Note: Descriptions are shown in the official language in which they were submitted.
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A method to increase the excretion of non-sterol endogenous hydrophobic
substances by increasing excretion of fat via the faeces
Summary of the invention
The invention concerns a method to increase the excretion of non-sterol
endogenous hydrophobic substances or metabolic derivatives thereof by
increasing
excretion via the faeces of the hydrophobic substance or metabolic derivative
thereof.
Also the invention concerns a method for prevention or treatment of conditions
associated with the accumulation of non-sterol endogenous hydrophobic
substances or
metabolic derivatives thereof.
Background -of the invention
Disturbance of the homeostasis of non-sterol endogenous hydrophobic
compounds in mammals, specifically in humans, can lead to accumulation of
1 S detrimental amounts of these compounds.
One example of such a compound is bilirubin. Under physiological conditions,
bilirubin undergoes two conjugation reactions with glucuronic acid, derived
from UDP-
glucoronide, which results in the formation of bilirubin diglucuronide.
Bilirubin
diglucuronide is significantly more water-soluble than the parent compound,
unconjugated bilirubin (L1CB), and can be readily excreted via the bile into
the faeces.
The two conjugation reactions are catalysed by the hepatic enzyme uridine
diphosphoglucuronosyl transferase (h-UDPGTbiI, EC 2.4.1.17). In Crigler
Najjar's
disease (CN) the activity of h-UDPGTbiI is completely absent (CN type I) or
significantly reduced (CN type II), leading to increased serum concentrations
of UCB.
Increased serum levels of UCB are also found during the neonatal period,
especially in
preterms, during increased rates of haemoglobin degradation (for example
sickle cell
crisis, anaemic crisis in G6PD-deficient individual, ABO-antagonism or other
forms of
immune or non-immune hemolysis), or during impaired hepatic conjugation
efficiency
(for example viral infections, metabolic diseases, and others)(for review, see
Chowdhury et al., Hereditary jaundice and disorders of bilirubin metabolism.
In:
Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular
Basis of
Inherited Disease. New York: McGraw-Hill, Inc. 1995:2161-2208).
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High serum concentrations of UCB are associated with accumulation in other
organs of
the body, among which the central nervous system, and with toxic effects on
the central
nervous system. In order to keep serum concentrations of UCB below 250 pmol/L,
unconjugated hyperbilirubinemia is conventionally treated with phototherapy
for many
hours daily. Phototherapy (wavelength 400-460 nm) results in the formation of
a
variety of photoproducts which can be secreted into the bile, however, only at
a
relatively slow rate. After biliary secretion, the configurational isomers can
spontaneously revert to the normal configuration and be absorbed from the
intestinal
lumen. These features of the confi,gurational isomers obviously decrease the
efficacy of
phototherapy. Whereas Crigler-Najjar's disease patients (in particular type I)
are usually
treated lifelong by phototherapy at home, neonates with unconjugated
hyperbilirubinemia are generally admitted to hospitals for phototherapeutic
treatment.
If phototherapy fails to lead to clinically acceptable serum concentrations of
UCB,
patients may need to undergo one or more exchange transfusions, which
comprises a
high-risk therapy with considerable morbidity and even mortality.
Alternative strategies for the treatment of unconjugated hyperbilirubinemia
involve the capture of UCB or of its photoisomers in the intestinal lumen,
thereby
preventing their intestinal uptake and enterohepatic circulation. The first
results of the
intestinal UCB capture approach date back to 1983. It was demonstrated that
the enteral
administration of agar could serve as an adjunct to phototherapy in neonates
with
unconjugated hyperbilirubinemia (Odell et al., Pediatr Res 1983;17:810-814).
Also, the
oral administration of activated charcoal to Gunn rats was associated with a
decrease in
serum bilirubin concentration (Davis et al., Pediatr Res 1983;17:208-209). The
capture
of bilirubin in the intestinal lumen has also been attempted with
cholestyramine, but
only a modest benefit was obtained (Nicolopoulos et al., J Pediatr 1978;93:684-
688,
Tan et al., J Pediatr 1984;104:284-286). Nagyvary described and patented the
use of
chitosan (a polymer of N-acetyl-D-glucosamine units) to treat
hyperbilirubinemia,
based on the intraluminal binding of bilirubin in the intestine (US
4,363,801).
