U.S. patent application number 15/768101 was filed with the patent office on 2018-10-18 for prophylactic and therapeutic agent for rett syndrome (rtt) comprising ghrelin as active ingredient.
This patent application is currently assigned to KURUME UNIVERSITY. The applicant listed for this patent is KURUME UNIVERSITY. Invention is credited to Munetsugu HARA, Masayasu KOJIMA, Toyojiro MATSUISHI, Yushiro YAMASHITA, Kotaro YUGE.
Application Number | 20180296647 15/768101 |
Document ID | / |
Family ID | 58518184 |
Filed Date | 2018-10-18 |
United States Patent
Application |
20180296647 |
Kind Code |
A1 |
MATSUISHI; Toyojiro ; et
al. |
October 18, 2018 |
PROPHYLACTIC AND THERAPEUTIC AGENT FOR RETT SYNDROME (RTT)
COMPRISING GHRELIN AS ACTIVE INGREDIENT
Abstract
A prophylactic and therapeutic agent for Rett Syndrome (RTT) is
provided, a pharmaceutical composition comprising ghrelin and a
pharmaceutically acceptable carrier and a prophylactic and
therapeutic agent for RTT comprising a therapeutically effective
amount of ghrelin. The side chain of serine at the 3rd position of
ghrelin is modified with octanoic acid.
Inventors: |
MATSUISHI; Toyojiro;
(Kurume-shi, Fukuoka, JP) ; KOJIMA; Masayasu;
(Kurume-shi, Fukuoka, JP) ; YUGE; Kotaro;
(Kurume-shi, Fukuoka, JP) ; HARA; Munetsugu;
(Kurume-shi, Fukuoka, JP) ; YAMASHITA; Yushiro;
(Kurume-shi, Fukuoka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KURUME UNIVERSITY |
Fukuoka |
|
JP |
|
|
Assignee: |
KURUME UNIVERSITY
Fukuoka
JP
|
Family ID: |
58518184 |
Appl. No.: |
15/768101 |
Filed: |
April 20, 2016 |
PCT Filed: |
April 20, 2016 |
PCT NO: |
PCT/JP2016/062553 |
371 Date: |
April 13, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/22 20130101;
A61P 25/00 20180101 |
International
Class: |
A61K 38/22 20060101
A61K038/22; A61P 25/00 20060101 A61P025/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 14, 2015 |
JP |
2015-202751 |
Claims
1. A pharmaceutical composition comprising ghrelin and a
pharmaceutically acceptable carrier.
2. The pharmaceutical composition according to claim 1 wherein the
side chain of serine at the 3rd position of ghrelin is modified
with octanoic acid.
3. A prophylactic and therapeutic agent for Rett Syndrome (RTT)
comprising a therapeutically effective amount of ghrelin.
4. The prophylactic and therapeutic agent according to claim 3
wherein the side chain of serine at the 3rd position of ghrelin is
modified with octanoic acid.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel use of ghrelin in
the clinical field. Specifically, the present invention relates to
a prophylactic and therapeutic agent for Rett Syndrome (RTT)
comprising ghrelin as an active ingredient.
[0002] Rett Syndrome (RTT) is a neurodevelopmental disorder which
begins during infancy, characterized by intellectual disability,
autistic behavior, epilepsy, etc. with various other symptoms such
as autonomic dysfunction, gastrointestinal and respiratory
problems. RTT was first described by Austrian pediatric neurologist
Andreas Rett in 1966. It received global attention after a report
by the Swedish, Bengt Hagberg et al. in 1983. The prevalence of RTT
is estimated to be 0.9 per 10,000 in females before adulthood in
Japan. An estimate in Japan is that there are at least 1,000
patients with RTT but actual number of patients is thought to be
more. As RTT occurs almost exclusively in females, it was proposed
as caused by an X-linked dominant mutation. Exclusion mapping
studies mapped the locus to Xq28 on the long arm of the X
chromosome in 1996. In 1999, a causative gene mutation was
discovered in methyl-CpG binding protein 2 (MECP2) (MECP2
gene).
[0003] RTT develops mainly in female infants, who exhibit hypotonia
and autistic tendencies in early infancy and then impaired
locomotion, such as crawling and walking, retardation of language
development and, finally, severe intellectual disabilities. From
infancy to early childhood, there is a loss of purposeful motor
functions of the hand, such as in hand washing, kneading,
handwringing or bringing one hand to the mouth while pounding on
the chest, with distinctive hand stereotypies. These symptoms are
almost always present in typical RTT cases. Among other clinical
manifestations of RTT are stagnated head circumference development
and acquired microcephaly, early childhood hypertonia, dystonia,
grinding of the teeth, respiratory abnormalities such as
hyperventilation and apnea, constipation, autonomic nervous
dysfunctions, such as in cold sensation, small feet and frequent
occurrences of epilepsy. However, these symptoms are not always
present as essential criteria. An important aspect of the disease
is that the symptoms occur in an age-dependent manner. In 1999,
Amir et al. found that a mutation in the methyl-CpG-binding protein
2 gene (MECP2 gene) was causative in RTT. It was reported that the
MECP2 gene mutation was identified in 90% or more of typical cases.
