(12) INTERNATIONAL APPLICATION PUBLISHED UNDER mE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I RUI 11~11 II BR~ IUD ~II I~ m 11111 w111m1 11111111111110 llU HI llB
`
`(43) International Publication Date
`8 February 2001 (08.02.2001)
`
`PCT
`
`(10) International Publication Number
`WO 01/08679 Al
`
`(51) International Patent Classification': A61K 311335,
`3ln15
`
`(21) International Application Number: PCT/NL99/00492
`
`(22) lnlemational. Filing Date:
`
`30 July 1999 (30.07.1999)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(71) Applicant and
`(NUNL];
`(72) Inventor:
`VERKADE, Hendrik. Jan
`Froukemaheerd 63, NL-9736 RE Groningen (NL).
`
`ES, Fl, GB, GD, GE, GH. GM, HR, HU, ID, IL, IN, IS, JP;
`KE, KG, KP. KR, KZ. LC, LK, LR,t.S. LT. LU, LV, MO,
`MG. MK. MN. MW, MX. NO. NZ, PL, PT. RO. RU. so;
`SE, SG, SI, SK. SL, TJ, TM, 'ffi, TT, UA, UG, US,.UZ.
`VN, YU, ZA, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM.
`KE. LS, MW, SD, SL. SZ. UG, ZW); Eurasian patent (AM,
`AZ. BY. KG, KZ, MD, RU, TJ, TM). European patent (AT,
`BE. CH. CY, DE. DK. ES. Fl. fR. GB, GR, IE. IT, LU. MC,
`NL. PT, SE), OAPI patent (BF. BJ. CF, CG, CI, CM, GA,
`GN, GW. ML, MR. NE. SN. TD, TG).
`
`(74) Agent: DE BRUIJN, Leendert, C.; Nederlandsch Published:
`With international search report.
`Octrooibureau, Scheveningseweg 82, P.O. Box 29720,
`NL-2502 LS The Hague (NL).
`
`FOi' two-Leiter codes and other abb~iations. refer to the "Guid(cid:173)
`ance Notes on Code1 and Abbreviations n appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`(81) Designated States (national): AE, AL, AM. AT, AU, AZ.
`BA, BB, BG, BR. BY, CA, CH, CN. CU, CZ. DE, DK, EE.
`
`----~· -;;;;;;;;;;;;;;
`;;;;;;;;;;;;;; ----
`iiiiiiiiii -== -;;;;;;;;;;; -
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`_..
`<
`O"\ I ' (54) Title: A MEIHOD TO INCREASE 1RE EXCRETION OF NON-STEROL ENDOOENOUS HYDROPHOBIC
`\C SUBSTANCES BY INCREASING EXCRETION OF FAT VIA THE FAECES
`QO = -- (57) Abstract: The subject invention concerns a method to increase the excretion of non-sterol endogenous hydrophobic substances
`S or metabolic derivatives thereof by increasing excretion via the faeces of the hydrophobic substance or metabolic derivative thereof
`0 associated with the accumulation of non-sterol endogenous hydrophobic substances or metabolic derivatives thereof, such substances
`:;;;;.... being unconjugaled bilirubin and protoporphyrin. More particularly, lipstatin, orlistat, tetrahydrolipstatin." polyol fauy acids like
`~ olestra, and non-conventional doses of dietary jet are used 10 treat neonatal jaundice, haemolytic or erytbropoietic prolOporphyria.
`
`characterisedin thal the excretion of fat via the faeces is increased. The purpose of the invention is to prevent or treat conditions
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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
`
`5
`
`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 · condition5.
`
`associated with the accwnulation of non-sterol endogenous hydrophobic substances or·
`10 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 accwnulation of
`detrimental amounts of these compounds.
`
`15
`
`One example of such a compound is bilirubin. Under physiological conditions,
`bilirubin undergoes two conjugation reactions with glucuronic acid, derived from UDP(cid:173)
`
`glucoronide, which results in the formation of bilirubin diglucuronide. Bilirubin
`
`20
`
`is significantly more water-soluble
`diglucuronide
`the parent compound,
`than
`unconjugated bilirubin (UCB), 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-UDPGThil, EC 2.4.1.17). In Crigler Najjar's
`disease (CN) the activity of h-UDPGThil is completely absent (CN type I) or
`significantly reduced (CN type II), leading to increased serum concentrations of UCB.