Disadvantage of the use of these hydrophilic resins or resin-like materials is
that these
will bind a great variety of other useful components, which subsequently will
be
excreted. Other patents on the application of UCB adsorption to treat
hyperbilirubinemic states include US 5,200,181, in which a bilirubin
converting
enzyme is used; US 4,593,073, in which amino acid containing polymers are
used, US
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5,804,218 in which zinc salts are used. None of these alternative strategies
has resulted
in a practically used therapy.
Another example of a non-sterol endogenous hydrophobic compound in
mammals, whose accumulation can lead to detrimental consequences, is
protoporphyrin. Protoporphyrin (PP) is a hydrophobic intermediate in the
biosynthesis
of heme. Heme is an iron-containing, prosthetic group in many proteins, which
function
in for example oxygen en electron transport, H202 generation and degeneration,
and
nitric oxide synthesis. Catalysed by the enzyme ferrochelatase (EC 4.99.1.1 ),
PP is
converted into heme through the addition of a Fe2+-atom. Although all
mammalian cells
synthesise heme, the major site is the bone marrow, where approximately 85% of
the
body's heme is produced for the formation of hemoglobin. The second major site
of
heme synthesis in mammalians is the liver (see for review Kappas et al., The
Porphyrias. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic
and
Molecular Basis of Inherited Disease. New York: McGraw-Hill, Inc. 1995:2103-
2159).
Under several pathophysiological conditions, for example in the disease
erythropoietic
protoporphyria (EPP), PP accumulates in body. EPP is an autosomal dominant,
inherited disease, which is characterised by a strongly reduced activity of
the
ferrochelatase enzyme. Under the pathophysiological condition of PP
accumulation,
particularly in erythrocytes, liver and faeces increased PP concentrations are
found
(Romslo et al., Arch Dermatol 1982;118:668-671, Beukeveld et al., Clin Chem
1987;33:2164-2170). The clinical consequences of increased concentrations of
PP in
the body can be exemplified by the symptoms of EPP. At young age, EPP patients
have
a cutaneous photosensitivity in light-exposed areas. The mechanism of the
photosensitivity involves the generation of free oxygen radicals from
accumulated PP
in the skin, under influence of light (wavelength 400-410 nm). The reactive
oxygen
radicals damage primarily the mitochondria and cellular membranes, leading to
severely discomforting skin lesions (burn-like lesions, itching, oedema,
scarring).
The disposal from the body of PP involves biliary secretion and subsequent
loss via the
stools. It is not known whether the highly hydrophobic parent molecule PP can
be
reabsorbed by the intestinal mucosa and undergoes enterohepatic cycling.
Strong,
indirect support for this possibility can be derived from the observed
beneficial effects
of cholestyramine on PP accumulation in EPP. It appeared that the
administration of
cholestyramine, in analogy to its use in hyperbilirubinemia, improved
photosensitivity
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and reduced hepatic PP content (Tishler et al., Methods Find Exp Clin
Pharmacol
1985;7:485-491, McCullough et al., Gastroenterology 1988;94:177-181).
Description of the invention
The present invention is directed at a novel mechanism of intestinal capture
of
non-sterol endogenous hydrophobic compounds, specifically the induction of
increased
fat excretion via the faeces (steatorrhoea).
The capture of non-sterol endogenous hydrophobic compounds by luminal fat
differs from the previous approaches. Calcium phosphate and activated charcoal
are
suggested to bind UCB for instance or its photoisomers by an adsorption
process.
Cholestyramine, applied in conditions in which UCB, PP and plant sterols were
increased, is applied as a resin, which binds the respective molecules. The
same is true
for the fibrous material chitosan.
By increasing the lipophilic phase, or amount of fat, in the intestinal lumen,
as
disclosed according to the invention, hydrophobic compounds will dissolve or
diffuse
into the generated apolar phase. The apolar, lipophilic phase persists
throughout the
digestive tract and will drag hydrophobic compounds along the intestinal
tract, which
eventually will be excreted. The hydrophobic phase is virtually impermeable
for polar
detergents, such as bile salts. It can be expected that the absorption of fat-
soluble
compounds such as fat soluble vitamins, such as vitamin A, vitamin D, vitamin
E, and
vitamin K, will also be inhibited. To compensate for this, an increased
dietary intake,
either in natural or in water-soluble form, may be warranted. The induction of
increased
faecal fat excretion by any means increases the disposal of endogenous
hydrophobic
substances such as UCB and PP from the body, at least under conditions of
their
previous accumulation. Thus the present invention provides a method to prevent
or
treat conditions such as neonatal jaundice, haemolytic jaundice and
erythropoietic
protoporphyria.