In some cases, psychomotor function is ameliorated at certain
clinical stages and a pseudo-stable is observed. RTT is understood
to be a neurodevelopmental, rather than a neurodegenerative,
disorder. Angela J McArthur et al., Developmentulhledicitie &
Cbild Neirrology 1998, 40, 186-192 (Non-patent reference 1);
Carolyn Ellaway et al., Brain & Development 23 (2001) S101-S103
(Non-patent reference 2); Yoshiko Nomura, Brain & Development
27 (2005) S35-S42 (Non-patent reference 3); Deidra Young et al.,
Brain & Development 29 (2007) 609-616 (Non-patent reference 4);
Meir Lotan et al., The Scientific World Journal (2006) 6, 1737-1749
(Non-patent reference 5); Flavia Schwartzman et al., Arq
Gastroenterol v. 45, no. 4, Jul. set. 2008 (Non-patent reference
6); Kathleen J. Motil et al., JPGN Volume 55, Number 3, September
2012 (Non-patent reference 7).
[0004] Uniform diagnostic criteria for RTT were not established
until recently. It is important that diagnostic criteria for
typical RTT of female infant meet all of the following, consisting
of essential requirements and exclusion criteria for typical
RTT.
Essential Requirements:
[0005] (1) Partial or complete loss of acquired purposeful hand
skills (2) Partial or complete loss of acquired spoken language (3)
Gait abnormalities: impaired (dyspraxic) or absent abilities (4)
Stereotypic hand movements
Exclusion Criteria for Typical RTT:
[0006] Brain injury secondary to trauma (perinatally or
postnatally), neurometabolic diseases or severe infection causing
neurological problems, grossly abnormal psychomotor development in
the first six months of life
[0007] For RTT, there is not yet an established effective therapy.
Jacky Guy et al. reported that about 70% of the symptoms of RTT
were partially reversed in a conditional knockout mouse when
conditioning knockout mouse model of the causative gene MECP2 of
Rett Syndrome was prepared and administered with tamoxifen (TM) at
3-4 weeks and 12-17 weeks, at which the symptoms of Rett Syndrome
occur, so as to let the MECP2 be expressed (SCIENCE VOL 315 23 Feb.
2007 (Non-patent reference 8)).
[0008] Daniela Tropea et al. reported the results of Phase I study
where extended lifespan, improved locomotor function, and
amelioration in respiratory and cardiac function were observed when
Rett Syndrome model mice were administered with IGF-1 (PNAS Feb.
10, 2009 vol. 106, no. 6, 2029-2034 (Non-patent reference 9)).
[0009] Maria C. N. Marchetto et al. described iPS cells established
from fibroblasts harvested from patients with RTT. After extensive
study, they found that iPS cells from patients with RTT had fewer
synapses, lower neurite densities, smaller cell sizes, altered
calcium signaling and electrophysiological defects (Cell 143,
527-539, Nov. 12, 2010 (Non-patent reference 10)). Therapeutic
candidates were studied by treating the iPS cells with various
agents and it was shown that IGF-1 treatment led to neurite
elongation, suggesting a potential therapeutic approach.
[0010] Ruben Deogracias et al. reported that fingolimod, a modifier
of the sphingosine-1 phosphate receptor, increased (BDNF) levels
and ameliorated symptoms in RTT model mice (PNAS Aug. 28, 2012 vol.
109 no. 35, 14230-14235 (Non-patent reference 11)).
[0011] Noel C. Derecki et al. reported that wild microglia cells,
when bone-marrow was transplanted to mouse model animal, could
increase lifespan, ameliorate respiratory disorder, decrease apnea,
ameliorate weight gain and locomotion activity, close to those of
wild mice (Nature Vol 4844, 5 Apr. 2012, 105-111 (Non-patent
reference 12)).
[0012] Giorgio Pini et al. reported that IGF-1 was administered to
six patients with RTT, ages 4-11 years. The IGF-1 was administered
twice per day for 6 months at 0.05 mg/kg. The International
Severity Score was ameliorated in three patients, the test could be
performed safely and drug tolerability was confirmed (Autism
Research and Treatment, Volume 2012, Article ID 679801, 14 pages
(Non-patent reference 13)).
[0013] Laura Ricceri et al. reviewed clinical and neurobiological
aspects of RTT and then discussed clinical therapeutic candidates
targeting GABA, neurotoxin-related material, NMDA receptor,
acetylcholine, biogenic amines, neurotrophic factors including
BDNF, IGF1 and carnitine, corticosterone and stress coping,
RhoGTPase and glial cells (Neuropharmacology 68 (2013) 106-115
(Non-patent reference 14)).
[0014] Ito M et al. reviewed recent clinical trials in RTT,
evaluating IGF-1, the tricyclic antidepressant desipramine,
antitussive dextromethorphan (inhibiting activity against
non-selective reuptake of serotonin and an NMDA-type glutamate
receptor inhibitor), bone marrow transplantation and fingolimod,
which increases BDNF expression (SRL Hokan, Vol. 34, No. 2, 2013
28-39 (Non-patent reference 15)).
[0015] Omar S. Khwaja et al. reported that recombinant human IGF-1,
mecasermin, was administered to 9 patients with RTT at 40 to 120
.mu.g/kg twice daily. The investigators analyzed subjects based on
cerebrospinal fluid samples, electroencephalogram, cardiac function
and respiration. The results showed that IGF-1, known not to cross
the blood-brain barrier, could be administered safely. However,
they observed increased IGF-1 levels in cerebrospinal fluid.