`
`25
`
`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
`
`inunune or non-immune hemolysis), or during impaired hepatic conjugation efficiency
`(for example viral
`infections, metabolic diseases, and others)(for review, see
`
`30 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 accwnulation in other organs of
`
`the body, among which the ceniral nervous system, and with toxic effects on the central
`
`nervous system. In order to keep serum concentrations of UCB below 250 Jimol/L, ·
`
`unconjugated hyperbilirubinemia is conventionally treated with phototherapy for many
`
`5
`
`hours daily. Phototherapy (wavelength 400-460 run) 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 configurational isomers obviously decrease the efficacy of
`
`10
`
`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
`
`IS
`
`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
`
`20
`
`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
`
`25
`
`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 hyperbiHrubinemia,
`
`based on the intralwninal 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
`
`30 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
`
`5
`
`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, H20i 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 cell~
`synthesise heme, the major site is the bone marrow, where approximately 85% of the
`
`/
`
`10
`
`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).
`
`15 Under several patbophysiological 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,
`
`20
`
`25
`
`particularly in erythrocytes, liver and faeces increased PP concentrations. are found
`(Romslo et al., Arch Dermatril 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
`
`light-exposed areas. The mechanism of the
`in
`a cutaneous photosensitivity
`photosensitivity involves the generation of free oxygen radicals from ~ccumulated PP
`in the skin, under influence of light (wavelength 400-410 run)~ The reactive oxygen
`radicals damage primarily the mitochondria and cellular membranes, .leading to
`severely discomforting skin lesions (bum-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,
`
`30
`
`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 Phannacol
`
`1985;7:485-491, McCullough et al., Gastroenterology 1988;94:177-181).
`
`4
`
`Description of the invention
`
`5
`
`The present invention is directed at a novel mechanism of intestinal capture of
`non-sterol endogenous hydrophobic compounds, specifically the induction of increased
`
`IO
`
`15
`
`fat excretion via the faeces (steatorrhoea).
`The capture of non-sterol endogenous hydrophobic compounds by :Juminal 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 lwnen, 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
`
`20
`
`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 di~tary intake,
`--either-in natural or in·water-soluble form, may be warranted. The induction of incr~d
`
`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
`
`25
`
`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 concentratio_n 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 ass.ociated with an.
`improved plasma lipid and lipoprotein profile (J andacek. Int J Obes t 984;& Suppl: 13;,,
`
`10
`
`21, Jandacek et al., Metabolism 1990;39:848-852, US 3,954,976, US 4,005,195, US
`
`4,005, l 96). 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 indu8trial· chemicals
`
`(for example, polychlorinated biphenyls (PCB's), polybrominated biphenols (PBB's))
`
`t 5
`
`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
`
`20
`
`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
`
`protoporpbyria have been occupied with totally different concepts of treatment.
`
`Several strategies to induce increased faecal fat excretion can be envisaged for
`
`25
`
`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 oflipolysis: Dietary fat consists for 92-96% of triacylglycerols, wh;ch ha\'e
`
`30
`
`to be hydrolysed by lipolytic eru:ymes 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 (Yosbinari 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). Inhibitio.n 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 {Hogal) et al., Int
`
`5
`
`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, Hauptinan-et al.,
`Am J Clin Nutr l 992;55:309S-313S, Reitsma et al., Metabolism 1994;43:293-298, Islet
`
`et al., Br J Nutr 1995;73:851-862).
`
`10
`
`- inactivation of fatty acid and monoacylglycerol solubilisation: Long-chain :fatty acids
`and monoacylglycerols, the metabolic products of intralurnenal lipolysis, are hardly
`soluble in the aqueous environment of the small intestine. Under physiological
`
`circwnstances, 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,
`
`15
`
`phospholipids, cholesterol. fu.terference 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 wi~ 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
`
`20
`
`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,
`
`-Camielli 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
`
`25
`
`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 effects of low
`
`calorie fat materials (EP 236288).
`
`- inhibition of lipid translocation across the apical membrane of the intestinal mucosa!
