Title: Specific pancreatic lipase inhibitors and their
United States Patent: 6,432,400
Issued: August 13, 2002
Inventors: Chapus; Catherine (Marseilles, FR)
Assignee: Laboratoire Laphal (Laboratoire de Pharmacologie
Appliquee) (Allauch, FR)
Appl. No.: 341184
Filed: September 27, 1999
PCT Filed: January 6, 1998
PCT NO: PCT/FR98/00009
371 Date: September 27, 1999
102(e) Date: September 27, 1999
PCT PUB.NO.: WO98/30588
PCT PUB. Date: July 16, 1998
The invention concerns specific pancreatic lipase inhibitors and their
applications in the treatment and prevention of cardiovascular diseases, of
lyperlipemia and of obesity, as well as diagnostic reagent and as regulating
agent in a process of controlled lipolysis of triglycerides. Said inhibitors
correspond in particular to a peptide consisting of a C-terminal fragment of
pancreatic lipase including the recognition site of a colipase.
Description of the Invention
The present invention relates to specific pancreatic lipase inhibitors
and to their applications in the treatment and prevention of cardiovascular
diseases, of hyperlipemia and of obesity, and also as diagnostic reagent and
as regulating agent in a method of controlled lipolysis of triglycerides.
Dietary fats represent an efficient source of energy for the body. Indeed,
the quantity of energy metabolized from lipids is significantly greater than
that metabolized from carbohydrates or proteins. However, given that
practically all the lipids ingested are assimilated by the body, a dietary
excess of lipids can cause substantial health disorders: cardiovascular
disorders, hyperlipemias and obesity.
These disorders are frequently encountered in industrialized countries where
the populations often have diets which are too high in saturated fats.
In order to combat these various pathological conditions, a dietary care
consisting in limiting the ingestion of fatty substances is necessary but
not always sufficient. Indeed, while some fatty substances are easy to
detect and to remove from the diet (butter, oils and the like), this is
considerably less true of others by virtue of their integration into the
foods (meats, dairy products and the like).
In the cases where the introduction of a dietary regime proves insufficient,
pharmacological treatments are then proposed. Most of the current treatments
are intended in particular to combat hyperlipemias by using, inter alia,
inhibitors of the synthesis of cholesterol.
However, current research is increasingly geared towards products which
induce a more general inhibition of the lipolytic activity.
In such a perspective, one of the orientations which has been particularly
studied is that of the inhibition of pancreatic lipase, which is a key
enzyme in the digestion of dietary triglycerides; the digestion of the
latter, major constituents of lipids (about 95%), which is started in the
upper digestive tract by lipases of preduodenal origin (gastric lipase in
humans), is essentially carried out in the intestine, under the action of
pancreatic lipase. The latter converts the triglycerides to free fatty acids
and to 2-monoglycerides, more polar products of hydrolysis, which are
capable of crossing the enterocyte brush border membrane, after
incorporation into mixed micelles of bile salts and phospholipids.
Pancreatic lipase therefore plays a role in the emergence of diseases linked
to the presence of an excess of lipids, such as cardiovascular diseases,
hyperlipemias and obesity, by allowing the assimilation of practically all
the triglyercides ingested. In addition, it promotes the intestinal
absorption of cholesterol, since the solubility of cholesterol is increased
in the mixed micelles, which are high in fatty acids.
The action of pancreatic lipase comprises several stages: adsorption of the
enzyme, in the intestine, onto the lipid interface, in the presence of bile
salts and of a colipase, whose role is to anchor the lipase onto the
interface (C. Chapus et al., FEBS Letters, 1975, 58, 1, 155-158), followed
by the hydrolysis of the sn-1,3 ester bonds of the triacylglycerols.
Thus, the digestion of triglycerides involves lipase/colipase interactions
regulated by a lipid interface.
Porcine lipase, for example, is a glycoprotein containing 449 amino acids,
whose glycan chains are linked to asparagine (Asn) at position 166; it
contains two domains separated by the Phe336 -Ala337 bond (M.
Bousset-Risso et al., FEBS Letters, 1985, 182, 2, 323-326). Each domain
carries a recognition site, namely: an interfacial recognition site
(N-terminal domain), site of the hydrolysis per se, and a recognition site
for its protein partner (C-terminal domain), the colipase. The N-terminal
domain (residues 1-335), which carries the active centre of the enzyme, is
separated from the C-terminal domain (residues 336-449) by a narrow region
which is very resistant to proteolysis. This organization into two domains
corresponds to the two specific functions listed above: the N-terminal
domain is responsible for catalysis, whereas the C-terminal domain is
involved in the recognition of the colipase (A. Abousalham et al., Protein
Engineering, 1992, 5, 1, 105-111).
The colipase is a small molecule (10 kDa) which is highly cross-linked
because of the presence of 5 disulphide bridges. It carries three
recognition sites which are essential for its function, namely: an
interfacial recognition site (H. van Tilbeurgh et al., Nature, 1993, 362,
814-820), a lipase recognition site (C. Chaillan et al., FEBS Letters, 1989,
257, 2, 443-446; H. van Tilbeurgh et al., Nature, 1992, 359, 159-162) and a
micellar recognition site (J. Hermoso et al., EMBO J., 1997, 16, 18,
5531-5536). These three sites are topologically distinct.