In the examples it is described that in Gunn rats the induction of fat
malabsorption, and thus, of increased faecal fat excretion, was dose-
dependently related
to decreased plasma UCB concentrations. A strong, inverse correlation was
observed
between the amount of faeces produced and the plasma UCB concentration in the
rats
studied. Based on the nature of the effect, namely hydrophobic diffusion, it
can be
anticipated that the same strategy would allow increasing the disposal from
the body of
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other hydrophobic compounds upon their accumulation under pathophysiological
circumstances:
Admittedly, parts of the presently proposed concept have been appreciated
quite
some time previously when addressing other problems In particular is referred
to
5 strategies to decrease plasma levels of cholesterol. The plasma cholesterol
concentration is related to the intestinal absorption of cholesterol, derived
either from
the diet or from the bile. As exemplified by studies employing sucrose fatty
acid
polyesters, decreasing the intestinal {re)absorption of cholesterol is
associated with an
improved plasma lipid and lipoprotein profile (Jandacek, Int J Obes 1984;8
Supp1:13-
21, Jandacek et al., Metabolism 1990;39:848-852, US 3,954,976, US 4,005,195,
US
4,005,196). The specific use of sucrose polyesters to detoxify humans and
lower
animals after accidental or chronic ingestion of lipophilic toxins such as
insecticides
(for example, DDT, Kepone), herbicides (for example, PCP) or industrial
chemicals
(for example, polychlorinated biphenyls (PCB's), polybrominated biphenols
(PBB's))
has also been patented (US 4,241,054).
The present invention however is directed at the general approach to increase
the
disposal from the body of non-sterol endogenous hydrophobic compounds, such as
UCB and PP, namely by inducing/increasing the faecal excretion of a
hydrophobic
(lipophilic) phase, through whatever mechanism. The previous disclosures are
directed
at elimination of a different category of compounds to the invention, using
different
means. The previous disclosures have been available in the prior art for a
long time and
nevertheless the skilled person in the field of for instance
hyperbilirubinemia or
protoporphyria have been occupied with totally different concepts of
treatment.
Several strategies to induce increased faecal fat excretion can be envisaged
for
carrying out the method according to the invention. The various physiological
processes involved in fat absorption can be influenced for example in the
following
ways:
- inhibition of lipolysis: Dietary fat consists for 92-96% of
triacylglycerols, which have
to be hydrolysed by lipolytic enzymes before they can be absorbed.
Interference with
the lipolysis can be obtained by inhibition of the lipase enzymes, operational
in the gut
lumen, for example by orlistat (Hochuli et al., J Antibiot (Tokyo)
1987;40:1086-1091,
Weibel et al., J Antibiot (Tokyo) 1987;40:1081-1085) or functionally and/or
structurally related compounds (Yoshinari et al., J Antibiot (Tokyo)
1994;47:1376-
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1384) (see also for example US. 4,598, 089, EP 185 359 and EP 444 482).
Inhibition of
lipolysis could also be achieved by using compounds from the oxetanone group
(US
4,931,463) or the esterastin group (US 4,202,824). The validity of the concept
to induce
fat malabsorption by this principle has been described in the prior art (Hogan
et al., Int
J Obes 1987;11 Suppl 3:35-42, Fernandez and Borgstrom, Biochim Biophys Acta
1989;1001:249-255 and Biochim Biophys Acta 1989;1001:249-255, Hauptman et al.,
Am J Clin Nutr 1992;55:3095-3135, Reitsma et al., Metabolism 1994;43:293-298,
Isler
et al., Br J Nutr 1995;73:851-862).
- inactivation of fatty acid and monoacyJelycerol solubilisation: Long-chain
fatty acids
and monoacylglycerols, the metabolic products of intralumenal lipolysis, are
hardly
soluble in the aqueous environment of the small intestine. Under physiological
circumstances, bile components (bile salts, phospholipids) increase their
aqueous
solubility (a process also known as solubilisation), by the formation of
complex
aggregates {micelles, vesicles), consisting of fatty acids, monoacylglycerols,
bile salts,
phospholipids, cholesterol. Interference with this process of solubilisation,
for example
by inhibiting the enteral influx of bile or by decreasing the soluble
("active")
concentration of bile salts, has been demonstrated to be associated with
faecal fat
excretion and, thus, to impair intestinal fat absorption (Poley et al.,
Gastroenterology
1976;71:38-44, Graham and Sackman, Gastroenterology 1982;83:638-644, DeVizia
et
al., Pediatr Res 1985;19:800-806, Chappell et al., J Pediatr 1986;108:439-447,
Potter et
al., Nutrition 1990;6:309-312, Sandberg et al., Am J Clin Nutr 1994;60:751-
756,
Carnielli et al., Am J Clin Nutr 1995;61:1037-1042, Mabayo et al., Lipids
1995;30:839-
845, Xu C et al., J Dairy Sci 1998;81:2173-7). Increasing the viscosity of the
lumenal
phase, such as by carboxymethylcellulose, will also impair fat absorption and
thus
increase faecal fat excretion (Smits et al., Poult Sci 1998;77:1534-9).