Anxiety and mood improvements were indicated by the reversal of
right frontal band asymmetry of the alpha wave of EEGs. However,
the subsequent phase II trial failed to show significant difference
(PNAS Mar. 25, 2014 vol. 111, no. 12, 4596-4601 (Non-patent
reference 16)).
[0016] Previous trials for RTT, to date, tested a morphinan
compound (JP 2010-526089 (Patent reference 1), JP 2012-131815
(Patent reference 2), JP 2012-503009 (Patent reference 3), JP
2014-196331 (Patent reference 4), JP 2015-145407 (Patent reference
5)), glycyl-L-2-methylpropyl-L-glutamic acid (JP 2014-508744
(Patent reference 6)) and tyrosine kinase receptor B (TrkB) binding
molecule (JP 2011-501760 (Patent reference 7)).
[0017] Although various trials for treating human RTT have been
performed, no effective therapy has yet been established. Gene
therapy and bone marrow transplantation ameliorated symptoms and
increased lifespan in an animal model of RTT. However, these
approaches were considered to be potentially difficult for treating
human RTT. IGF-1 showed no side effects but no significant efficacy
was observed. BDNF (brain derived neurotrophic factor), glutamate
receptor antagonists, antidepressants and other drug classes are
potential candidates for clinical trials in RTT, with some trials
already underway (in the United States and Europe) (Non-patent
reference 17).
[0018] Ghrelin is a peptide hormone first detected in the stomach
as an endogenous ligand of GHS receptor, an orphan receptor without
known ligand (Kojima M et al., Nature 402, 656-660 (1999)
(Non-patent reference 18); Kojima M et al., Trends Endocrinol Metab
12, 118-122 (2001) (Non-patent reference 19). Human ghrelin is a 28
amino acid peptide in which the side chain of the 3rd amino acid
residue, serine, is modified with the fatty acid octanoic acid
(N-GSSFLSPEHQRVQQRKESKKPPAKLQPR-C). This octanoic acid modification
is essential for its biological activity. Namely, ghrelin is
transformed into an active form by octanoylation to exhibit its
physiologic activities. Ghrelin was identified in fish, amphibian,
birds and many mammalian species and has a fatty acid at the 3rd
serine or threonine residue. It is a peptide hormone with potent
growth hormone secretion promoting and feeding stimulating
activities which regulates endocrine and energy metabolism (Takaya
K et al., J Clin Endocrinol Metab 85, 4908 (2000) (Non-patent
reference 20); Tschop M et al., Nature 407, 908 (2000) (Non-patent
reference 21); Nakazato M et al., Nature 409, 194 (2001)
(Non-patent reference 22)).
[0019] The ghrelin producing cells in the stomach are called
X/A-like cells, function of which is not known up till the present.
Besides in the stomach, production of ghrelin, though in lesser
amounts, is observed in tissues including the intestinal tract,
hypothalamus, pituitary gland, pancreas, kidney, placenta and
testes. Plasma ghrelin levels increase with fasting and decrease
with food intake. Plasma ghrelin levels are lower in obese
individuals and higher under lean conditions. Ghrelin acts in the
pituitary gland to stimulate GH secretion. This activity is
synergistic with that of growth hormone-releasing hormone (GHRH).
Ghrelin also acts in the hypothalamus to stimulate food intake and,
thus, acts as a feeding promoting peptide. Weight gain and
increased adipose tissue is observed after ghrelin administration.
Therefore, ghrelin is considered to be a hormone antagonistic to
leptin, an anti-obesity hormone produced in fat cells.
[0020] As described above, ghrelin is mainly produced in gastric
endocrine cells and has an important activity in regulating energy
metabolism such as feeding promotion, weight gain and regulation of
gastrointestinal function. It is, so far, the only peptide hormone
produced in the periphery with feeding promoting activity. Ghrelin
is secreted in the stomach when hunger signals are transmitted to
the brain via the afferent vagus nerve. Ghrelin acts in the
hypothalamus, which is the central region for regulating feeding
and GH secretion.
[0021] Ghrelin is present in the a cells which produce glucagon in
the pancreatic islets. The ghrelin receptor gene is expressed in
both .alpha. and .beta. cells. Ghrelin at physiological
concentrations (10.sup.-12 to 10.sup.-11 M) increases intracellular
Ca.sup.2+ levels in pancreatic .beta. cells isolated from rats
under hyperglycemic conditions, promoting insulin secretion. On the
other hand, under hypoglycemic conditions, ghrelin does not affect
intracellular Ca.sup.2+ levels or insulin secretion in pancreatic
.beta. cells. Another possible effect is that ghrelin modifies
insulin activity in the liver and is involved in glucose
metabolism. Ghrelin, when intravenously administered to healthy
subjects, decreases average arterial blood pressure without
changing heart rate and increases cardiac output. When ghrelin is
continually administered in a rat model for heart failure after
cardiac infarction, ghrelin levels are increased in the left
ventricular ejection fraction together with an increase in serum GH
and alleviation of cachexia. These observations suggested the
usefulness of ghrelin as a drug to treat heart failure because of
its amelioration of cardiac dysfunction and malnutrition.