`
`cells: Carrier-mediated uptake mechanisms for lipids have been hypothesised and
`
`30
`
`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 J 996;2l2:15-23,
`Schulthess et al.. J Lipid Res 1996;37:2405-2419, Boffelli et al., Biochemistry
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`1997;36: 10784-l 0792). Candidate proteins involved in these processes have been .
`
`indicated and it is reasonable to assume that specific inhibitors of this (these) earner
`
`system(s) 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
`
`5
`
`Stremmel et al. (J Clin Invest l 985;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 absomtion: fatty acid/monoacylglycerol
`
`reacylation. chylomicron assembly. and/or basolateral chylomicron secretion: After
`
`10
`
`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
`
`15
`
`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 Trigl:yceriqe 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~ induce
`
`20
`
`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 involv~ 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
`
`25
`
`inhibitors to remove plant sterols from the body has for instance been descriPed 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
`
`30
`
`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., Arn J Clin Nutr 1983;37:339-346, Jandacek et al., Metabolism
`
`l 990;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
`
`5
`
`l 988;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 irivolves the
`
`administration of supraphysiological amounts of conventional dietary fats (Fomon et
`
`10
`
`al., Am J Clin Nutr 1970;23:1299-1313) which ca.Dnot be absorbed quantitatively.
`
`Endogenous hydrophobic compounds dissolve in dietary fats and are concomitantly
`
`excreted.
`
`The present invention is also directed at phannaceutical compositions comprising
`
`the compound to be administered as active compound in an effective amount according
`
`15
`
`to any of the abovementioned strategies for . prevention or treatment of. conditions
`
`associated with the accumulation of non-sterol endogenous hydrophobic sub~ces or
`
`metabolic derivatives thereof to_gether with a phannaceutically 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
`
`20
`
`unconjugated bilirubin or protoporphyrin, such as neonatal jaundice, haemolytic
`
`jaundice or erythropoietic protoporphyria.
`
`-----·Tue 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
`
`25
`
`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 _ th_e
`
`30
`
`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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`9
`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
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`body mass ratio and the condition the recipient is suffering or to be prevented from
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`suffering.
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`5
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`The following examples illustrate the feasibility of the subject invention :and are
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`not intended to limit the scope of the invention. The references cited are incorporated
`
`by citation in the subject description.
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`10
`
`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 Fanns, Woerden, The
`15 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
`
`mglkg). 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)~
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`20
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`Bilirubin concentration in plasma was determined by automated analysis, based on the
`
`diazo method-{Novros etal., Clin Chim-Acta 25:1891-9; 1979). The results are
`
`depicted in Fig. 1. A significant decrease of the plasma concentration ofunconjugated
`
`bilirubin as a result of an orlistat containing diet was observed.
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`25
`
`Example2
`
`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 Fanns, Woerden, The
`
`30 Netherlands). After two weeks, arumals were divided in three groups (each n=S). Each
`
`group received for another 6 days grounded and dried high fat diet, supplemented
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`without (control) or with the lipase inhibit or orlistat (Xenical®, 200 mg/kg; 800
`
`mglkg). From day 4 till day 6, faeces was quantitatively collected and weighed. Faeces
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`10
`production was compared with plasma bilirubin concentrations at day 6 (see Fig. 1) in
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`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 ofunconjugated bilirubin was inversely related to •
`
`5
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`the amount of faeces production as a result of an orlistat containing diet..
`
`Description of the figures
`
`Fig. I: The effect of dietary supplementation with orlistat for 6 days on plasma
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`10
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`concentration ofunconjugated bilirubin in male Gunn rats (each group n=S).
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`Fig.2: Inverse relation of the plasma concentration ofunconjugated bilirubin and
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`the amount of faeces production in Gunn rats on orlistat-containing diets.
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`Claims
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`PCT/NL99/00492
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`11
`
`1. Method to increase the excretion of non-sterol endogenous hydrophobic substances
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`or metabolic derivatives thereof by increasing excretion via the faeces of· the
`
`hydrophobic substance or metabolic derivative thereof characterised in that the
`
`5
`
`excretion of fat via the faeces is increased.
