U.S. patent application number 11/913455 was filed with the patent office on 2009-08-13 for fatty acid analogues, i.e. including dha derivatives for uses as a medicament.
Invention is credited to Morten Bryhn, Anne Kristin Holmeide, Jan Kopecky.
Application Number | 20090203778 11/913455 |
Document ID | / |
Family ID | 43955576 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090203778 |
Kind Code |
A1 |
Bryhn; Morten ; et
al. |
August 13, 2009 |
FATTY ACID ANALOGUES, I.E. INCLUDING DHA DERIVATIVES FOR USES AS A
MEDICAMENT
Abstract
A compound of formula (I) wherein R.sub.1 and R.sub.2 are
different and each is chosen from a methyl group and a hydrogen
atom; wherein X is chosen from a carboxylic acid group, a
carboxylate group, a carboxamide group; or any pharmaceutically
acceptable salt, solvate, complex, or pro-drug of said compound. A
pharmaceutical composition and a lipid composition comprising a
compound of formula (I) is also disclosed. A method for the
treatment of obesity, diabetes mellitus, amyloidos-related
diseases, cardiovascular-diseases, and cerebrovascular diseases is
also disclosed. ##STR00001##
Inventors: |
Bryhn; Morten; (Svelvik,
NO) ; Holmeide; Anne Kristin; (Oslo, NO) ;
Kopecky; Jan; (Praha, CZ) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Family ID: |
43955576 |
Appl. No.: |
11/913455 |
Filed: |
May 4, 2006 |
PCT Filed: |
May 4, 2006 |
PCT NO: |
PCT/IB06/01164 |
371 Date: |
August 14, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60677350 |
May 4, 2005 |
|
|
|
60677351 |
May 4, 2005 |
|
|
|
Current U.S.
Class: |
514/549 ;
514/560; 514/627; 554/224; 554/35 |
Current CPC
Class: |
A61P 3/06 20180101; C07D
263/22 20130101; C07C 69/618 20130101; A61P 3/04 20180101; A61P
25/28 20180101; C07D 263/24 20130101; C07C 57/03 20130101; C07C
69/587 20130101; C07C 69/65 20130101; A61P 9/10 20180101; C07C
69/732 20130101; A61P 29/00 20180101; A61P 3/00 20180101; C07C
235/28 20130101; C07D 209/48 20130101; A61P 3/10 20180101; A61P
9/00 20180101; C07C 229/30 20130101; A61P 43/00 20180101; C07C
233/09 20130101; C11C 3/00 20130101; C07C 69/734 20130101; C07C
317/44 20130101; C07C 323/54 20130101; C07C 237/16 20130101 |
Class at
Publication: |
514/549 ; 554/35;
554/224; 514/560; 514/627 |
International
Class: |
A61K 31/232 20060101
A61K031/232; C07C 233/09 20060101 C07C233/09; C07C 57/02 20060101
C07C057/02; A61K 31/202 20060101 A61K031/202; A61K 31/164 20060101
A61K031/164; A61P 3/00 20060101 A61P003/00; A61P 9/00 20060101
A61P009/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 4, 2005 |
SE |
0501044-2 |
May 4, 2005 |
SE |
0501045-9 |
Claims
1-37. (canceled)
38. A compound of formula (I): ##STR00005## wherein R.sub.1 and
R.sub.2 are different and each is chosen from a methyl group and a
hydrogen atom; and wherein X is chosen from a carboxylic acid
group, a carboxylate group, a carboxamide group; or any
pharmaceutically acceptable salt, solvate, complex, or pro-drug of
said compound.
39. The compound according to claim 38, wherein the carboxylate
group is chosen from ethyl carboxylate, methyl carboxylate,
n-propyl carboxylate, isopropyl carboxylate, n-butyl carboxylate,
sec-butyl carboxylate, and n-hexyl carboxylate.
40. The compound according to claim 39, wherein the carboxylate
group is ethyl carboxylate.
41. The compound according to claim 38, wherein the carboxamide
group is chosen from a primary carboxamide, N-methyl carboxamide,
N,N-dimethyl carboxamide, N-ethyl carboxamide, and N,N-diethyl
carboxamide.
42. The compound according to claim 38, wherein the compound is
present in the form of a phospholipid, a triglyceride, a
diglyceride, a monoglyceride, or a free acid.
43. The compound according to claim 38, wherein the compound of
formula (I) is present in racemic form.
44. The compound according to claim 38, wherein the compound of
formula (I) is present as the R stereoisomer.
45. The compound according to claim 38, wherein the compound of
formula (I) is present as the S stereoisomer.
46. A composition comprising at least one compound of claim 42 or a
mixture thereof.
47. A lipid composition comprising a compound of formula (I):
##STR00006## wherein R.sub.1 and R.sub.2 are different and each is
chosen from a methyl group and a hydrogen atom; and wherein X is
chosen from a carboxylic acid group, a carboxylate group, a
carboxamide group; or any pharmaceutically acceptable salt,
solvate, complex, or pro-drug of said compound; and a
pharmaceutically acceptable antioxidant.
48. The lipid composition according to claim 47, wherein the
compound of formula (I) comprises at least 60% by weight of the
total composition.
49. The lipid composition according to claim 48, wherein the
compound of formula (I) comprises at least 90% by weight of the
total composition.
50. The lipid composition according to claim 47, wherein the
composition further comprises at least one fatty acid chosen from
(all-Z)-5,8,11,14,17-eicosapentaenoic acid (EPA),
(all-Z)-6,9,12,15,18-heneicosapentaenoic acid (HPA),
(all-Z)-7,10,13,16,19-docosapentaenoic acid (DPA), and derivative
forms thereof.
51. The lipid composition according to claim 47, wherein the
antioxidant is tocopherol.
52. A pharmaceutical composition comprising a compound of formula
(I): ##STR00007## wherein R.sub.1 and R.sub.2 are different and
each is chosen from a methyl group and a hydrogen atom; and wherein
X is chosen from a carboxylic acid group, a carboxylate group, a
carboxamide group; or any pharmaceutically acceptable salt,
solvate, complex, or pro-drug of said compound; and at least one of
component chosen from a pharmaceutically acceptable carrier,
diluent, and excipient.
53. The pharmaceutical composition according to claim 52 formulated
for oral administration.
54. The pharmaceutical composition according to claim 52 formulated
as a capsule or sachet.
55. The pharmaceutical composition according to claim 52 formulated
to provide a daily dosage of 10 mg to 10 g of the compound of
formula (I).
56. The pharmaceutical composition according to claim 55,
formulated to provide a daily dosage of 100 mg to 1 g of the
compound of formula (I).
57. A method of treatment for at least one disorder or disease in a
human or animal patient in need thereof, wherein the at least one
disorder or disease is chosen from obesity, diabetes mellitus,
amyloidos-related diseases, cardiovascular diseases, and
cerebrovascular diseases comprising: administering to the human or
animal patient in need thereof a pharmaceutically effective amount
of a compound of formula (I): ##STR00008## wherein R.sub.1 and
R.sub.2 are different and each is chosen from a methyl group and a
hydrogen atom; and wherein X is chosen from a carboxylic acid
group, a carboxylate group, a carboxamide group; or any
pharmaceutically acceptable salt, solvate, complex, or pro-drug of
said compound.
58. The method of treatment according to claim 57, wherein the at
least one diabetes mellitus disorder or disease is diabetes
mellitus type 2.
59. The method of treatment according to claim 57, wherein the at
least one cardiovascular disorder or disease is chosen from
elevated blood lipid levels.
60. The method of treatment according to claim 57, wherein the at
least one cerebrovascular disorder or disease is chosen from
stroke, cerebral ischaemic attacks related to atherosclerosis of
several arteries, or transient ischaemic attacks related to
atherosclerosis of several arteries.
61. A method of treatment for body weight reduction and prevention
of body weight gain in a human or animal patient in need thereof,
comprising: administering to the human or animal patient in need
thereof a pharmaceutically effective amount of a compound of
formula (I): ##STR00009## wherein R.sub.1 and R.sub.2 are different
and each is chosen from a methyl group and a hydrogen atom; and
wherein X is chosen from a carboxylic acid group, a carboxylate
group, a carboxamide group; or any pharmaceutically acceptable
salt, solvate, complex, or pro-drug of said compound.
Description
TECHNICAL FIELD
[0001] The present invention relates to compounds of the general
formula (I):
##STR00002##
for use as a medicament, in particular for the treatment of
diabetes mellitus, type 2, and pre-stages thereof. It also relates
to a pharmaceutical composition comprising a compound of formula
(I), as well as to a fatty acid composition comprising a compounds
of formula (I).
BACKGROUND OF THE INVENTION
[0002] The increasing incidence of type 2 diabetes mellitus
worldwide poses an immense public health and medical challenge for
the implementation of successful preventive and treatment
strategies. The concurrent rise in overweight and obesity, which is
tightly correlated to type 2 diabetes, interferes with diabetes
treatment and increases the likelihood of hypertension,
dyslipidemia, and atherosclerosis related diseases.