Furthermore, when ghrelin was administered to patients with chronic
heart failure, increased cardiac index and improved hemodynamics
were reported.
[0022] Munetsugu Hara et al. measured plasma ghrelin levels in 27
patients with RTT and 53 healthy controls. The plasma levels of
both total ghrelin and active ghrelin with octanoyl modification
were lower in patients with RTT than in healthy controls (Int. J.
Devl Neuroscience 29 (2011) 899-902 (Non-patent reference 23)). In
addition, plasma levels of total ghrelin were positively correlated
with serum IGF-1 levels and head circumference. There was also a
correlation between decreased plasma levels of total and active
ghrelin and eating difficulties and constipation, as well as
between decreased plasma levels of active ghrelin and eating
difficulties.
[0023] C. Caffarelli et al. reported comparison between 123 female
RTT patients and 55 healthy controls in Italy (Bone 50 (2012)
830-835 (Non-patent reference 24)). In a study of ghrelin and bone
density, adolescent patients with RTT had higher ghrelin levels,
lower total bone densities and a lower total bone mineral
content/height ratio. In adolescent female patients with RTT, serum
ghrelin levels were inversely correlated with bone age and BMI.
[0024] Munetsugu Hara et al. reported comparison between plasma
levels of ghrelin, GH and IGF-1 with anthropometric data (weight,
height, BMI and head circumference) in 22 patients with RTT,
associated with MECP2 gene mutations, and 14 age- and
gender-matched healthy controls (Brain & Development 36 (2014)
794-800 (Non-patent reference 25)). To subdivide the patients with
(RTT) and without (non-RTT) MeCP2 mutations in the group with
epilepsy and intellectual disabilities, those with RTT had
significantly lower BMI values and heights than those in the
non-RTT group. More significantly, there was an inverse correlation
between plasma ghrelin levels and head circumference in the RTT
group.
[0025] Toyojiro Matsuishi et al. reviewed an interim report that
plasma levels of total ghrelin were lower in patients with RTT than
in healthy controls. This suggested that ghrelin plays an important
role in the pathophysiology of RTT (Brain & Development 33
(2011) 627-631 (Non-patent reference 26)).
[0026] Toyojiro Matsuishi reviewed the discovery of RTT, discovery
of its causative gene, establishment of an animal model and current
therapeutic candidates identified using model animals and presented
therapeutic agents and future perspectives animals or human
(Japanese Journal of Clinical Medicine, Vol. 71, No. 11 (2013)
2043-2053) (Non-patent reference 27)).
[0027] To date, there have only been a few studies on ghrelin and
RTT. However, there remains a lack of understanding and the
potential use of ghrelin as a therapeutic agent for RTT was not
known.
PRIOR ART
Patent Reference
[0028] Patent reference 1: JP 2010-526089 [0029] Patent reference
2: JP 2012-131815 [0030] Patent reference 3: JP 2012-503009 [0031]
Patent reference 4: JP 2014-196331 [0032] Patent reference 5: JP
2015-145407 [0033] Patent reference 6: JP 2014-508744 [0034] Patent
reference 7: JP 2011-501760
Non-Patent Reference
[0034] [0035] Non-patent reference 1: Angela J McArthur et al.,
Developmentulhledicitie & Cbild Neirrology (1998) 40, 186-192
[0036] Non-patent reference 2: Carolyn Ellaway et al., Brain &
Development (2001) 23, S101-S103 [0037] Non-patent reference 3:
Yoshiko Nomura, Brain & Development (2005) 27, S35-S42 [0038]
Non-patent reference 4: Deidra Young et al., Brain &
Development (2007) 29, 609-616 [0039] Non-patent reference 5: Meir
Lotan et al., The Scientific World JOURNAL (2006) 6, 1737-1749
[0040] Non-patent reference 6: Flavia Schwartzman et al., Arq
Gastroenterol (2008) v. 45, no. 4, jul. set. [0041] Non-patent
reference 7: Kathleen J. Motil et al., JPGN (2012) Volume 55,
Number 3, September [0042] Non-patent reference 8: Jacky Guy et
al., SCIENCE (2007) 315, 23 FEBRUARY [0043] Non-patent reference 9:
Daniela Tropea et al., PNAS (2009) Feb. 10, 106, no. 6, 2029-2034
[0044] Non-patent reference 10: Maria C. N. Marchetto et al., Cell
(2010) Nov. 12, 143, 527-539 [0045] Non-patent reference 11: Rube'n
Deogracias et al., PNAS (2012) Aug. 28, 109, no. 35, 14230-14235
[0046] Non-patent reference 12: Noel C. Derecki et al., Nature
(2012) 5 Apr., 484, 105-111 [0047] Non-patent reference 13: Giorgio
Pini et al., Autism Research and Treatment (2012), 2012, Article ID
679801, 14 pages [0048] Non-patent reference 14: Laura Ricceri et
al., Neuropharmacology (2013) 68, 106-115 [0049] Non-patent
reference 15: Ito et al., SRL Hokan (2013) 34, No. 2 [0050]
Non-patent reference 16: Omar S. Khwaja et al., PNAS (2014) Mar.
