EP0795011A1 - Technique et compositions permettant de reduire l'absorption de cholesterol - Google Patents
Technique et compositions permettant de reduire l'absorption de cholesterolInfo
- Publication number
- EP0795011A1 EP0795011A1 EP95943643A EP95943643A EP0795011A1 EP 0795011 A1 EP0795011 A1 EP 0795011A1 EP 95943643 A EP95943643 A EP 95943643A EP 95943643 A EP95943643 A EP 95943643A EP 0795011 A1 EP0795011 A1 EP 0795011A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- bal
- tail
- cholesterol
- composition
- intestinal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/0493—Steroids, e.g. cholesterol, testosterone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention is in the field of dietary science and more particularly relates to the use of bile salt-activated lipase to reduce cholesterol uptake in the intestine.
- Hyperlipidemias particularly hypercholesterolemia and the hyperlipoproteinemias, are among the most potent risk factors in the causation of atherosclerosis. Hyperlipoproteinemias are also implicated in the development of pancreatitis. A long-established theory suggests that the higher the circulating levels of cholesterol, usually in the form of low density lipoproteins (LDLs) containing cholesterol, the more likely it is to gain entrance to the arterial wall and cause atherosclerosis.
- LDLs low density lipoproteins
- Cardiovascular disease is the leading cause of death in women and middle-aged American men. In 1988, more than 41,000 U.S. residents died of cardiovascular disease before the age of 50. Atherosclerosis, however, which is known to contribute to cardiovascular disease and stroke, begins at a much earlier age. Fatty streaks are common in the arterial walls of children, and a high prevalence of coronary-artery lesions has been found in young men who die accidentally or violently. Children and adolescents with elevated serum cholesterol levels are more likely than their counterparts with normal cholesterol levels to have parents with coronary heart disease. Higher serum cholesterol levels in childhood have been associated with aortic atherosclerosis at autopsy in adolescents
- Cholesterol is used by the body in the synthesis of the steroid hormones by certain endocrine glands and of bile acids by hepatocytes, and is an essential constituent of cell membranes. It is found only in animals. Related sterols occur in plants, but plant sterols are not absorbed from the gastrointestinal tract. Most of the dietary cholesterol is contained in egg yolks and animal fat.
- Cholesterol that is taken up in the intestine is derived directly from the diet and from cholesterol-containing bile salt and acids and free cholesterol synthesized in the liver and secreted into the intestine via bile ducts. Cholesterol esters from the bile and diet are absorbed from the lumen of the small intestine by the intestinal epithelial lining cells, and incorporated intracellularly into chylomicrons and, in minor amounts, incorporated into very low density lipoproteins (VLDLs), both of which are secreted into lymphatics that ultimately join the bloodstream.
- VLDLs very low density lipoproteins
- the chylomicrons and VLDLs deliver their triacyglycerols and some of their cholesterol to cells in endothelial, muscle, and adipose tissue.
- the cholesterol-enriched chylomicron remnants and VLDLs then deliver cholesterol back to the hepatocytes and to other cells of the vascular wall along the way (Ganong, Review of Medical Physiology 249-250 (Lange Medical Publications, 1985).
- the VLDLs from intestinal and liver cells can be converted to low density lipoproteins (LDLs) by discharge of their triacyglycerols. LDLs contain three-fourths of the total plasma cholesterol.
- hypercholesterolemia the increase in the blood cholesterol level is associated mainly with a rise in LDL concentrations.
- the specific causes of hypercholesterolemia are complicated and varied.
- At least one kind of hypercholesterolemia is caused by a mutation in the gene for the LDL receptor that moves cholesterol out of the blood, primarily in
- hyperlipidemia including hypercholesterolemia
- hypercholesterolemia Some forms of hyperlipidemia, including hypercholesterolemia, are potentially partially reversible with current techniques of preventive management. However, none of the current techniques is completely successful and many are associated with unwanted side effects and complications. Taking cholesterol-lowering drugs can result in a twenty percent reduction in serum cholesterol.
- hypolipemic drugs such as Lovastatin, mevastatin, cholestyramine (Questran), Clofibrate, Probucol, and nicotinic acid
- Dietary therapy is usually recommended for all patients with hypercholesterolemia but is not always effective.
- compositions and methods of use in lowering serum cholesterol in a patient in need thereof are provided.
- compositions derived from all or a portion of the carboxy terminal region of human bile salt-activated lipase (BAL) are described, which, when orally ingested, compete with native BAL in binding to the intestinal surface, thus reducing the physiological role of BAL in mediating the transfer of cholesterol into the intestinal cells, and, as a result, reducing the amount of cholesterol absorbed from the intestine into the blood stream.
- BAL human bile salt-activated lipase
- Useful derivatives of the carboxy terminal region of BAL are derived from all or portion of the region containing amino acid residues 539 to 722, and have a mucin-like structure containing at least three of the repeating proline-rich units of eleven amino acid residues each.
- Preferred proline-rich units have the consensus sequence PVPPTGDSGAP (Sequence ID No. 6).
- Figure 1 shows the proposed binding of BAL to intestinal endothelium cells via the C-tail O-glycosylated carbohydrate binding to a lectin-like receptor, binding of cholesterol and cholesterol ester to BAL, hydrolyzing cholesterol ester by BAL, followed by transfer of enzyme bound cholesterol into cells.
- Figure 2 shows the elution of endogenous BAL lipolytic activity, measured in ⁇ mol/g/h from mouse intestinal mucosa using either isotonic phosphate buffer, fucose (0.1 M), galactose (0.1 M), heparin (10 mg/ml), or NaCl (0.3 M).