[0003] The pathophysiologic condition preluding the development of
type 2 diabetes is related to reduced effects of insulin on
peripheral tissues, called insulin resistance. These tissues are
mainly muscle, fat and liver. Muscle tissue is the main tissue
concerned by insulin resistance in type 2 diabetes. The syndrome
characterised by insulin resistance, hypertension, dyslipidemia and
a systemic proinflammatory state, is referred to as metabolic
syndrome. The prevalence of metabolic syndrome in the adult
population in developed countries is 22-39% (Meighs 2003)
[0004] Currently the most promising approach to mitigate and deter
the metabolic syndrome is lifestyle intervention with weight
reduction, decreased consumption of saturated fat, increased
physical activity in combination with appropriate pharmacotherapy.
Healthy diets that avoid excess energy intake encompass
substitution of mono and polyunsaturated fatty acids in exchange
for saturated fat. In particular the long-chain omega-3 fatty acids
from fatty fish, namely eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) have proven beneficial in prevention of
type 2 diabetes.
[0005] EPA and DHA have effects on diverse physiological processes
impacting normal health and chronic disease, such as the regulation
of plasma lipid levels, cardiovascular and immune function, insulin
action and neural development and visual function. Firm evidence
exist for their beneficial role in the prevention and management of
coronary heart disease, dyslipidemias, type 2 diabetes, insulin
resistance, and hypertension (Simonopoulos 1999; Geleijnse 2002;
Storlien 1998).
[0006] Recent studies suggest that omega-3 fatty acids serve as
important mediators of gene expression, working via nuclear
receptors like the peroxisome proliferator-activated receptors
(PPARs) controlling the expression of the genes involved in the
lipid and glucose metabolism and adipogenesis (Jump 2002). PPARs
are nuclear fatty acid receptors that have been implicated to play
an important role in obesity-related metabolic diseases such as
hyperlipidemia, insulin resistance, and coronary heart disease.
[0007] The three subtypes, .alpha., .gamma., and .delta., have
distinct expression pattern and evolved to sense components of
different lipoproteins and regulate lipid homeostasis based on the
need of a specific tissue. PPAR.alpha. potentiates fatty acid
catabolism in the liver and is the molecular target of the
lipid-lowering fibrates. PPAR.gamma. on the other hand is essential
for adipocyte differentiation and mediates the activity of the
insulin-sensitizing thiazolidinedions (the glitazones) through
mechanisms not fully understood. (Chih-Hao 2003; Yki-Jarvinen
2004)
[0008] Recently, pharmaceuticals acting as ligands to the
PPAR.gamma. receptor have been introduced as treatment of type 2
diabetes (Yki-Jarvinen 2004). These compounds called
thiazolidinediones or glitazones are drugs that reverse insulin
resistance which is the pathophysiologic basis for development of
the metabolic syndrome and type 2 diabetes. These compounds, of
which rosiglitazone and pioglitazone have been launched as
pharmaceuticals, lower fasting and postprandial glucose
concentrations (which is being manifest as a pathologic glucose
tolerance test), plasma insulin as well as free fatty acid
concentrations. In this respect the glitazones act as insulin
sensitizers.
[0009] However, these improvements are generally accompanied by
weight gain and an increase in the subcutaneous adipose-tissue mass
(Adams 1997). The use of thiazolidinediones is not only associated
with weight gain but a subgroup of patients also have fluid
retention and plasma volume expansion, leading to peripheral
oedema. The increase in body weight and oedema has been associated
with an increase in the incidence of heart failure, which is the
reason why the Food and Drug Administration has included a warning
in the prescription information for rosiglitazone (provided by
Avandia) and pioglitazone (provided by Takeda). These adverse
effects restrict the use of the glitazones especially in patients
with coronary heart conditions. Clearly there is a potential for
new drugs with positive effects on insulin resistance but with
weight reduction activity and no fluid retention tendency.
[0010] The effect of the poly-unsaturated fatty acids (PUFAs) on
PPARs are not only a result of fatty acid structure and affinity to
the receptor. Factors contributing to the composition of the
intracellular non-esterified fatty acids (NEFA) levels are also
important. This NEFA pool is affected by the concentration of
exogenous fatty acids entering the cell and the amount of
endogenous synthesised fatty acids, their removal via incorporation
into lipids as well as their oxidation pathways. (Pawar 2003)
[0011] Although omega-3 fatty acids are weak agonists of PPARs,
when compared with pharmacological agonists like the
thioglitazones, these fatty acids have demonstrated improvement in
glucose uptake and insulin sensitivity (Storlien 1987). It has been
reported that adipocytes were more insulin sensitive and
transported more glucose when the polyunsaturated to saturated
fatty acid ratio in the diet was increased (Field 1990).
Collectively, these data indicate that the 20- and 22-carbon fatty
acids, namely EPA and DHA could play a preventive role in the
development of insulin resistance.
[0012] Due to their limited stability in vivo and their lack of
biological specificity, PUFAs have not achieved widespread use as
therapeutic agents. Chemical modifications of the n-3
polyunsaturated fatty acids have been performed by several research
groups in order to change or increase their metabolic effects.
[0013] For example, the hypolipidemic effects of EPA was
potentiated by introducing methyl or ethyl in .alpha.- or
.beta.-position of EPA. (Vaagenes 1999). The compounds also reduced
plasma free fatty acid while EPA EE had no effect.
[0014] In a recent work published by L. Larsen (Larsen 2005) the
authors show that the .alpha.-methyl derivatives of EPA and DHA
increased the activation of the nuclear receptor PPAR.alpha. and
thereby the expression of L-FABP compared to EPA/DHA. EPA with an
ethyl group in the .alpha.-position activated PPAR.alpha. with
equal strength as .alpha.-methyl EPA. The authors suggest that
delayed catabolism of these .alpha.-methyl FA may contribute to
their increased effects due to decreased .beta.-oxidation in
mitochondria leading to peroxisomal oxidation.
[0015] Alpha-methyl EPA has been shown to be a stronger inhibitor
of platelet aggregation than EPA, both in vitro (Larsen 1998) and
in vivo (Willumsen 1998).
[0016] Patent Abstract of Japan, publication number 05-00974
discloses DHA substituted in alpha-position with an OH-group,
however only as an intermediate. No examination as to possible
pharmaceutical effects of this compound is disclosed.
[0017] Laxdale Limited has also described the use of alpha
substituted derivatives of EPA in the treatment of psychiatric or
central nervous disorders (U.S. Pat. No. 6,689,812).
##STR00003##
[0018] The vast research in the field of substituted fatty acids
demonstrates the great interest in finding appropriate medical and
pharmaceutical applications. However, so far the practical
applications have been very limited, and there is thus a continuing
need for finding useful application areas for fatty acid
derivatives.
SUMMARY OF THE INVENTION
[0019] One aim of the present invention is to provide a useful
medical application of DHA-derivatives. Accordingly, the present
invention provides a compound of formula (I);
##STR00004## [0020] wherein one of R.sub.1 and R.sub.2 is a methyl
group and the other of R.sub.1 and R.sub.2 is a hydrogen atom;
[0021] wherein X represents a carboxylic acid group, a carboxylate
group, or a carboxamide group; or any pharmaceutically acceptable
salt, solvate, complex or pro-drug thereof, for use as a
medicament. The alpha-substituted DHA-derivative according to the
invention has very surprisingly shown excellent results with regard
to pharmaceutical activity. In particular, the fatty acid
derivative according to the present invention possess a huge
potential to be used in the treatment and/or prevention of diabetes
and pre-stages thereof.
[0022] The carboxylate group may be selected from the group
consisting of ethyl carboxylate, methyl carboxylate, n-propyl
carboxylate, isopropyl carboxylate, n-butyl carboxylate, sec.-butyl
carboxylate, and n-hexyl carboxylate. Preferably, the carboxylate
group is ethyl carboxylate.
[0023] The carboxamide group may be selected from the group
consisting of primary carboxamide, N-methyl carboxamide,
N,N-dimethyl carboxamide, N-ethyl carboxamide, and N,N-diethyl
carboxamide.
[0024] The compounds of formula (I) are capable of existing in
stereoisomeric forms. It will be understood that the invention
encompasses all optical isomers of the compounds of formula (I) and
mixtures thereof including racemates for use as a medicament.
[0025] The compound of formula (I) may also exist in the form of a
phospholipid, a tri-, di- or monoglyceride, or in the form of a
free acid.
[0026] Another aspect of the present invention relates to a
pharmaceutical composition comprising a compound of formula (I) as
an active ingredient. The pharmaceutical composition may further
comprise a pharmaceutically acceptable carrier. Suitably, a
pharmaceutical composition according to the invention is formulated
for oral administration, e.g. in the form of a capsule or a sachet.