25, 111, no. 12, 4596-4601 [0051] Non-patent reference 17: Kojima M
et al., Nature (1999) 402, 656 [0052] Non-patent reference 18:
Kojima M et al., Trends Endocrinol Metab (2001) 12, 118 [0053]
Non-patent reference 19: Takaya K et al., J Clin Endocrinol Metab
(2000) 85, 4908 [0054] Non-patent reference 20: Tschop M et al.,
Nature (2000) 407, 908 [0055] Non-patent reference 21: Nakazato M
et al., Nature (2001) 409, 194 [0056] Non-patent reference 22:
Munetsugu Hara et al., Int. J. Devl Neuroscience (2011) 29,899-902
[0057] Non-patent reference 23: C. Caffarelli et al., Bone (2012)
50, 830-835 [0058] Non-patent reference 24: Munetsugu Hara et al.,
Brain & Development (2014) 36, 794-800 [0059] Non-patent
reference 25: Toyojiro Matsuishi et al., Brain & Development
(2011) 33, 627-631 [0060] Non-patent reference 26: Munetsugu Hara
et al., Brain & Development (2013) article in press [0061]
Non-patent reference 27: Toyojiro Matsuishi, Japanese Journal of
Clinical Medicine, (2013-11) 71, No. 11, 2043-2053
DISCLOSURE OF THE INVENTION
Technical Problem to be Solved by the Invention
[0062] Until now, no effective therapeutic methods for RTT were
established. RTT is a neurodevelopmental disorder with various
symptoms such as neurological symptoms including dystonia,
seizures, sleep disturbances, eating disorders and emaciation. A
therapeutic agent for ameliorating these symptoms is needed.
Means for Solving the Problem
[0063] The present inventors conducted research to identify an
effective therapeutic method for RTT. As a result, they found that
ghrelin levels decreased in patients with RTT in an age-dependent
manner and were correlated with gastrointestinal symptoms such as
feeding and constipation, and autonomic nervous symptoms, thereby
completing the present invention.
[0064] Thus, the present invention relates to pharmaceutical
composition comprising ghrelin and a pharmaceutically acceptable
carrier. In particular, the present invention relates to a
prophylactic and therapeutic agent for RTT comprising a
therapeutically effective amount of ghrelin. In accordance with the
present invention, ghrelin was actually administered to patients
with RTT to prove its effectiveness.
Effects of the Invention
[0065] The prophylactic and therapeutic agent for RTT of the
present invention comprising a therapeutically effective amount of
ghrelin can be administered to patients safely without severe side
effects and exerts the effects of increased GH secretion and
amelioration of constipation, sleep, muscle tone and dystonia.
BRIEF DESCRIPTION OF DRAWINGS
[0066] FIG. 1-1 shows amelioration symptoms in patients with RTT
after administration of ghrelin.
[0067] FIG. 1-2 shows amelioration of symptoms in patients with RTT
after administration of ghrelin.
[0068] FIG. 1-3 shows amelioration of symptoms in patients with RTT
after administration of ghrelin.
[0069] FIG. 2 shows changes in plasma ghrelin levels with the
passage of time in patients with RTT after administration of
ghrelin.
[0070] FIG. 3 shows changes in growth hormone secretion with the
passage of time in patients with RTT after administration of
ghrelin.
[0071] FIG. 4 shows changes in blood glucose with the passage of
time in patients with RTT after administration of ghrelin.
[0072] FIG. 5-1 shows time-dependent changes in thermography in
patients with RTT after administration of ghrelin.
[0073] FIG. 5-2 shows time-dependent changes in surface temperature
in patients with RTT after administration of ghrelin.
[0074] FIG. 5-3 shows time-dependent changes in surface temperature
and deep body temperature in patients with RTT after administration
of ghrelin.
[0075] FIG. 6 shows chest and abdominal movements in patients with
RTT after administration of ghrelin.
[0076] FIG. 7 shows the results of a test investigating effects on
breathing before and after intravenous administration of ghrelin in
patients with RTT.
[0077] FIG. 8-1 shows the results of autonomic analysis (Holter
electrocardiography) in patients with RTT after administration of
ghrelin.
[0078] FIG. 8-2 shows the results of autonomic analysis (Holter
electrocardiography) in patients with RTT after administration of
ghrelin.
[0079] FIG. 9 shows that cortisol awaking response (CAR) was
ameliorated by administration of ghrelin in patients with RTT.
[0080] FIG. 10 shows the results of melatonin measurements in the
saliva of patients with RTT after administration of ghrelin.
[0081] FIG. 11 illustrates symptomatic progress in patients of
interest with RTT, shown in the chronological order of symptom
development.
[0082] FIG. 12-1 shows time-dependent changes in VAS in patients
with RTT after administration of ghrelin.
[0083] FIG. 12-2 shows time-dependent changes in VAS in patients
with RTT after administration of ghrelin.
[0084] FIG. 13 shows amelioration of dystonia by VAS in patients
with RTT after administration of ghrelin.
[0085] FIG. 14-1 shows the sleep diary of a patient (Case 1) with
RTT before and after administration of ghrelin.
[0086] FIG. 14-2 shows the sleep diary of a patient (Case 4) with
RTT before and after administration of ghrelin.