`
`I
`
`2. Method according to claim 1, for prevention or treatment of conditions associated
`
`with the accumulation of non-sterol endogenous hydrophobic substances or metabolic
`derivatives thereof.
`
`3. Method according to claim 1 or 2, characterised in that the non-sterol endogenous
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`10
`
`hydrophobic substances are unconjugated bilirubin or protopotphyrin.
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`4. Method according to claim 2 or 3, characterised in that the conditions to be
`
`prevented or treated are associated with the accumulation of unconjugated bilirubin or
`protoporphyrin, such as neonatal jaundice, haemolytic jaundice or erythropoietic
`protoporphyria.
`
`15
`
`5. Method according to any of claims 1-4, comprising administering a compound that
`is an inhibitor of lipolysis such as oxetanone derivatives, esterastin derivatives, lipstatin
`
`or orlistat, tetrahydrolipstatin and functionally and/or structurally related compounds
`6. Method according to any of claims 1-4, comprising administering a compound that
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`20
`
`inactivates fatty acid and monoacylglyerol solubilisation.
`7. Method according to any of claims 1-4, comprising administering a compound that
`is an inhibitor of the translocation of fatty acids from the intestinal lumen· across .the
`.·-apical-membrane of the intestinal mucosal cells.
`
`8. Method according to any of claims 1-4, comprising administering a compound that
`
`is an inhibitor of any intracellular event of fat absorption, such as fatty acid and/or
`25 monoatylglycerol reacylation and/or the assembly and/or secretion of chylomicrons
`from the intestinal mucosal cells, such compounds for example being MTP inhibitors.
`
`9. Method according to any of claims 1-4, comprising administering a compound: that
`
`is a non-digestible hydrophobic compound such as a polyol fatty acid ester, such a.
`compound for example being a sucrose fatty acid ester with at least 4 fatty acid esters
`or for example olestra.
`
`30
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`10. Method according to any of claims 1-4, comprising administering a non(cid:173)
`conventional dosage of dietary fat or dietary fat containing food products as
`supplement to a conventional diet.
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`12
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`I I. Pharmaceutical composition comprising
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`the compound
`
`to be administered
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`according to any of claims S-10 as active compound in an effective amount for
`
`.prevention or treatment of conditions associated with the accumwation of non-sterol
`endogenous hydrophobic substances or metabolic derivatives thereof together with a
`
`5
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`pharmaceutically acceptable carrier.
`
`12. Pharmaceutical composition according to claim I I in which the conditions to be
`
`prevented or treated are associated with the accumulation of unconjugated bilirubin or
`protoporphyrin, such as neonatal jaundice, . haemolytic jaundice or erythropoietic
`
`protoporphyria.
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`10
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`13. Pharmaceutical composition according to claim I I or 12 supplemented with
`additives that readily dissolve in fat, such as fat soluble vitamins, such as vitamin E,
`
`vitamin A, vitamin D and vitamin K.
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`14. Use of any compound being the compound to be administered according to any of
`
`claims 5-10 as active compound for 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.
`
`15. Use of any compound according to claim 14 in which the conditions to be
`
`prevented or treated are associated with the accumulation of unconjugated bilirubin or
`protoporphyrin, such as neonatal jaundice, haemolytic jaundice or erythropoietic
`protoporphyria.
`
`.. ..
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`15
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`20
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`1/2
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`Fig.1
`
`........
`
`"'O
`Q)
`
`Q)
`0
`
`...J -0
`::L. -. c:
`E 300
`c
`:s
`0
`:J ~
`·= ~ 200
`:s ..... c:
`.....
`c m 0 100
`·-c: E
`Cl 0
`:J m
`0
`0
`c: m
`:J a.
`
`U)
`
`0
`
`800 mg
`0 mg (control) 200 mg
`orlistat content (kg chow)
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`212
`
`Fig. 2
`
`0
`E
`
`30
`
`200
`
`100
`
`-_J -
`•
`-c.5
`:i. ~ •
`..
`•
`o..c
`•
`cu ·=
`~ :::J
`•
`~-c:::c
`B -o
`cS
`cu
`0
`0 C>
`cu.~
`E c
`rn 8
`~c
`c.. :::J
`
`