A suitable daily dosage of a compound of formula (I) according to
the present invention is 10 mg to 10 g, in particular 100 mg to 1 g
of said compound per 24 hours.
[0027] In addition, the present invention relates to a fatty acid
composition comprising a compound of formula (I). At least 60%, or
at least 90% by weight of the fatty acid composition may be
comprised of said compound. The fatty acid composition may further
comprise (all-Z)-5,8,11,14,17-eicosapentaenoic acid (EPA),
(all-Z)-4,7,10,13,16,19-docosahexaenoic acid (DHA),
(all-Z)-6,9,12,15,18-heneicosapentaenoic acid (HPA), and/or
(all-Z)-7,10,13,16,19-docosapentaenoic acid (DPA). The fatty acids
may be present in the form of derivatives. A fatty acid composition
according to the present invention may further comprise a
pharmaceutically acceptable antioxidant, e.g. tocopherol. Within
the scope of the present invention is also a fatty acid composition
described above, for use as a medicament.
[0028] In a further aspect, the present invention relates to the
use of a compound according to formula (I) for the manufacture of a
medicament for controlling body weight reduction and/or for
preventing body weight gain; for the manufacture of a medicament
for the treatment and/or the prevention of obesity or an overweight
condition; for the manufacture of a medicament for the prevention
and/or treatment of diabetes in an animal, in particular type 2
diabetes; for the manufacture of a medicament for the treatment
and/or prevention of amyloidos-related diseases; for the
manufacture of a medicament for the treatment or prophylaxis of
multiple risk factors for cardiovascular diseases, preferably for
the treatment of elevated blood lipids for the manufacture of a
medicament for prevention of stroke, cerebral or transient
ischaemic attacks related to atherosclerosis of several
arteries.
[0029] In addition, the present invention relates to a method for
controlling body weight reduction and/or for preventing body weight
gain; a method for the treatment and/or the prevention of obesity
or an overweight condition; a method for the prevention and/or
treatment of diabetes, in particular type 2 diabetes; a method for
the treatment and/or prevention of amyloidos-related diseases; a
method for the treatment or prophylaxis of multiple risk factors
for cardiovascular diseases; a method for the prevention of stroke,
cerebral or transient ischaemic attacks related to atherosclerosis
of several arteries, wherein a pharmaceutically effective amount of
a compound of formula (I) is administered to a human or an animal.
Suitably, the compound of formula (I) is administered orally to a
human or an animal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows the structural formula of alpha-methyl DHA
ethyl ester.
[0031] FIG. 2 is a schematic overview of the free fatty acid pool
theory.
[0032] FIG. 3 depicts the release of luciferase from transfected
cells treated with the compound according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0033] In the research work leading to the present invention, it
was found that the alpha-methyl-DHA shows excellent pharmaceutical
activity.
[0034] Fatty acids enter cells passively or trough G-protein
coupled transporter systems, such as fatty acid transport proteins.
Well inside the cells they are temporarily bound by binding
proteins (Fatty acid binding proteins, FABP), which play an
important role in directing fatty acids to various intracellular
compartments for metabolism and gene expression (Pawar & Jump
2003). (FIG. 2 liver cell).
[0035] Esterification of fatty acids into triglycerides, polar
lipids, and cholesterol esters and their beta-oxidation
(mitochondrial and peroxisomal) requires conversion of fatty acids
to acyl CoA thioesters. Other pathways, like microsomal
NADPH-dependent mono-oxidation and eikosanoids synthesis, utilise
non-esterified fatty acids as substrates. All these reactions are
likely to influence cellular levels of free fatty acids
(non-esterfified) and thereby the amount and type of fatty acids
which could be used as ligands to nuclear receptors. Because PPARs
are known to bind non-esterified fatty acids it is reasonable to
expect that the composition of the free fatty acid pool is an
important determinant in the control of PPAR activity.
[0036] The composition of the free fatty acid pool is affected by
the concentration of exogenous fatty acids entering the cells, and
their rate of removal via pathways listed above. Since short and
medium chain fatty acids are effectively recruited to these
pathways, in practice only the long-chain polyunsaturated fatty
acids will be available for liganding to nuclear receptors. In
addition, fatty acid structure may also be an important
determinant. Even if a series of mono and polyunsaturated fatty
acids demonstrated affinity to the PPAR.alpha. receptor, EPA and
DHA demonstrated the highest binding capacity in experiments with
rat liver cells (Pawar & Jump 2003).
[0037] Searching for fatty acid candidates available for genetic
modification of proteins by interaction with nuclear receptors like
the PPARs, it is important to verify that the respective fatty
acids will be enriched in the free fatty acid pool.
[0038] DHA which enter cells are rapidly converted to fatty
acyl-CoA thioesters and incorporated into phospholipids and due to
this, the intracellular DHA level is relatively low. These DHA-CoA
are also substrate for .beta.-oxidation primarily in the
peroxisomes that lead to retroconvertion of DHA to EPA, see FIG. 2.
Because of the rapid incorporation into neutral lipids and the
oxidation pathway DHA will not stay long in the free fatty acid
pool. Due to this the effect of DHA on gene expression is probably
limited.
[0039] The present invention aims at achieving an accumulation of
fatty acid derivatives in the free fatty acid pool, rather than
incorporation into phosholipids. The present inventors have
surprisingly found that the introduction of a methyl substituent in
the .alpha.-position of DHA will lead to a slower oxidation rate in
addition to less incorporation into neutral lipids. This will lead
to an increased effect on gene expression, since the DHA derivative
will accumulate in the tissue particular within liver, muscle, and
adipose cells and trigger local nuclear receptor activity to a
greater extent than DHA.
[0040] EPA (all-Z)-5,8,11,14,17-eicosapentaenoic acid) has earlier
been alkylated in .alpha.- and .beta.-position to inhibit
mitochondrial .beta.-oxidation. DHA is not oxidised in the
mitochondria, but rather incorporated into phospholipids. In the
peroxisomes though some DHA is retroconverted to EPA. A substituent
in the .alpha.-position of EPA and DHA will due to this affect
different metabolic pathways. It has earlier been shown that
.alpha.-methyl EPA and .beta.-methyl EPA is incorporated into
phospholipids and triglycerids while .alpha.-ethyl EPA is not
(Larsen 1998). In this study the derivatives were tested as
substrates and/or inhibitors of enzymes involved in the eicosanoid
cascade. Since most of the substrates for these enzymes are fatty
acids liberated from phospholipids it was desired that the
derivatives were incorporated into phospholipids. In contrast to
this, as mentioned before, this invention aims at providing a
derivative that will not incorporate into lipids, but rather
accumulate in the NEFA pool.
[0041] It is to be understood that the present invention
encompasses any possible pharmaceutically acceptable salts,
solvates, complexes or prodrogs of the compounds of formula
(I).
[0042] "Prodrugs" are entities which may or may not possess
pharmacological activity as such, but may be administered (such as
orally or parenterally) and thereafter subjected to bioactivation
(for example metabolized) in the body to form the agent of the
present invention which is pharmacologically active.
[0043] Where X is a carboxylic acid, the present invention also
includes salts of the carboxylic acids. Suitable pharmaceutically
acceptable salts of carboxy groups includes metal salts, such as
for example aluminium, alkali metal salts such as lithium, sodium
or potassium, alkaline metal salts such as calcium or magnesium and
ammonium or substituted ammonium salts.
[0044] A "therapeutically effective amount" refers to the amount of
the therapeutic agent which is effective to achieve its intended
purpose. While individual patient needs may vary, determination of
optimal ranges for effective amounts of each nitric oxide adduct is
within the skill of the art. Generally the dosage regimen for
treating a condition with the compounds and/or compositions of this
invention is selected in accordance with a variety of factors,
including the type, age, weight, sex, diet and medical condition of
the patient.
[0045] By "a medicament" is meant a compound according to formula
(I), in any form suitable to be used for a medical purpose, e.g. in
the form of a medicinal product, a pharmaceutical preparation or
product, a dietary product, a food stuff or a food supplement.
[0046] In the context of the present specification, the term
"therapy" also includes "prophylaxis" unless there are specific
indications to the contrary. The terms "therapeutic" and
"therapeutically" should be constructed accordingly.
[0047] Treatment includes any therapeutic application that can
benefit a human or non-human animal. The treatment of mammals is
particularly preferred. Both human and veterinary treatments are
within the scope of the present invention. Treatment may be in
respect of an existing condition or it may be prophylactic. It may
be of an adult, a juvenile, an infant, a foetus, or a part of any
of the aforesaid (e.g. an organ, tissue, cell, or nucleic acid
molecule). By "chronic treatment" is meant treatment that continues
for some weeks or years.
[0048] "A therapeutically or a pharmaceutically active amount"
relates to an amount that w.+-.1 lead to the desired
pharmacological and/or therapeutic effects.