BEST MODE FOR IMPLEMENTING THE INVENTION
[0087] Currently, ghrelin is undergoing clinical testing for
gastrointestinal symptoms such as anorexia nervosa and
cardiovascular disorders and for use after total gastrectomy. These
tests are performed without showing significant side effects. In
accordance with the present invention, amelioration of eating
disorders, constipation, autonomic symptoms and central nervous
system manifestations--which impair quality of life of, and are
involved in lifespan prognosis of, patients with RTT--is expected
after ghrelin administration.
[0088] Ghrelin as used herein is active ghrelin showing its
original physiological activities wherein the side chain of the 3rd
amino acid residue in a peptide consisting of 28 amino acid
residues is modified with octanoic acid. Ghrelin as used herein may
be a ghrelin derivative where any one to several of the 28 amino
acid residues is deleted, substituted or added, a ghrelin
derivative substituted with, lauric acid or palmitic acid in place
of octanoic acid, a ghrelin derivative substituted with an
unsaturated fatty acid or a branched fatty acid (e.g. 3-octenoyl
(C8:1) or 4-methylpentanoyl) in place of octanoic acid, or a
ghrelin derivative substituted with an aromatic amino acid,
tryptophan, in place of octanoic acid, insofar as its physiological
activity is maintained.
[0089] Ghrelin as used herein may be obtained by isolating the
natural peptide or may be prepared in a conventional manner using a
peptide synthesizer. A method for preparing ghrelin as used herein
is not particularly limited. For instance, ghrelin as used herein
may be prepared by isolation from ghrelin producing cells of the
human gastric corpus or by a gene recombination technique. In case
of isolation from ghrelin producing cells, ghrelin may be purified
by chromatography. For instance, ghrelin may be purified by gel
filtration, two ion exchange HPLCs, and reverse phase HPLC. Ghrelin
as used herein may also be purified by affinity chromatography
using a suitable carrier to which an antibody to ghrelin is
bound.
[0090] After purification, ghrelin is dissolved in a solvent and
the solution is aseptically filtered and transferred to an ample or
vial to prepare the composition of the present invention. For a
solvent, distilled water for injection, saline, 0.01 M to 0.1 M
phosphate buffer and the like may be used, if necessary, in
admixture with ethanol, glycerol, etc. Furthermore, after ghrelin
is dissolved in a solvent, the solution is aseptically filtered,
transferred to an ample, a vial etc. and lyophilized to prepare the
pharmaceutical composition of the present invention.
[0091] The therapeutic agent of the present invention may also be
mixed with sugars such as mannitol, glucose and lactose, salts such
as common salt and sodium phosphate, and the like, as additives.
The pharmaceutical composition of the present invention in a
dissolved state usually has a pH ranging from 6.8 to 7.5,
preferably 7.3 to 7.4, more preferably 7.35.
[0092] The route of administration of the pharmaceutical
composition of the present invention is not particularly limited
and is in accordance with common practice including, for instance,
oral administration, intraperitoneal injection, intratracheal
injection, intrabronchial injection and direct intrabronchial
instillation, subcutaneous injection, transdermal delivery,
intra-arterial injection, intravenous injection, nasal
administration and the like. However, it is preferably administered
via parenteral administration, namely, subcutaneous, intradermal or
intravenous injection. The pharmaceutical composition for
parenteral administration includes a solution of ghrelin of the
present invention dissolved in a commonly acceptable carrier,
preferably an aqueous carrier. A variety of aqueous carriers, all
known in the art, may be used, including, for instance, water,
buffer, brine, glycine and the like. These solutions are sterilized
and generally contain no particulate substances. These
pharmaceutical compositions may be sterilized by a method for
sterilization well known in the art. The composition of the present
invention may be supplemented with a commonly used additive, for
instance, a stabilizing agent (arginine, polysorbate 80, macrogol
4000, etc.), an excipient (mannitol, sorbitol, sucrose, etc.) and
the like, and formulated for injection or in preparations that can
be administered transmucosally (nasally, orally or sublingually) by
procedures such as sterile filtration, dispersion, lyophilization,
etc.
[0093] In accordance with the present invention, a therapeutically
effective amount of ghrelin may vary depending on the severity of
disease state, age, body weight etc. of the subject and is
ultimately determined at the discretion of the physician. Normally,
ghrelin may be administered once at a dose of 0.03 .mu.g/kg/day to
10 .mu.g/kg/day, preferably 1 .mu.g/kg/day to 5 .mu.g/kg/day, more
preferably 1 .mu.g/kg/day to 3 .mu.g/kg/day. A dose of 3
.mu.g/kg/day is the most preferable. A skilled person could
determine the necessary procedural regimen, depending on the
severity of a specific disease and condition to be treated, using a
standard pharmacological method.
[0094] For assessment of RTT, four international scales, the Rett
Syndrome Behavioral Questionnaire (RSBQ), the Anxiety Depression
and Mood Scale (ADAMS), Burke-Fahn-Marsden Dystonia Rating Scale
(BFMDRS) and Pittsburgh Sleep Quality Index (PSQI) for caregivers,
and the Neurological Test Chart developed by the Japanese Society
of Child Neurology are known. A visual analog scale (VAS) with 5 as
the baseline, 10 as the best and 0 as the worst may be used by
parents or physical therapists. In the present invention, dystonia
was assessed using the international BFMDRS. The VAS assesses
appetite, bowel movement, dystonia, vasomotor reflex, sleep,
swallowing etc.
