors. Endocrinology 129,
1481-1488 (1991).
[0147] 13. P. Gandolfo et al., The triakontatetraneuropeptide TTN
increases [CA2+]i in rat astrocytes through activation of
peripheral-type benzodiazepine receptors. Glia 35, 90-100
(2001).
[0148] 14. C. Patte et al., The endozepine ODN stimulates
polyphosphoinositide metabolism in rat astrocytes. FEBS Lett 362,
106-110 (1995).
[0149] 15. P. Gandolfo et al., The stimulatory effect of the
octadecaneuropeptide (ODN) on cytosolic Ca2+in rat astrocytes is
not mediated through classical benzodiazepine receptors. Eur J
Pharmacol 322, 275-281 (1997).
[0150] 16. J. Leprince et al., Synthesis, conformational analysis
and biological activity of cyclic analogs of the
octadecaneuropeptide ODN. Design of a potent endozepine antagonist.
Eur J Biochem 268, 6045-6057 (2001).
[0151] 17. Z. Farzampour, R. J. Reimer, J. Huguenard, Endozepines.
Adv Pharmacol 72, 147-164 (2015).
[0152] 18. N. Mizushima, A. Yamamoto, M. Matsui, T. Yoshimori, Y.
Ohsumi, In vivo analysis of autophagy in response to nutrient
starvation using transgenic mice expressing a fluorescent
autophagosome marker. Mol Biol Cell 15, 1101-1111 (2004).
[0153] 19. W. Fan, J. Cheng, S. Zhang, X. Liu, Cloning and
functions of the HBxAg-binding protein XBP1. Mol Med Rep 7, 618-622
(2013).
[0154] 20. E. Conti et al., Reduced fasting plasma levels of
diazepam-binding inhibitor in adolescents with anorexia nervosa.
Int J Eat Disord 46, 626-629 (2013).
[0155] 21. D. Lanfray et al., Gliotransmission and brain glucose
sensing: critical role of endozepines. Diabetes 62, 801-810
(2013).
[0156] 22. F. Guillebaud et al., Glial Endozepines Inhibit
Feeding-Related Autonomic Functions by Acting at the Brainstem
Level. Front Neurosci 11, 308 (2017).
[0157] 23. D. Landrock et al., Acyl-CoA binding protein gene
ablation induces pre-implantation embryonic lethality in mice.
Lipids 45, 567-580 (2010).
[0158] 24. D. Neess et al., Disruption of the acyl-CoA-binding
protein gene delays hepatic adaptation to metabolic changes at
weaning. J Biol Chem 286, 3460-3472 (2011).
[0159] 25. S. Langaa et al., Mice with targeted disruption of the
acyl-CoA binding protein display attenuated urine concentrating
ability and diminished renal aquaporin-3 abundance. Am J Physiol
Renal Physiol 302, F1034-1044 (2012).
[0160] 26. M. Bloksgaard et al., The acyl-CoA binding protein is
required for normal epidermal barrier function in mice. J Lipid Res
53, 2162-2174 (2012).
[0161] 27. D. Neess et al., Delayed hepatic adaptation to weaning
in ACBP-/- mice is caused by disruption of the epidermal barrier.
Cell Rep 5, 1403-1412 (2013).
[0162] 28. M. Bloksgaard, D. Neess, N. J. Faergeman, S. Mandrup,
Acyl-CoA binding protein and epidermal barrier function. Biochim
Biophys Acta 1841, 369-376 (2014).
[0163] 29. S. Bek et al., Compromised epidermal barrier stimulates
Harderian gland activity and hypertrophy in ACBP-/- mice. J Lipid
Res 56, 1738-1746 (2015).
[0164] 30. D. Neess, S. Bek, H. Engelsby, S. F. Gallego, N. J.
Faergeman, Long-chain acyl-CoA esters in metabolism and signaling:
Role of acyl-CoA binding proteins. Prog Lipid Res 59, 1-25
(2015).
[0165] 31. K. Bouyakdan et al., A novel role for central ACBP/DBI
as a regulator of long-chain fatty acid metabolism in astrocytes. J
Neurochem 133, 253-265 (2015).
[0166] 32. L. Budry et al., DBI/ACBP loss-of-function does not
affect anxiety-like behaviour but reduces anxiolytic responses to
diazepam in mice. Behav Brain Res 313, 201-207 (2016).
[0167] 33. S. F. Gallego et al., Quantitative lipidomics reveals
age-dependent perturbations of whole-body lipid metabolism in ACBP
deficient mice. Biochim Biophys Acta 1862, 145-155 (2017).