[0049] A compound according to the present invention may for
example be included in a food stuff, a food supplement, a
nutritional supplement, or a dietary product
[0050] Alpha-substituted DHA derivatives and EPA (or DHA for that
matter) can be bound together and combined on triglyceride form by
an esterification process between a mixture of alpha-derivatives,
EPA and glycerol catalysed by Novozym 435 (a commersially available
lipase from Candida antarctica on immobilised form).
[0051] The compound of formula (I) has activity as pharmaceuticals,
in particular as triggers of nuclear receptor activity. Thus, the
present invention also relates to the compound of formula (I),
pharmaceutically acceptable salts, solvates, complexes or pro-drugs
thereof, as hereinbefore defined, for use as a medicament and/or
for use in therapy. Preferably, the compound of formula (I), or
pharmaceutically acceptable salts, solvates, complexes or pro-drugs
thereof, of the invention may be used: [0052] for the prevention
and/or treatment of diabetes mellitus in humans or animals; [0053]
for controlling body weight reduction and/or for preventing body
weight gain; [0054] for the prevention and/or treatment of obesity
or an overweight condition in humans or in an animal; [0055] for
the treatment and/or prevention of amyloidos-related diseases;
[0056] for the treatment or prophylaxis of multiple risk factors
for cardiovascular diseases; [0057] for the prevention of stroke,
cerebral or transient ischaemic attacks related to atherosclerosis
of several arteries. [0058] for the treatment of TBC or HIV.
[0059] There are two major forms of diabetes mellitus. One is type
1 diabetes, which is known as insulin-dependent diabetes mellitus
(IDDM), and the other one is type 2 diabetes, which is also known
as non-insulin-dependent diabetes mellitus (NIDDM). Type 2 diabetes
is related to obesity/overweight and lack of exercise, often of
gradual onset, usually in adults, and caused by reduced insulin
sensitivity, so called periferral insulin resistance. This leads to
a compensatory increase in insulin production. This stage before
developing full fetched type 2 diabetes is called the metabolic
syndrome and characterized by hyperinsulinemia, insulin resistance,
obesity, glucose intolerance, hypertension, abnormal blood lipids,
hypercoagulopathia, dyslipidemia and inflammation, often leading to
atherosclerosis of the arteries. Later when insulin production
seizes, type 2 diabetes mellitus develops.
[0060] In a preferred embodiment, the compound according to formula
(I) may used for the treatment of type 2 diabetes. The compound
according to formula (I) may also be used for the treatment of
other types of diabetes selected from the group consisting of
metabolic syndrome, secondary diabetes, such as pancreatic,
extrapancreatic/endocrine or drug-induced diabetes, or exceptional
forms of diabetes, such as lipoatrophic, myatonic or a disease
caused by disturbance of the insulin receptors. The invention also
includes treatment of type 2 diabetes. Suitably, the compound of
formula (I), as hereinbefore defined, may activate nuclear
receptors, preferably PPAR (peroxisome proliferator-activated
receptor) .alpha. and/or .gamma..
[0061] The compound of formula (I) may also be used for the
treatment and/or prevention of obesity. Obesity is usually linked
to an increased insulin resistance and obese people run a high risk
of developing type 2 diabetes which is a major risk factor for
development of cardiovascular diseases. Obesity is a chronic
disease that afflict an increasing proportion of the population in
Western societies and is associated, not only with a social stigma,
but also with decreasing life span and numerous problems, for
instance diabetes mellitus, insulin resistance and hypertension.
The present invention thus fulfils a long-felt need for a drug that
will reduce total body weight, or the amount of adipose tissue, of
preferably obese humans, towards their ideal body weight without
significant adverse side effects.
[0062] The compound according to formula (I) may also be used for
the prevention and/or treatment of amyloidos-related diseases.
Amyloidos-related conditions or diseases associated with deposition
of amyloid, preferably as a consequence of fibril or plaque
formation, includes Alzheimer's disease or dementia, Parkinson's
disease, amyotropic lateral sclerosis, the spongiform
encephalopathies, such as Creutzfeld-jacob disease, cystic
fibrosis, primary or secondary renal amyloidoses, IgA nephropathy,
and amyloid depostion in arteries, myocardium and neutral
tissue.
[0063] These diseases can be sporadic, inherited or even related to
infections such as TBC or HIV, and are often manifested only late
in life even if inherited forms may appear much earlier. Each
disease is associated with a particular protein or aggregates of
these proteins are thought to be the direct origin of the
pathological conditions associated with the disease. The treatment
of a amyloidos-related disease can be made either acutely or
chronically.
[0064] The compound of formula (I) may also be used for the
treatment due to reduction of amyloid aggregates, prevention of
misfolding of proteins that may lead to formation of so called
fibrils or plaque, treatment due to decreasing of the production of
precursor protein such as A.beta.-protein (amyloid beta protein),
and prevention and/or treatment due to inhibiting or slow down the
formation of protein fibrils, aggregates, or plaque. Prevention of
fibril accumulation, or formation, by administering a compound of
formula (I), as hereinbefore defined, is also included herein. In
one embodiment, the compound of formula (I), pharmaceutically
acceptable salts, solvates, complexes or pro-drugs thereof, as
hereinbefore defined, are used for the treatment of TBC
(tuberculosis) or HIV (human immunodeficiency virus).
[0065] Further, the compound of formula (I) may be administered to
patients with symptoms of atherosclerosis of arteries supplying the
brain, for instance a stroke or transient ischaemic attack, in
order to reduce the risk of a further, possible fatal, attack.
[0066] The compound of formula (I) may also be used for the
treatment of elevated blood lipids in humans.
[0067] Additionally, the compound of formula (I), as hereinbefore
defined, are valuable for the treatment and prophylaxis of multiple
risk factors known for cardiovascular diseases, such as
hypertension, hypertriglyceridemia and high coagulation factor VII
phospholipid complex activity. Preferably, the compound of formula
(I) is used for the treatment of elevated blood lipids in
humans.
[0068] The compound of formula (I) and pharmaceutically acceptable
salts, solvates, pro-drugs or complexes thereof may be used on
their own but will generally be administered in the form of a
pharmaceutical composition in which the compound of formula (I)
(the active ingredient) are in association with a pharmaceutically
acceptable adjuvant, diluent or carrier.
[0069] The present invention thus also provides a pharmaceutical
composition comprising a therapeutically effective amount of the
compound of formula (I) of the present invention and a
pharmaceutically acceptable carrier, diluent or excipients
(including combinations thereof).
[0070] This is a composition that comprises or consists of a
therapeutically effective amount of a pharmaceutically active
agent. It preferably includes a pharmaceutically acceptable
carrier, diluent or excipients (including combinations thereof).
Acceptable carriers or diluents for therapeutic use are well known
in the pharmaceutical art. The choice of pharmaceutical carrier,
excipient or diluent can be selected with regard to the intended
route of administration and standard pharmaceutical practice. The
pharmaceutical compositions may comprise as--or in addition to--the
carrier, excipient or diluent any suitable binder(s), lubricant(s),
suspending agent(s), coating agent(s), solubilising agent(s).
[0071] Pharmaceutical compositions within the scope of the present
invention may include one or more of the following: preserving
agents, solubilising agents, stabilising agents, s wetting agents,
emulsifiers, sweeteners, colourants, flavouring agents, odourants,
salts compounds of the present invention may themselves be provided
in the form of a pharmaceutically acceptable salt), buffers,
coating agents, antioxidants, suspending agents, adjuvants,
excipients and diluents.
[0072] A pharmaceutical composition according to the invention is
preferably formulated for oral administration to a human or an
animal. The pharmaceutical composition may also be formulated for
administration through any other route where the active ingredients
may be efficiently absorbed and utilized, e.g. intravenously,
subcutaneously, intramuscularly, intranasally, rectally, vaginally
or topically.
[0073] In a specific embodiment of the invention, the
pharmaceutical composition is shaped in form of a capsule, which
could also be microcapsules generating a powder or a sachet. The
capsule may be flavoured. This embodiment also includes a capsule
wherein both the capsule and the encapsulated fatty acid
composition according to the invention is flavoured. By flavouring
the capsule it becomes more attractive to the user. For the
above-mentioned therapeutic uses the dosage administered will, of
course, vary with the compound employed, the mode of
administration, the treatment desired and the disorder
indicated.
[0074] The pharmaceutical composition may be formulated to provide
a daily dosage of 10 mg to 10 g. Preferably, the pharmaceutical
composition is formulated to provide a daily dosage between 50 mg
and 5 g of said composition. Most preferably, the pharmaceutical
composition is formulated to provide a daily dosage between 100 mg
and 1 g of said composition. By a daily dosage is meant the dosage
per 24 hours.