Relevant to this
approach is the described use of such products in elimination of laxative
elects of low
calorie fat materials (EP 236288).
- inhibition of lipid translocation across the apical membrane of the
intestinal mucosal
cells: Carrier-mediated uptake mechanisms for lipids have been hypothesised
and
partially identified at the level of the brush border membrane of the small
intestinal
mucosa (Compassi et al., Biochemistry 1995;34:16473-16482, Schoeller et al.,
Clin
Invest Med 1995;18:380-388, Fitscher et al., Proc Soc Exp Biol Med 1996;212:15-
23,
Schulthess et al., J Lipid Res 1996;37:2405-2419, Boffelli et al.,
Biochemistry
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1997;36:10784-10792). Candidate proteins involved in these processes have been
indicated and it is reasonable to assume that specific inhibitors of this
(these) carrier
systems) would inhibit the absorption of lumenal lipids, leading to increased
faecal fat
excretion. An example of this phenomenon can be derived from the studies by
Stremmel et al. (J Clin Invest 1985;75:1068-1076), in which the translocation
of a fatty
acid across brush border membrane vesicles could be inhibited by the
incubation with a
specific antibody.
- inhibition of an intracellular event of fat absorption' fatty
acid/monoacylelvcerol
reacylation chylomicron assembly and/or basolateral chvlomicron secretion:
After
lipids have entered the small intestinal mucosal cell, they are reassembled
into
chylomicrons, either with (monoacylglycerols, fatty acids, lysophospholipids,
unesterified sterols) or without (fraction of unesterified sterols,
phospholipids) prior to
reacylation. The intracellular events in fat absorption and chylomicron
assembly are
only partially understood (see for review Tso P. Intestinal lipid absorption.
In: Johnson
LR, ed. Physiology of the gastrointestinal tract. New York: Raven Press,
1994:1867-
1907). One of the factors which recently has been identified to be of crucial
importance
for proper chylomicron assembly and secretion is the Microsomal Triglyceride
Transfer
Protein (Wetterau et al., Science 1992;258:999-1001, Wetterau et al., Biochim
Biophys
Acta 1997;1345:136-150). Inhibition of this protein has been demonstrated to
induce
net fat malabsorption, by means of impaired assembly and secretion of
chylomicrons,
and the subsequent shredding of the lipid-loaded mucosal cell into the
intestinal lumen.
This form of strategy, in which an intracellular event involved in fat
absorption is
inhibited, would also be applicable to induce an increased faecal fat
excretion with the
aim to increase disposal of unconjugated bilirubin or protoporphyrin. The use
of MTP
inhibitors to remove plant sterols from the body has for instance been
described in WO
98 31225.
The purpose to increase faecal fat excretion cannot only be accomplished by
interference with the physiological processes involved in fat absorption.
Other
approaches are the administration of non-absorbable hydrophobic compounds or
the
administration of hydrophobic compounds in a non-absorbable amount.
Non-absorbable hydrophobic compounds include for example (poly)esters of
fatty acids and sugars or alcohols. Administration of olestra, one particular
type of
sucrose polyester, has been demonstrated to decrease intestinal absorption and
decrease
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body disposal of the hydrophobic sterol cholesterol in hypercholesterolemic
man
(Mellies et al., Am J Clin Nutr 1983;37:339-346, Jandacek et al., Metabolism
1990;39:848-852) or of hydrophobic environmental pollutants in gerbils
(Jandacek et
al., Drug Metab Rev 1982;13:695-714, Mutter et al., Toxicol Appl Pharmacol
1988;92:428-435). The specific use of sucrose polyesters to detoxify humans
and lower
animals after accidental or chronic ingestion of toxic lipophilic materials
has also been
patented (US 4,241,054).
An alternative method to obtain an increased faecal excretion of fat involves
the
administration of supraphysiological amounts of conventional dietary fats
(Fomon et
al., Am J Clin Nutr 1970;23:1299-1313) which cannot be absorbed
quantitatively.