[0095] Test items evaluated in patients with RTT are the following:
As test items, cortisol in saliva, ghrelin, growth hormone, IGF-1,
blood glucose in blood etc. were measured in addition to breathing
and cardiac function. Sleep was assessed with a sleep diary and by
actigraphy. Day-long EEG video monitoring was also used for
assessment, before and after ghrelin administration.
Breathing pattern: chest/abdominal sensor, SpO.sub.2 monitor
Circulation: 12-lead electrocardiogram Temperature: surface
temperature, deep body temperature, thermography Sleep: sleep
diary, actigraphy Blood test: ghrelin, growth hormone, blood
glucose, IGF-1 etc. Saliva: cortisol, melatonin, MHPG etc.
Electroencephalogram (EEG): 24-hour EEG video monitoring Autonomic
nervous system: Holter electrocardiography
[0096] Ghrelin has a variety of physiological activities besides
appetite promotion and GH secretion. It is known that ghrelin
alleviates and modulates sympathetic hypertonic autonomic nerves
and impaired autonomic nerve imbalances are observed in RTT.
Patients with RTT having eating disorders exhibit significantly
lower levels of both total ghrelin and active ghrelin than do
controls. Lower total ghrelin levels are significantly correlated
with constipation. RTT model animals have lower brain weights than
control animals, with microcephalic tendencies and also have lower
plasma ghrelin levels.
[0097] The present invention is explained in more detail in the
following examples but is not limited thereto.
Example 1
[0098] After approval of the ethics committee of Kurume University,
the test was conducted after obtaining Informed Consent (IC) from
the patients' parents. Four patients aged 21 years old (case 1), 12
years old (case 2), 22 years old (case 3) and 32 years old (case
4), each with the MECP2 mutation and a definitive diagnosis, were
subjected to the test (Table 1). Ghrelin was intravenously
administered in the morning, after fasting for 3 days. For clinical
data, RTT Behavioral Questionnaire, Burke-Fahn-Marsden Dystonia
Rating Scale, VAS (Visual Analog Scale; 5: baseline before
treatment; 10: markedly improved; 0: markedly worsened) evaluated
by their parents, and a sleep diary were used. For biochemical
data, plasma levels of ghrelin, GH, blood glucose, saliva cortisol
and melatonin were obtained. For physiological data,
electroencephalogram, respiration sensor, Holter
electrocardiography and actigraphy were used.
TABLE-US-00001 TABLE 1 List of backgrounds of patients with ghrelin
administration Case: Age Case1: 21 years old Case2: 12 years old
Case3: 22 years old Case4: 32 years old Genetic mutation c.C4767
c.6232A c.C883T c.1196_1200delCCACC ADL Bedridden Able to roll over
Bedridden Able to walk alone Sleep disturbance Yes None Yes Yes
Epilepsy None Yes Yes Yes Hyperventilation/ None Yes Yes None Apnea
Abnormal Mild long QT VPC None None electro cardiogram Dystonia Yes
Yes Yes Yes Scoliosis Yes None Yes Yes Alimentation Oral +
Gastrostomy Oral Oral Oral Bowel movement Diarrhea Constipation
Constipation Constipation after gastrostomy Medication CBZ, VPA,
CLB, LEV CBZ, VPA, CLB CBZ, VPA, CLB Flunitrazepam Pramipexole
Magnesium oxide Aripiprazole Eperisone Piperidone Melatonin
Magnesium oxide Alfacalcidol Magnesium oxide Sennoside Calcium
lactate Ramelteort Suvorexant
[0099] The protocol for the study was as follows:
On the 1st day in the hospital: wear actigraphy sensor (return it
on leaving the hospital), saliva sampled every 3 hours after 12
o'clock. In the hospital (ghrelin administration once daily, in the
morning):
[0100] In the morning without breakfast
[0101] Intravenous line (saline with heparin lock) inserted before
breakfast
[0102] Administration of ghrelin (3 .mu.g/kg/day), at 9 a.m. for 3
consecutive days
[0103] After administration, allowed to eat normally Ghrelin blood
sampling: before, immediately after, 30 minutes after and 60
minutes after ghrelin administration GH blood sampling: before,
immediately after, 30 minutes after, 60 minutes after and 90
minutes after ghrelin administration
Saliva sampling: at bedtime of the 1st day in the hospital and
immediately after awakening on the 2nd day, thereafter every 3
hours until bedtime Assessment: neurological examination on
admission to the hospital Central nervous system: seizure
frequency, dystonia Neurological Test Chart check, etc. Autonomic
nervous system: Holter electrocardiography: RR interval,
sympathetic thermography, deep body temperature measurement Others:
constipation status, duration of eating (mean duration of eating
time in the morning, afternoon and evening), sleep, respiratory
abnormalities, scoliosis, grinding of the teeth, developmental
psychomotor milestones, and other clinical parameters
[0104] The patients were admitted to the hospital in the morning
after their histories were taken, wore actigraphy devices until
dinner, and had saliva sampled with cotton swabs and sample tube
for collecting and storing samples. Fasting before breakfast,
venous line maintained with saline (with heparin lock) was inserted
and ghrelin was administered at 3 .mu.g/kg/day. Blood sampling for
ghrelin and growth hormone (GH) measurements was performed and
saliva was successively sampled to measure cortisol,
neurotransmitter and the like. Ghrelin was administered before
breakfast on 3 consecutive days, a total of 3 administrations. The
amount of blood sampled for ghrelin measurements was 2 ml each, a
total of 8 ml, and sampling was done before, immediately after, 30
minutes after and 60 minutes after administration of ghrelin, a
total of 4 times. GH sampling was conducted a total of 5 times on
the first day in the hospital. The amount of blood collected for GH
measurements was 0.5 ml whole blood per sampling, a total of 2.5
ml, and blood samples were collected once between 9:00 and 9:30
a.m. before the initiation of the test, and immediately after, 30
minutes after, 60 minutes after and 90 minutes after ghrelin
administration, a total of 5 times. Saliva samples were collected
30 minutes after awakening and then 3 hours later before bedtime at
21 o'clock in the evening. The amount of each saliva sample was
approximately 500 al. On the day of release from the hospital, the
short-term effects of ghrelin were assessed and each patient was
informed of the next appointment day.