[0075] The dosage administered will, of course, vary with the
compound employed, the mode of administration, the treatment
desired and the disorder indicated. Typically, a physician will
determine the actual dosage which will be most suitable for an
individual subject. The specific dose level and frequency of dosage
for any particular patient may be varied and will depend upon a
variety of factors including the activity of the specific compound
employed, the metabolic stability and length of action of that
compound, the age, body weight, general health, sex, diet, mode and
time of administration, rate of excretion, drug combination, the
severity of the particular condition, and the individual undergoing
therapy. The agent and/or the pharmaceutical composition of the
present invention may be administered in accordance with a regimen
of from 1 to 10 times per day, such as once or twice per day. For
oral and parenteral administration to human patients, the daily
dosage level of the agent may be in single or divided doses.
[0076] A further aspect of the present invention relates to a fatty
acid composition comprising a compound of formula (I). A fatty acid
composition comprising a compound of formula (I) increases the
natural biological effects of DHA that are a result of regulation
of gene expression, and the derivatives according to the present
invention will accumulate in the free fatty acid pool.
[0077] The fatty acid composition may comprise in the range of 60
to 100% by weight of the compound of formula (I), all percentages
by weight being based on the total weight of the fatty acid
composition. In a preferred embodiment of the invention, at least
80% by weight of the fatty acid composition is comprised of a
compound of formula (I). More preferably, the compound of formula
(I) constitute at least 90% by weight of the fatty acid
composition. Most preferably, the compound of formula (I)
constitutes more than 95% by weight of the fatty acid
composition.
[0078] The fatty acid composition may further comprise at least one
of the fatty acids (all-Z)-5,8,11,14,17-eicosapentaenoic acid
(EPA), (all-Z)-4,7,10,13,16,19-docosahexaenoic acid (DHA),
(all-Z)-6,9,12,15,18-heneicosapentaenoic acid (HPA), and
(all-Z)-7,10,13,16,19-docosapentaenoic acid (DPAn-3),
(all-Z)-8,11,14,17-eicosatetraenoic acid (ETAn-3), or combinations
thereof. Further, the fatty acid composition may comprise
(all-Z)-4,7,10,13,16-Docosapentaenoic acid (DPAn-6) and/or
(all-Z)-5,8,11,14-eicosatetraenoic acid (ARA), or derivatives
thereof. The fatty acid composition may also comprise at least
these fatty acids, or combinations thereof, in the form of
derivatives. The derivatives are suitably substituted in the same
way as the DHA derivative of formula (I), as hereinbefore
defined.
[0079] The fatty acid composition according to the invention may
comprise (all-Z omega-3)-6,9,12,15,18-heneicosapentaenoic acid
(HPA), or derivatives thereof, in an amount of at least 1% by
weight, or in an amount of 1 to 4% by weight.
[0080] Further, the fatty acid composition according to the
invention may comprise omega-3 fatty acids other than EPA and DHA
that have 20, 21, or 22 carbon atoms, or derivatives thereof, in an
amount of at least 1.5% by weight, or in an amount of at least 3%
by weight.
[0081] In specific embodiments of the invention, the fatty acid
composition is a pharmaceutical composition, a nutritional
composition or a dietary composition.
[0082] The fatty acid composition may further comprise an effective
amount of a pharmaceutically acceptable antioxidant. Preferably,
the antioxidant is tocopherol or a mixture of tocopherlos. In a
preferred embodiment the fatty acid composition further comprises
tocopherol, or a mixture of tocopherols, in an amount of up to 4 mg
per g of the total weight of the fatty acid composition.
Preferably, the fatty acid composition comprises an amount of 0.2
to 0.4 mg per g of tocopherols, based on the total weight of the
composition.
[0083] Another aspect of the invention provides a fatty acid
composition, or any pharmaceutically acceptable salt, solvate,
pro-drug or complex thereof, comprising a compound of formula (I),
as hereinbefore defined, for use as a medicament and/or in therapy.
Such a fatty acid composition may be used to prevent and/or treat
the same conditions as outlined for the compound of formula (I)
above.
[0084] When the fatty acid composition is used as a medicament, it
will be administered in a therapeutically or a pharmaceutically
active amount.
[0085] In a preferred embodiment, the fatty acid composition is
administered orally to a human or an animal.
[0086] The present invention also provides the use of a compound of
formula (I), or a pharmaceutically acceptable salt, solvate,
pro-drug or complex thereof, as hereinbefore defined, for the
manufacture of a medicament for controlling body weight reduction
and/or for preventing body weight gain; for the manufacture of a
medicament for the treatment and/or the prevention of obesity or an
overweight condition; for the manufacture of a medicament for the
prevention and/or treatment of diabetes in a human or animal; for
the manufacture of a medicament for the treatment and/or prevention
of amyloidos-related diseases; for the manufacture of a medicament
for the treatment and prophylaxis of multiple risk factors known
for cardiovascular diseases, such as hypertension,
hypertriglyceridemia and high coagulation factor VII phospholipid
complex activity; for the manufacture of a medicament for the
treatment of TBC or HIV; for the manufacture of a medicament for
prevention of stroke, cerebral or transient ischaemic attacks
related to atherosclerosis of several arteries; for the
manufacturing of a medicament for lowering triglycerides in the
blood of mammals and/or evelating the HDL cholesterol levels in the
serum of a human patients; or for the manufacturing of a medicament
for the treatment and/or prevention of the multi metabolic syndrome
termed "metabolic syndrome". All these embodiments also include the
use of a fatty acid composition, as hereinbefore defined,
comprising a compound of formula (I) for the manufacture of
medicaments as outlined above.
[0087] The present invention also relates to a method for
controlling body weight reduction and for preventing body weight
gain, wherein a fatty acid composition comprising at least a
compound of formula (I), as hereinbefore defined, is administered
to a human or an animal.
[0088] Further, the invention relates to a method for the treatment
and/or the prevention of obesity or an overweight condition,
wherein a fatty acid composition comprising at least a compound of
formula (I), as hereinbefore defined, is administered to a human or
an animal.
[0089] In a preferred embodiment of the invention, the present
invention relates to a method for the prevention and/or treatment
of diabetes mellitus, wherein a fatty acid composition comprising
at least a compound of formula (I), as hereinbefore defined, is
administered to a human or an animal. Preferably, diabetes mellitus
is a type 2 diabetes.
[0090] Other aspects of the present invention relate to; [0091] a
method for the treatment and/or prevention of amyloidos-related
diseases; [0092] a method for the treatment or prophylaxis of
multiple risk factors for cardiovascular diseases; [0093] a method
for prevention of stroke, cerebral or transient ischaemic attacks
related to atherosclerosis of several arteries; wherein a fatty
acid composition comprising at least a compound of formula (I), as
hereinbefore defined, is administered to a human or an animal.
[0094] The fatty acid derivative of formula (I) may be prepared
most effectively from DHA. If the start material is not pure DHA
(i.e. not 100% DHA) the final fatty acid composition will contain a
mixture of DHA derivatives, as hereinbefore defined, and an amount
of other fatty acids than DHA, wherein these fatty acids are
substituted in the same way as the novel fatty acid analogue of
formula (I). Such embodiments are also included herein.
[0095] In another embodiment of the invention, the compound of
formula (I) is prepared from
(all-Z)-4,7,10,13,16,19-docosahexaenoic acid (DHA), wherein said
DHA is obtained from a vegetable, a microbial and/or an animal
source, or combinations thereof. Preferably, said DHA is obtained
from a marine oil, such as a fish oil.
[0096] The fatty acids in the composition may also be obtained from
a vegetable, a microbial or an animal source, or combinations
thereof. Thus, the invention also includes a fatty acid composition
prepared from a microbial oil.
[0097] DHA is produced from biological sources like marine,
microbial or vegetable fats. All possible raw materials are
mixtures of fatty acids on triglyceride form where DHA constitutes
only a fraction of the fatty acids. Typical DHA concetrations are
40% in microbial fats and 10-25% in marine fats. DHA-containing
vegetable fats are during development and fats with high DHA
concentrations are expected in the future.
[0098] The first process step will always be conversion of the
triglycerides to free fatty acids or monoesters. Preferable esters
are methyl or ethyl esters, but other esters are possible. In this
way the fatty acids bound together three by three on triglycerides
are separated from each other and thereby making separation
possible. Several methods of separating DHA from other fatty acids
are available, the most common ones being short path distillation
separating the fatty acids by volatility, and urea precipitation
separating the fatty acids by degree of unsaturation. Other methods
reported are silver nitrate complexation also separating the fatty
acids on degree on unsaturation, esterification reactions catalysed
by fatty acid selective lipases in combination with short path
distillation and countercurrent extraction with supercritical
carbon dioxide.
[0099] The most important challenges connected to production of
pure DHA is to separate it from the other C20-22 highly unsaturated
fatty acids present in all available sources. These fatty acids
have properties so similar to DHA that none of the methods
mentioned above provide sufficient degree of separation. For some
microbial high DHA fats, which have very low levels of C20-22
highly unsaturated fatty acids, short path distillation alone or in
combination of other methods mentioned may provide more that 90%
purity.