Endogenous hydrophobic compounds dissolve in dietary fats and are
concomitantly
excreted.
The present invention is also directed at pharmaceutical compositions
comprising
the compound to be administered as active compound in an effective amount
according
to any of the abovementioned strategies for prevention or treatment of
conditions
associated with the accumulation of non-sterol endogenous hydrophobic
substances or
metabolic derivatives thereof together with a pharmaceutically acceptable
carrier.
According to the invention the pharmaceutical compositions are directed at the
prevention or treatment of conditions that are associated with the
accumulation of
unconjugated bilirubin or protoporphyrin, such as neonatal jaundice,
haemolytic
jaundice or erythropoietic protoporphyria.
The present invention is also directed at the use of any compound being the
compound to be administered as active compound according to any of the
abovementioned strategies for the manufacture of a pharmaceutical composition
for
prevention or treatment of conditions associated with the accumulation of non-
sterol
endogenous hydrophobic substances or metabolic derivatives thereof, such as
the
accumulation of unconjugated bilirubin or protoporphyrin, possibly leading to,
for
example, neonatal jaundice, haemolytic jaundice or erythropoietic
protoporphyria.
The active compound according to the invention has to be delivered in the
intestinal lumen in a therapeutically effective amount. The form of
admistration of the
active compound or pharmaceutical composition containing the active compound
according to the invention can be any suitable form known to one of ordinary
skill in
the art for administering a therapeutically active agent to the intestinal
lumen, e.g. oral,
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enteral or rectal adminstration. Selection of the dosage is to be made by
anyone of
ordinary skill in the art of medicine, giving consideration to age, sex, size,
fat mass/lean
body mass ratio and the condition the recipient is suffering or to be
prevented from
suffering.
The following examples illustrate the feasibility of the subject invention and
are
not intended to limit the scope of the invention. The references cited are
incorporated
by citation in the subject description.
Examples
Example 1
Male Gunn rats (body weight 300-350 g) were fed a high-fat (16 wt%) chow diet
for 2 weeks. Major long-chain fatty acid composition of the diet: palmitic
acid, 31.9%;
stearic acid, 5.2%; oleic acid, 32.7%; linoleic acid 30.2%; Hope Farms,
Woerden, The
Netherlands). After two weeks, animals were divided in three groups (each
n=5). Each
group received for another 6 days grounded and dried high fat diet,
supplemented
without (control) or with the lipase inhibitor orlistat (Xenical~, 200 mg/kg;
800
mg/kg). Blood samples were collected obtained in EDTA-containing cups by tail
bleeding at day 6, and plasma was obtained by centrifugation ( 10 min, 2000
rp, 4 °C)
and stored under light-protected conditions until analysis (within hours after
sampling).
Bilirubin concentration in plasma was determined by automated analysis, based
on the
diazo method (Novros et al., Clin Chim Acta 25:1891-9; 1979). The results are
depicted in Fig. 1. A significant decrease of the plasma concentration of
unconjugated
bilirubin as a result of an orlistat containing diet was observed.
Example 2
Male Gunn rats (body weight 300-350 g) were fed a high-fat (16 wt%) chow diet
for 2 weeks. Major long-chain fatty acid composition of the diet: palmitic
acid, 31.9%;
stearic acid, 5.2%; oleic acid, 32.7%; linoleic acid 30.2%; Hope Farms,
Woerden, The
Netherlands). After two weeks, animals were divided in three groups (each
n=5). Each
group received for another 6 days grounded and dried high fat diet,
supplemented
without (control) or with the lipase inhibit or orlistat (Xenical~, 200 mg/kg;
800
mg/kg). From day 4 till day 6, faeces was quantitatively collected and
weighed. Faeces
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production was compared with plasma bilirubin concentrations at day 6 (see
Fig. 1 ) in
individual animals. The results are represented in Fig.2. The correlation line
was
characterised by the equation: Y = 293.6 - 9.9.X; in which Y = unconjugated
bilirubin
concentration in plasma, and X = total faeces production, R = -0.67, P =
0.006. It was
found that the plasma concentration of unconjugated bilirubin was inversely
related to
the amount of faeces production as a result of an orlistat containing diet.
Description of the figures
Fig.l : The effect of dietary supplementation with orlistat for 6 days on
plasma
10 concentration of unconjugated bilirubin in male Gunn rats (each group n=5).
Fig.2: Inverse relation of the plasma concentration of unconjugated bilirubin
and
the amount of faeces production in Gunn rats on orlistat-containing diets.