[0105] In the 21 year old female (case 1), dystonia and sleep
disturbances were markedly alleviated and melatonin, which has been
taken orally in a large amount for a long time, could be
discontinued (FIGS. 1-1 and 1-2), provided that, about 3 weeks
after ghrelin administration, the symptoms had returned to those
before the treatment. Improved conditions were maintained after
consecutive administration of ghrelin for 2 days every 3 weeks and
alleviation of the symptoms continued for about 1 year without side
effects. In the 32 year old female (case 4), dystonia and sleep
disturbances were markedly alleviated and improved conditions were
maintained after consecutive ghrelin administration for 2 days
every 3 weeks and the alleviation of symptoms continued for about 4
months without side effects (FIG. 1-3).
[0106] The plasma ghrelin levels increased immediately after
administration and returned to baseline levels after about 60
minutes (FIG. 2). GH levels reached a peak of secretion (30 to 175
times higher than baseline level) at 30 minutes after ghrelin
administration (G in the figure) at 3 .mu.g/kg (FIG. 3). Blood
glucose levels increased slightly at 30 to 60 minutes-after ghrelin
administration (FIG. 4). Ghrelin administration did not affect
surface temperature or deep body temperature (FIG. 5). The
temperature tended to once decrease and then increase. It was
difficult to keep environmental conditions constant, such as room
temperature, clothes and bed/couch, making these temperature
assessments difficult. Most of the patients with RTT showed
respiratory abnormalities such as hyperventilation and/or apnea.
There were no significant changes in breathing or chest movements
after ghrelin administration (FIGS. 6 and 7). Breathing was
monitored in the waking state and compared before and after
intravenous ghrelin administration. Apnea for 10 seconds or more,
its frequency and total time, hypopnea for 10 seconds or less, its
frequency and total time, were assessed but no significant effects
on breathing abnormalities were observed. The results of autonomic
analysis (Holter electrocardiography) are shown in FIG. 8. Cortisol
awaking response (CAR), reflecting awaking rhythm and the autonomic
nerves, is seen in healthy adults at 30 minutes after awakening and
is regarded as an important reaction predominantly of the
sympathetic nervous system. CAR was not observed in the patients
with RTT (FIG. 9). After ghrelin administration, these CAR deficits
appeared to be alleviated. The results of saliva melatonin
measurements are shown in FIG. 10. Ghrelin administration induced
peaks of melatonin. FIG. 11 illustrates symptomatic progress in one
of the patients. Time-dependent changes in VAS and amelioration of
dystonia by VAS are shown in FIGS. 12-1 and 12-2 and FIG. 13,
respectively. A sleep diary before and after ghrelin administration
is shown in FIG. 14. Improvement of sleep rhythms was reported in
the sleep diary.
[0107] After the therapy in accordance with the present invention,
the mother of the 21 year old female patient made the following
remarks: "she became calm and peaceful; I'm glad that she looks
merry; she got to be able to sleep, no longer needs melatonin, and
more active during the day; her duration of eating became shorter;
she got to have formed stool (I was afraid of worsening of
diarrhea); dystonia was ameliorated; she got to be able to open the
mouth at the dental checkup; PT was surprised; tremor of the head
decreased and hair sprouted; vasomotor nerve reflex was ameliorated
(frequency became lowered, time was reduced by half (from 20
minutes to 10 minutes), she got no longer sweaty)." In the
32-year-old female, dystonia was ameliorated, it became easier for
her to open her mouth and she became able to eat large amounts of
solid foods. Hypertonia of the cheeks and around the mouth,
shoulder and abdomen was ameliorated. Sleep was also improved. She
had difficulty sleeping before treatment even with two tablets of
suvorexant and two tablets of ramelteon and, thus, had also taken 5
mg of diazepam. After treatment, she became able to sleep with only
one tablet each of suvorexant and ramelteon and the number of days
when a hypnotic agent was also required were decreased by half.
INDUSTRIAL APPLICABILITY
[0108] The prophylactic and therapeutic agent for RTT of the
present invention comprising a therapeutically effective amount of
ghrelin can be administered to patients safely without severe side
effects and can be used effectively for therapy of RTT, such as
increasing GH secretion and ameliorating constipation, sleep,
muscle tone and dystonia.
* * * * *