[0100] Most DHA containing fats also contain considerable amounts
of C20-22 highly unsaturated fatty acids, e.g. EPA (20:5n-3),
n-3DPA (22:5n-3), HPA (21:5n-3) and others. The only available
method for separating DHA from such fatty acids is preparative High
Performance Liquid Chromatography, the stationary phase being
silica gel or silver nitrate impregnated silica gel, the moblie
phase being selected organic solvents or supercritical carbon
dioxide. With this method DHA with more than 97% purity is
available. However, it has to be noted that the production costs
increases strongly with concentration, as an example is production
cost for 97% DHA more 5 times higher than for 90% DHA.
[0101] DHA having a purity of 90, 95 eller 97% contains small
amounts of other fatty acids. As an example, DHA having a purity of
97% contains n-3DPA (22:5n-3), but also long chain fatty acids,
e.g. EPA (20:5n-3), HPA (21:5n-3), and others. However, the other
fatty acids will react in a way similar to DHA and provide
alpha-substituted derivatives.
[0102] Organic synthesis may provide a purification method since
DHA and n-6DPA (and 22:5n-6 which normally is present in very low
concentrations) are the only known fatty acids that can provide
gamma-lactones by cyclisation with the first double bond.
Lactonisation followed by purification and hydrolysis back to DHA
may be a possibility, but it is expected that this pathway is even
more expensive than HPLC.
EXAMPLES
[0103] The invention will now be described in more detail by the
following example, which is not to be constructed as limiting the
invention.
Synthesis Protocol
[0104] Preparation of .alpha.-methyl DHA EE (PRB-1)
[0105] Butyllithium (228 ml, 0.37 mol, 1.6 M in hexane) was added
dropwise to a stirred solution of diisopropylamine (59.5 ml, 0.42
mol) in dry THF (800 ml) under N.sub.2 at 0.degree. C. The
resulting solution was stirred at 0.degree. C. for 30 min., cooled
to -78.degree. C. and stirred an additional 30 min. before dropwise
addition of DHA EE (100 g, 0.28 mol) in dry THF (500 ml) during 2
h. The dark-green solution was stirred at -78.degree. C. for 30
min. before MeI (28 ml, 0.45 mol) was added. The solution was
allowed to reach -20.degree. C. during 1.5 h, then poured into
water (1.5 l) and extracted with heptane (2.times.800 ml). The
combined organic phases were washed with 1 M HCl (1 l), dried
(Na.sub.2SO.sub.4), filtered and evaporated in vacuo. The product
was purified by dry flash chromatography on silica gel eluting with
heptane/EtOAc (99:1) to give 50 g (48%) of the titled compound as a
slightly yellow oil;
[0106] .sup.1H-NMR (200 MHz, CDCl.sub.3) .delta. 1.02 (t, J 7.5 Hz,
3H), 1.20 (d, J 6.8 Hz, 3H), 1.29 (t, J 7.1 Hz, 3H), 2.0-2.6 (m,
5H), 2.8-3.0 (m, 10H), 4.17 (t, J 7.1 Hz, 2H), 5.3-5.5 (m,
12H);
[0107] MS (electrospray); 393 [M+Na].
[0108] The enantiomeric pure compounds can be prepared by resolving
a racemic compound of formula (I), as hereinbefore defined. The
resolution of a compound of formula (I) may be carried out using
known resolution procedures, for example by reacting the compound
of formula (I) with an enantiomerically pure auxiliary to provide a
mixture of diastereomers that can be separated by chromatography.
Thereafter the two enantiomers of compound (I) may be regenerated
from the separated diastereomers by conventional means, such as
hydrolysis.
[0109] There is also a possibility to use stoichiometric chiral
auxiliaries to effect an asymmetric introduction of the
substituent, as hereinbefore defined, in the .alpha.-position of
DHA. The use of chiral oxazolidin-2-ones has proved to be a
particularly effective methodology. The enolates derived from
chiral N-acyloxazolidines can be quenched with a variety of
electrophiles in a highly stereoregulated manner (Ager, Prakash,
Schaad, Chem. Rev. 1996, 96, 835).
Experiments
[0110] A number of experiments has been performed in order to
demonstrate that the compound according to the invention is
effective in particular for the treatment and/or prevention of
diabetes mellitus.
[0111] In the following experiments, alpha-methyl-DHA EE is denoted
"PRB-1".
Example 1
Analysis of Intracellular Free Fatty Acids (Non-Esterified Fatty
Acids) in Liver Cells
Background
[0112] Liver tissue from animals fed PRB-1 was analysed with
respect to free unesterified fatty acids. The animals were
recruited from Experiment 4 (pharmacodynamic effects of DHA
derivatives in an animal model of metabolic syndrome). The animals
had been given DHA (15% of fat content of the diet) or the
DHA-derivative (1.5% of the fat content in their diet) for 8 weeks
and were supposed to be in a steady-state situation with stable
levels of DHA and the DHA-derivative intracellularly. Liver tissue
was chosen due to the fact that the metabolisation rate is very
high in liver.
Method
[0113] The liver samples were homogenized in cold PBS buffer, and
extracted immediately with chloroform:methanol (2:1) containing 0.2
mM butylated hydroxytoluene (BHT) using cis-10-heptadecenoic acid
as internal standard. The organic phases were dried under nitrogen,
re-dissolved in acetonitrile with 0.1% acetic acid and 10 .mu.M BHT
for RP-HPLC MS/MS analysis. Total protein content was measured
using Bio-Rad method after homogenization.
[0114] Agilent 1100 system was used for reverse phase column
(Supelco Ascentis C.sub.18 column, 25 cm.times.4.6 mm, i.d. 5
.mu.m) separation within 22 min. The flow phase was iso-gradient
acetonitrile-H.sub.2O (87+13, v/v) containing 0.1% acetic acid. The
column oven temperature was set at 35.degree. C. The column elute
was identified and quantified in the negative electrospray
ionisation applying multiple reaction monitoring mode by triple
tandem quadrapole mass/mass (ABI Qtrap-4000). The parent-daughter
ion pairs were 341.3/341.3 (PRB-1), under unit resolution. The
signal collection dwell time was all 100 msec except for FA 17:1
which was set at 200 msec. Accurate verification of isomeric PRB
compounds was done by combination of the retention time and
characteristic mass/charge ratio. The quadratic regression standard
curve was used for quantification after internal standard
calibration.
Results
[0115] The concentration of the DHA-derivative according to the
invention was about 10 .mu.g per g of total amount of protein in
the liver cells. This means that PRB-1 will be available as a
ligand to nuclear receptors, a pattern which could be translated
into therapeutic effects in handling of blood glucose and blood
lipids.
Example 2
Computer Based Affinity Testing
Background
[0116] Nuclear receptors have been sequenced and the amino acid
sequence is known for the PPARs and other relevant receptors
engaged in the genetic control of glucose and fat. X-ray
crystallography and NMR spectroscopy of the PPAR receptors are
available and computerised affinity testing of fatty acids
liganding to the receptors can be used to estimate binding
kinetics. The binding geometrics, often called binding modes or
poses, include both positioning of the ligand relative to the
receptor and the conformational state of the ligand and the
receptor. Effective ligand docking can therefore be analysed.
[0117] Affinity of the ligand to the receptor is defined by two
different parameters: docking of the ligand (DHA derivative) into
the binding site of the receptor and electrostatic bonding between
certain amino acids of the receptor and the carboxyl group or side
chains in the head of the fatty acid. (Krumrine).
[0118] As previously known, the PPAR.alpha. receptor is more
promiscuous compared to PPAR.gamma., meaning that PPAR.alpha. will
accept more fatty acids as ligands compared to PPAR.gamma..
However, since patients with metabolic syndrome or type 2 diabetes
are usually obese or overweight and have pathologic blood lipids,
mainly elevated triglycerides and low High-Density Cholesterol
(HDL-chol) activation of the PPAR.alpha. receptor is important. An
ideal drug for treatment of metabolic syndrome or type 2 diabetes
should act as ligand to both these receptors, preferably with the
highest affinity to the PPAR.gamma. receptor.
Method
[0119] Ranking of PRB-1 according to its binding affinity was
calculated and given as lowest binding affinity (LBA) and average
binding affinity (ABE).
[0120] PRB-1 was tested with the computerized docking method (both
r and s enantiomers). The PPAR.gamma. ligands rosiglitazone and
pioglitazone, both in the r and s form, were also tested for
comparison. These compounds are registered pharmaceuticals for
treatment of diabetes.
Results
[0121] The results are shown in Table 1, presenting the parameters
Lowest binding energy of single confirmation (LBE), average binding
energy (ABE) of the correctly posed confirmation and fraction of
correctly posed confirmation of the ICM-saved 20 lowest energy
confirmation (fbound) of the compounds tested. Affinity to the
RXR.alpha. was tested in the same setting. The RXR.alpha. receptor
interacts with the PPAR receptor forming a heterodimer by liganding
of a fatty acid.