[0168] 34. L. Semerano et al., Targeting VEGF-A with a vaccine
decreases inflammation and joint destruction in experimental
arthritis. Angiogenesis 19, 39-52 (2016).
[0169] 35. D. Neess et al., ACBP--a PPAR and SREBP modulated
housekeeping gene. Mol Cell Biochem 284, 149-157 (2006).
[0170] 36. L. Yang, P. Li, S. Fu, E. S. Calay, G. S. Hotamisligil,
Defective hepatic autophagy in obesity promotes ER stress and
causes insulin resistance. Cell Metab 11, 467-478 (2010).
[0171] 37. K. H. Kim, M. S. Lee, Autophagy--a key player in
cellular and body metabolism. Nat Rev Endocrinol 10, 322-337
(2014).
[0172] 38. G. Marino et al., Regulation of autophagy by cytosolic
acetyl-coenzyme A. Mol Cell 53, 710-725 (2014).
[0173] 39. A. F. Fernandez et al., Autophagy couteracts weight
gain, lipotoxicity and pancreatic beta-cell death upon hypercaloric
pro-diabetic regimens. Cell Death Dis 8, e2970 (2017).
[0174] 40. D. F. Egan et al., Phosphorylation of ULK1 (hATG1) by
AMP-Activated Protein Kinase Connects Energy Sensing to Mitophagy.
Science 331, 456 (2011).
[0175] 41. C. Kang, Y.-j. You, L. Avery, Dual roles of autophagy in
the survival of Caenorhabditis elegans during starvation. Genes
& Development 21, 2161-2171 (2007).
[0176] 42. Ida C. Elle et al., Tissue- and paralogue-specific
functions of acyl-CoA-binding proteins in lipid metabolism in
<em>Caenorhabditis elegans</em>. Biochemical Journal
437, 231 (2011).
[0177] 43. N. J. Palmisano, A. Melendez, Detection of Autophagy in
Caenorhabditis elegans Using GFP::LGG-1 as an Autophagy Marker.
Cold Spring Harbor protocols 2016, pdb.prot086496 (2016).
[0178] 44. A. Melendez et al., Autophagy Genes Are Essential for
Dauer Development and Life-Span Extension in <em>C.
elegans</em>. Science 301, 1387 (2003).
[0179] 45. J. Keane, L. Avery, Mechanosensory inputs influence
Caenorhabditis elegans pharyngeal activity via ivermectin
sensitivity genes. Genetics 164, 153-162 (2003).
Sequence CWU 1
1
2187PRTHomo sapiens 1Met Ser Gln Ala Glu Phe Glu Lys Ala Ala Glu
Glu Val Arg His Leu1 5 10 15Lys Thr Lys Pro Ser Asp Glu Glu Met Leu
Phe Ile Tyr Gly His Tyr 20 25 30Lys Gln Ala Thr Val Gly Asp Ile Asn
Thr Glu Arg Pro Gly Met Leu 35 40 45Asp Phe Thr Gly Lys Ala Lys Trp
Asp Ala Trp Asn Glu Leu Lys Gly 50 55 60Thr Ser Lys Glu Asp Ala Met
Lys Ala Tyr Ile Asn Lys Val Glu Glu65 70 75 80Leu Lys Lys Lys Tyr
Gly Ile 852634DNAHomo sapiens 2gctcgcccga gcagggttgg ggcgagtgga
ccgcgcctct aaaggcgctt gccagtgcaa 60tctgggcgat cgcttcctgg tcctcgcctc
ctccgctgtc tccctggagt tcttgcaagt 120cggccaggat gtctcaggct
gagtttgaga aagctgcaga ggaggttagg caccttaaga 180ccaagccatc
ggatgaggag atgctgttca tctatggcca ctacaaacaa gcaactgtgg
240gcgacataaa tacagaacgg cccgggatgt tggacttcac gggcaaggcc
aagtgggatg 300cctggaatga gctgaaaggg acttccaagg aagatgccat
gaaagcttac atcaacaaag 360tagaagagct aaagaaaaaa tacgggatat
gagagactgg atttggttac tgtgccatgt 420gtttatccta aactgagaca
atgccttgtt tttttctaat accgtggatg gtgggaattc 480gggaaaataa
ccagttaaac cagctactca aggctgctca ccatacggct ctaacagatt
540aggggctaaa acgattactg actttccttg agtagttttt atctgaaatc
aattaaaagt 600gtatttgtta ctttaaataa ctttaaaaaa aaaa 634
* * * * *
References