Extensive studies which have been carried out for many years have made it
possible to increase understanding of the structure/function relationships
in the pancreatic lipase/colipase system (C. Chaillan et al., 1989, cited
Additional structural studies relating to both human lipase (Winkler F. K.
et al., Nature, 1990, 343, 771-774) and horse lipase (B. Kerfelec et al.,
Eur. J. Biochem., 1992, 206, 279-287; Y. Bourne et al., J. Mol. Biol., 1994,
238, 709-732) have made it possible to confirm the existence of the
abovementioned two domains, in lipases of different origins.
The recognition between the lipase and the colipase involves, in particular,
hydrophobic interactions (N. Mahe-Gouhier et al., BBA, 1988, 962, 91-97) and
Covalent coupling experiments between the pancreatic lipase and colipase (C.
Chaillan et al., 1989, cited above), as well as the resolution of the
three-dimensional structure at 3.1 .ANG. of the lipase/colipase complex (H.
van Tilbeurgh et al., 1992, cited above), have led to the identification of
the recognition regions on the two molecules, in solution.
To inhibit pancreatic lipase and obtain a therapeutic activity on
hyperlipemias and obesity, various approaches have been proposed:
the use of covalent inhibitors, which bind to the active centre of the
enzyme; there may be mentioned, for example, tetrahydrolipstatin (THL),
[U.S. Pat. No. 4,598,089; P. Hadvary et al., J. Biol. Chemistry, 1991, 266,
4, 2021-2027; D. Hermier et al., FEBS Letters, 1991, 286, 1, 186-188]; a
complete inhibition of the lipolytic activity is obtained for doses of 1 mol
of THL/mol of enzyme (P. Hadvary et al., 1991, cited above) or doses of 10
to 400 mg, twice per day (J. Hauptman et al., Am. J. Clin. Nutr., 1992, 55,
309S-313S); such a method has a number of disadvantages, of which the main
one is the lack of specificity; tetrahydrolipstatin is indeed not specific
for pancreatic lipase and inhibits other lipases such as carboxylester
lipase, gastric lipase and lipase stimulated by the bile salts in human
the modification of the nature of the interface by addition of amphiphilic
proteins (Gargouri Y. et al., J. Biol. Chem., 1985, 260, 2268-2273); of
detergents (Gargouri Y. et al., J. Lip. Res., 1983, 24, 1336-1342) or of
fibres (Borel P. et al., Am. J. Clin. Nutr., 1989, 49, 1192-1202); such a
method has a very relative efficiency.
These two approaches comprise, in addition, a significant risk of
undesirable effects (nausea and the like).
Consequently, the applicant set itself the objective of providing a new
pancreatic lipase inhibitor which is better suited to the needs of practical
application than the prior art lipase inhibitors, in particular in that it
exhibits a real specificity of action towards pancreatic lipase.
The subject of the present invention is a peptide consisting of a C-terminal
fragment of a pancreatic lipase, including the recognition site for a
colipase (called hereinafter C-terminal peptide), for its use as medicament,
in particular for the treatment of hyperlipemias, of cardiovascular diseases
and of obesity.
According to an advantageous embodiment of the invention, the said peptide
is a C-terminal fragment of a pancreatic lipase selected from purified or
recombinant human, porcine or equine pancreatic lipases.
Unexpectedly, the C-terminal peptide of these various pancreatic lipases
effectively makes it possible for them to serve as a lure and to competition
between this peptide and the native lipase for the colipase and thus to
significantly reduce the lipolytic action of the said lipase.
The administration of such a peptide slows down the action of the pancreatic
lipase and surprisingly inhibits, at least in part, the hydrolysis of
dietary triglycerides, which will therefore not be absorbed (inhibitory
effect of the C-terminal peptide on the lipolysis).
Indeed, the affinity of this C-terminal peptide is, in vitro:
in solution, of the order of 106 M, towards the colipase, whereas
at the lipid interface, the affinity of the C-terminal peptide is of the
order of 2.times.108 M.
To obtain the desired action, that is to say an affinity at the interface of
the order of 2.times.108 M, the said C-terminal peptide is preferably
administered at doses of 0.5 to 10 mg/day distributed over 1 to 3 takings,
corresponding to doses of 0.2 to 10 mg per taking.
The subject of the present invention is also a pharmaceutical composition
comprising the C-terminal peptide of a pancreatic lipase, including the
recognition site for a colipase, as defined above and at least one
pharmaceutically acceptable vehicle.
The said pharmaceutical composition is advantageously provided in a unit
form capable of being administered by the oral route, selected from the
group consisting of soft or hard gelatin capsules, tablets, solutions,
suspensions and emulsions.
Such a pharmaceutical composition is preferably intended for oral
administration, in a gastroresistant form.
The subject of the present invention is also other applications of the said
C-terminal peptide of pancreatic lipase, for example as diagnostic reagent,
in particular in the carrying out of an immunoenzymatic test for the assay
of lipase by a competitive-type method and in the carrying out of methods of
controlled lipolysis of triglyceride substrates.
Claim 1 of 6 Claims
1. A method of treating disorders linked to an excess of lipids, comprising:
administering to a person in need thereof, an effective amount of a peptide
comprising a C-terminal fragment of a pancreatic lipase that contains a
recognition site for a colipase, wherein said colipase carries three
topologically distinct recognition sites: an interfacial recognition site, a
lipase recognition site and a micellar recognition site.
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