TABLE-US-00001 TABLE 1 PPAR.alpha. PPAR.gamma. RXR.alpha. LBE ABE
f.sub.bound LBE ABE f.sub.bound LBE ABE f.sub.bound DHA -16.14
-13.29 (0.47) 0.60 -11.34 -10.51 (0.21) 0.35 -12.15 -10.72 (0.29)
0.40 cr-PRB1 -16.29 -14.25 (0.53) 0.50 -12.96 -11.82 (0.38) 0.30
-15.68 -14.25 (0.35) 0.30 cs-PRB1 -15.97 -14.01 (0.30) 0.80 -12.74
-10.24 (0.48) 0.65 -17.17 -14.48 (0.44) 0.50 sROSI -9.47 -9.01
(0.17) 0.20 sROSI -10.05 -7.89 (0.91) 0.20 rPIO ND sPIO -7.59 -7.59
0.05 ND = Not docked, c = the double bonds in all-cis form. r = R
enantioisomer, s = S enantioisomer. ROSI = Rosiglitazone, PIO =
Pioglitazone
[0122] PRB-1 has a high LBE and ABE score for the PPAR.alpha. and
PPAR.gamma. receptors compared to the mother compound DHA but also
to the PPAR.gamma. ligands rosiglitazone and pioglitazone, both in
the r and s form. This is an interesting observation indicating
that PRB-1 could be promising competitors to the established
anti-diabetics rosiglitazone and pioglitazone.
[0123] In conclusion, the DHA-derivative according to the invention
demonstrated interesting affinities to the PPAR.alpha. and
PPAR.gamma. receptors with binding affinities better than
rosiglitazone and pioglitazone.
Example 3
Affinity Testing in Transfected Cells
Background
[0124] Release of luciferase is correlated to transcription of
genes. Binding of a ligand to a nuclear receptor such as
PPAR.gamma. induces transcription of the respective gene thereby
releasing luciferase. This technique therefore provides a measure
of ligand affinity to the receptor as well as activation of the
responsible gene.
Method
[0125] Transient transfection of COS-1 cells was performed in
6-well plates as described by Graham and van der Eb (Graham). For
full length PPAR transfection studies, each well received 5 .mu.g
reporter construct, 2.5 .mu.g pSV-.beta.-galactosidase as an
internal control, 0.4 .mu.g pSG5-PPAR.gamma.2. The cells were
harvested after 72 h, and the luciferase activity was measured
according to the protocol (Promega). The luciferase activity was
normalised against .beta.-galactosidase activity. The adipocytes
were transfected at D11 of differentiation using 16 .mu.L
LipofectaminPlus reagent, 4 .mu.l Lipofectamine (Life Technologies
Inc.), 0.2 .mu.g pSG5-PPAR.gamma., and 100 ng pTK Renilla
luciferase as control of transfection afficiency. Three hours after
transfection, cells were cultured in serum containing medium and
incubated for 48 hours in the same medium containing appropriate
agents. The luciferase activities were measured as recommended by
the manufacturer (Dual Luciferase assay, Promega). All
transfections were performed in triplicate.
[0126] Fatty acids (BRL or DHA) and PRB-1 (stock solutions) were
solubilized to 0.1 M final concentration in DMSO. Then, Fatty
solubilized to 10 mM in DMSO and stored in 1.5 ml tubes
(homopolymer, plastic tubes) flushed with argon and stored at
-20.degree. C. 10 .mu.M of PRB-1 or fatty acids and DMSO (control)
was added to the media 5 h after transfection. Transfected cells
were maintained for 24 h before lysis by reporter lysis buffer.
Binding of PRB-1 or fatty acids to the LBD of PPAR activates GAL4
binding to UAS, which in turn stimulates the tk promoter to drive
luciferase expression. Luciferase activity was measured using a
luminometer (TD-20/20 luminometer; Turner Designs, Sunnycvale,
Calif.) and normalized against protein content.
Results
[0127] FIG. 3 depicts the release of luciferase from transfected
cells treated with PRB-1. The results indicate that PRB-1 has a
high release of luciferase.
Example 4
Pharmacodynamic Effects of DHA Derivatives in an Animal Model of
Metabolic Syndrome
Background
[0128] An animal model of the metabolic syndrome using the adipose
prone mice of the C57BL/6J strain was used to document effects on
typical laboratory and pathological anatomical features common for
the metabolic syndrome. When given a high fat diet containing about
60% of fat, the animals are getting obese developing high insulin
plasma levels, pathological glucose tolerance test, elevated serum
triglycerides and non-esterified fatty acids, and fat liver.
Example 4a
Effect of the DHA Derivative According to the Invention in Adipose
Prone Mice During 4 Months of Dietary Interventions
Method
[0129] All experiments were performed on male C57BL/6 mice, either
a substrain C57BL/N (supplier: Charles River, Germany, n=160,
experiments A-C, see below), or a substrain C57BL/6J (supplier: the
Jackson laboratory, Bar Harbor, Me., USA, n=32, experiment D).
Total numbers of animals used were higher (n=170 and 36,
respectively), because of culling. In the latter case, animals were
bred for several generations (<20) at the Institute of
Physiology. At the beginning of the treatment, animals were
14-week-old and their body weight range was 23.6-27.1 g. One week
before the study start, animals were sorted according to their body
weight and assigned to subgroups (n=8) of similar mean body weight.
This method allowed for culling of about 5-10% of animals showing
the lowest and highest body weight, respectively. The animals
eliminated from the study at this stage were sacrificed by cervical
dislocation. Complete health check of mice was performed by the
supplier Charles River and at the start of study serological tests
were performed by ANLAB (Prague, Czech Republic). In addition,
regular health checks were performed in the animal house in
3-mo-intervals using sentinel mice and serological examinations
(ANLAB). In all the tests, the animals were free of specific
pathogens.
Diets
[0130] Animals were fed 3 types of experimental diets: [0131] (i)
Chow diet (ssniff RIM-H from SSNIFF Spezialdieten Gmbh, Soest,
Germany; see also http://ssniff.de) with protein, fat and
carbohydrate forming 33, 9, and 58 energy %, respectively [0132]
(ii) High-fat diet prepared in the laboratory (cHF diet) with
protein, fat and carbohydrate forming 15, 59, and 26 energy %,
respectively, and well characterized fatty acid composition (with
most of the lipids coming from corn oil; see Ruzickova 2004) [0133]
(iii) cHF diets in which 0.15, 0.5, and 1.5% of fat (specifically
the corn oil constituent) was replaced by various PRB-compounds,
namely PRB1, PRB2, PRB5, PRB7, and PRB8, or by DHA. All these
compounds were in the form of ethyl esters, provided by Pronova
Biocare a.s. in sealed containers. Chemical composition of the
PRB-compounds was unknown to the laboratory performing the
experiments (Institute of Physiology, Academy of Sciences Prague,
Czech Republic).
[0134] After arrival, the PRB-compound were stored in a
refrigerator in original containers. The containers were opened
just before preparation of the experimental diets. Diets were kept
in plastic bags flushed by nitrogen and stored at -70.degree. C. in
small aliquots sufficient for feeding animals for one week. Fresh
ratios were given in 2-day intervals or daily.
Outline of the Study
[0135] The study was based on 4 individual experiments. In each of
the experiments, PRB-1 (or DHA, respectively) admixed to cHF diet
in three different concentrations (0.15, 0.5, and 1.5% of the fat
content) were tested. In each experiment, a subgroup of plain cHF
diet-fed mice was included and served as a control. Mice were caged
in groups of 4 and fed standard chow diet until 3 mo of age, when
animals (n=8-13) were randomly assigned to the different test
diets. After 2 mo on this new diet (at 5 mo of age), animals were
fasted overnight and in the morning, intraperitoneal Glucose
Tolerance Test (GTT) was performed. Animals were sacrificed after 4
months on the experimental diets, at 7 mo of age, and the end-point
analysis were performed.
Study Parameters.
[0136] The parameters in the study were: Body weight gain (grams),
area under the curve (AUC) from intraperitoneal glucose tolerance
tests (mMol.times.180 min), plasma insulin (ng/ml), serum
triglycerides (TAGs, mmol/l), and non-esterified fatty acids (NEFA,
mmol/l).
Results
[0137] The results are shown in the following tables 2, 3 and 4.
(*=significant differencies compared to cHF diets (P<0.05).)
[0138] Table 2 shows the effects in animals given 1.5%
concentration of the PRB test compounds compared to animals given
standard chow (STD), composite high fat diet (cHF) or 97% DHA. A
pronounced reduction in AUC from glucose tolerance tests was seen
in the animals given PRB-1. Plasma insulin was low in the PRB-1
treated animals.
[0139] Table 3 shows the effects in animals given a lower
concentration, 0.5%, of the PRB test compounds compared to animals
given standard chow (STD), composite high fat diet (cHF) or 97%
DHA.
[0140] Table 4 shows the results from the lowest PRB concentration
given, 0.15%. Here, the differences were small. Weight gain was
somewhat lower in the PRB-1 group. Plasma insulin was lower in
PRB-1.
TABLE-US-00002 TABLE 2 The effect of PRB-1 after 4 months of
treatment with 1.5% concentration Parameter STD cHF PRB-1 DHA Body
weight 32.4 .+-. 0.7 49.6 .+-. 0.6 44.0 .+-. 1.5* 47.1 .+-. 0.7*
(grams) Body wt. gain 7.8 .+-. 0.4 25.2 .+-. 0.5 20.2 .+-. 1.3*
23.0 .+-. 0.8* (grams) Food intake 3.64 .+-. 0.04 2.70 .+-. 0.02
2.64 .+-. 0.03 2.63 .+-. 0.02 (grams/mouse/day) AUCglucose 1124
.+-. 57 1625 .+-. 151 913 .+-. 68* 2132 .+-. 288* (mM .times. 180
min) Fasted glucose 77 .+-. 3 145 .+-. 7 130 .+-. 14 138 .+-. 7
(mg/dL) Insulin 1.03 .+-. 0.09 5.35 .+-. 0.36 2.73 .+-. 0.33 6.55
.+-. 0.31 (ng/mL) TAGs 1.41 .+-. 0.09 1.45 .+-. 0.07 1.58 .+-. 0.08
1.91 .+-. 0.26* (mmol/L) NEFA 0.57 .+-. 0.05 0.61 .+-. 0.04 0.63
.+-. 0.03* 0.98 .+-. 0.07 (mmol/L)
TABLE-US-00003 TABLE 3 The effect of PRB-1 after 4 months of
dietary interventions: 0.5% concentration. Parameter STD CHF PRB-1
DHA Body weight 32.4 .+-. 0.7 49.6 .+-. 0.6 47.4 .+-. 0.6 46.9 .+-.
0.7* (grams) Body wt. gain 7.8 .+-. 0.4 25.2 .+-. 0.5 23.8 .+-. 0.5
22.9 .+-. 0.7* (grams) Food intake 3.64 .+-. 0.04 2.70 .+-. 0.02
2.67 .+-. 0.04 2.70 .+-. 0.03 (grams/mouse/day) AUCglucose 1124
.+-. 57 1625 .+-. 151 1596 .+-. 205 1816 .+-. 182 (mM .times. 180
min) Fasted glucose 77 .+-. 3 145 .+-. 7 131 .+-. 7 136 .+-. 8
(mg/dL) Insulin 1.03 .+-. 0.08 5.35 .+-. 0.36 3.93 .+-. 0.59 5.82
.+-. 0.47 (ng/mL) TAGs 1.41 .+-. 0.09 1.45 .+-. 0.07 2.03 .+-. 0.22
1.78 .+-. 0.08* (mmol/L) NEFA 0.57 .+-. 0.05 0.61 .+-. 0.04 0.73
.+-. 0.04* 0.89 .+-. 0.03 (mmol/L)
TABLE-US-00004 TABLE 4 The effect of PRB-1 after 4 months of
dietary interventions: 0.15% concentration. Parameter STD cHF PRB-1
DHA Body weight 32.4 .+-. 0.7 49.6 .+-. 0.6 47.2 .+-. 1.3 48.3 .+-.
0.6 (grams) Body wt. Gain 7.8 .+-. 0.4 25.2 .+-. 0.5 22.9 .+-. 1.1
24.3 .+-. 0.8 (grams) Food intake 3.64 .+-. 0.04 2.70 .+-. 0.02
2.63 .+-. 0.04 2.79 .+-. 0.03 (grams/mouse/day) AUCglucose 1124
.+-. 57 1625 .+-. 151 1291 .+-. 172 1477 .+-. 214 (mM .times. 180
min) Fasted glucose 77 .+-. 3 145 .+-. 7 126 .+-. 15 141 .+-. 10
(mg/dL) Insulin 1.03 .+-. 0.08 5.35 .+-. 0.36 3.50 .+-. 0.29 4.31
.+-. 0.39* (ng/mL) TAGs 1.41 .+-. 0.09 1.45 .+-. 0.07 1.75 .+-.
0.08 1.50 .+-. 0.13 (mmol/L) NEFA 0.57 .+-. 0.05 0.61 .+-. 0.04
0.62 .+-. 0.04* 0.96 .+-. 0.07 (mmol/L)
[0141] In conclusion, testing of PBR-1 during 4 months in adipose
prone animals with insulin resistance and metabolic syndrome
demonstrated a clear and unsuspected effect on insulin resistance
and symptoms of the metabolic syndrome such as weight reduction,
reduced AUC in the intraperitoneal glucose tolerance test, lower
insulin/plasma levels as well as reduced triglyceride and
non-esterified free fatty acids. Effects were observed in the dose
of 1.5% as well as in the 0.5% group. Some effects were even
noticed in the lowest concentration group of 0.15%.
Example 4b
Testing of DHA Derivatives on Liver Fat
Method
[0142] Tissue samples from animals in the experiments with DHA
derivatives was histologically analysed. After paraffination,
tissue samples from liver, adipose tissue, skeletal muscle,
pancreas, and kidney were stained with eosin-hematoxylin.
Results
[0143] There were no pathological findings in the tissues examined
with exception from liver. Control animals fed high fat diet had
developed fat liver (liver steatosis). Fat droplets in the liver
can easily be distinguished from normal liver cells. Animals
treated with PRB-1 had low degree of fat liver.
[0144] This is an extremely important finding and very relevant for
treatment of patients with insulin resistance, obesity and type 2
diabetes. Liver steatosis is a common finding in these patients
which is usually related to an overload of fatty acids and
triglycerides, biological markers present in the development of
insulin resistance and the metabolic syndrome. DHA-derivatives
reduce liver steatosis.
DISCUSSION AND CONCLUSIONS
[0145] The present application clearly shows that alpha-methyl-DHA
activatesu nuclear receptors, especially PPAR.gamma. and
PPAR.alpha., thereby offering a series of therapeutic effects in
the treatment of insulin resistance, the metabolic syndrome, type 2
diabetes, cardiovascular disease and other atherosclerotic related
diseases.
[0146] In testing of affinity to PPAR .gamma. and PPAR.alpha. using
computerized docking technology, the DHA-derivative according to
the present invention showed affinities to both receptors, not
least PPAR .gamma. which probably is the most important nuclear
receptor engaged in the activation of genes responsible for
metabolisation of blood glucose. Alpha-methyl DHA has two
stereoisomers, the r and the s form. Using the docking technology
both stereoisomers possessed about the same affinity to PPAR
.gamma. and PPAR.alpha. meaning that neither the r or the s form
should have advantages compared to the racemic form. In fact the
racemic form may have advantages over each one of the
stereoisomers.
[0147] When affinity was tested in transfected cells carrying the
nuclear receptor and the subsequent DNA response element, the
compound according to the invention demonstrated good affinity
measured as release of luciferase.
[0148] The DHA derivative according to the invention has been
tested in the C57BL/6 mouse model developing insulin resistance and
the metabolic syndrome when fed high fat diet. The derivative
demonstrated significant biological effects.
[0149] Comparing with pure DHA, alfa-methyl DHA (PRB-1) seems to be
more potent than DHA. These findings and the potency compared to
the mother molecule DHA are not predictable and highly
unexpected.
[0150] Since alfa-methyl DHA (PRB-1) seems to work by simultaneous
liganding to the nuclear receptors PPAR.alpha. and PPAR.gamma. the
compound would not only possess therapeutic interesting effects on
glucose and lipid metabolism, not least in patients with insulin
resistance, metabolic syndrome and type 2 diabetes but also have
weight reduction as well as a general anti-inflammatory effect.
Directly or through positive intervention on risk factors
alfa-methyl DHA (PRB-1) would have a preventive effect on the
development of cardiovascular disease such as myocardial infarction
and cerebral stroke as well as having a preventive effect on
cardio-vascular mortality.
[0151] Pharmaceuticals acting as PPAR.gamma. ligands are already on
the market but even if these compounds are having positive effects
on glucose metabolism, they are hampered by adverse effects such as
elevated triglycerides, weight increase and oedema. The
alfa-substituted DHA derivative presented in this application has a
combined PPAR.gamma. and PPAR.alpha. effect which is probably both
relevant and advantageous for patients with insulin resistance,
metabolic syndrome and type 2 diabetes. Furthermore, these
combinative actions should have important effects also on blood
lipids, inflammatory events, atherosclerosis, and thereby
cardiovascular disease.
[0152] The invention shall not be limited to the shown embodiments
and examples.
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