- Figure 3 shows the cholesterol uptake in nmol/g/h of radioactive cholesterol (oleate) ester in rat intestine either treated or untreated with Ethylene-bis(oxyethylenenitrilo)] tetraacetic acid (EGTA) to remove BAL.
- EGTA Ethylene-bis(oxyethylenenitrilo)] tetraacetic acid
- Figure 4 is a graph showing the cholesterol uptake rate (nmol/g/h) for 1 mM EGTA, 0.2 mg/ml C-tail, and the control in rat intestine.
- Figure 5 is a graph showing blood [ 3 H]-cholesterol levels (nmol/mL) versus time (hours) in rats fed with [ 3 H]-cholesterol oleate, with and without C-tail.
- the blood [ 3 H]-cholesterol levels of a rat fed C- tail isolated from human milk BAL are indicated by crosses.
- the blood [ 3 H]-cholesterol levels of a rat not fed C-tail are indicated by squares. Blood samples were taken at 1 hour intervals up to 5 hours after the feeding of [ 3 H]-cholesterol oleate.
- Figure 6 is a graph of the averaged (from 6 experiments) levels of
- [ 3 H]- cholesterol in the blood (nmol/mL) versus time (hours) in rats fed with [ 3 H]-cholesterol oleate, with (shaded) or without (unshaded) isolated C-tail from human milk BAL. Blood samples were taken at 1 hour intervals up to 5 hours after the initial tube feeding of radiolabelled cholesterol oleate. The horizontal lines on top of the data bars represent the standard errors (see Table XTV).
- Figure 7 is a graph of the rate of triolein uptake ( ⁇ mol/g/h) in rats whose intestines were washed with 1) an EGTA solution, to remove bound BAL prior to the uptake experiment, with 6 mM taurocholate (column 1), 2) buffer, so the bound BAL was not removed, without taurocholate (column 2), 3) buffer with 6 mM taurocholate (column 3), and 4) buffer with 30 mM taurocholate (column 4). Data shown are the averages of results from 5 experiments (see Table XV). All 4 groups of animals received emulsified radiolabelled triolein.
- Figure 8 is a graph of the rate of taurocholate uptake ( ⁇ mol/g/h) in rats whose intestines were washed with 1) an EGTA solution, to remove bound BAL prior to the uptake experiment (cross shaded), and 2) buffer so the bound BAL was not removed (shaded). Data shown are the averages of results from 5 experiments (see Table XVI). The left and right pairs of columns show uptake rates from 1 mM and 6 mM radiolabelled taurocholate, respectively.
- compositions including all or a portion of the carboxy terminal region of bile salt-activated lipase (BAL), or functional equivalents thereof, are described, which, in the intestine, compete with native BAL in binding to the intestinal surface to reduce the function of endogenous BAL to mediate the uptake of cholesterol eaters or free cholesterol in the form of free cholesterol taken into the blood stream.
- BAL bile salt-activated lipase
- BAL Bile salt-activated lipase
- pancreatic BAL The amino acid and cDNA sequences of human milk BAL are the same as those of pancreatic carboxy lesterase, and closely related to or the same as lipases referred to in the literature as lysophospholipase, cholesterol esterase, sterol ester hydrolase, non-specific lipase, lipase A, carboxyl ester lipase, and cholesterol ester hydrolase, with certain species differences, primarily with respect to the number of repeating units in the carboxy region (Wang and Hartsuck, Biochim. Biophys. Ada 1166:1-19 (1993)).
- Pancreatic BAL is distinct from other types of non-bile salt activated lipases, such as pancreatic lipase and phospholipase.
- BAL In the intestinal lumen, BAL becomes attached to the intestinal surfaces, most likely the surface of intestinal epithelial lining cells via a specific receptor. It can be released from the lumenal surface by EGTA, galactose and fucose, but not by heparin, isotonic buffer, or sodium chloride, as demonstrated below. BAL, in the required presence of bile salts, is essential for hydrolyzing cholesterol esters to free cholesterol or to bind free cholesterol in me food. Both of these processes are necessary
- BAL also hydrolyses carboxyl ester bonds of acylglycerols, phospholipid, and vitamin esters, forming fatty acids and glycerol, and can act on emulsified, micellar, or soluble substrates. It is thought that bile salt causes conformational change in BAL to provide active site access for the bulky substrate molecule and provides additional lipid binding capability in forming the enzyme-substrate complex. Additionally, it is thought that bile salt acts as a fatty acid acceptor during BAL catalysis. The proposed mechanism for the action of BAL is shown in Figure
- BAL binds via the C-tail O-glycosylated carbohydrates to a lectin-like receptor on the surface of intestinal endothelium cells.
- the catalytic unit of the enzyme remains away from the endothelium cells, with the heparin binding site and active site exposed. Free cholesterol or cholesterol ester is then bound to the active site, and, in the case of cholesterol ester, it is hydrolyzed to free cholesterol.
- the catalytic unit then binds to the heparin on the cell surface and transfers the cholesterol into the cells.
- Cholesterol, fatty acids, and monoacyglycerols derived from lipolysis by BAL in the intestinal lumen are taken up by the intestinal epithelial lining cell (mucosal cell), where these are reesterified to intracellular triacyglycerols. Cholesterol interacts in the cell with these reesterified triacylglycerols plus apolipoproteins and phospholipid to form chylomicrons and very low density lipoproteins, which are secreted into the lymphatics uiat ultimately join the blood vascular system for systemic circulation.
- the full, mature, human BAL contains 722 amino acid residues (Sequence ID No. 1).
- the carboxy terminal region of BAL refers to a region in the native BAL molecule including residues 539 to 722. This carboxy terminal region of BAL, along with derivatives of this region that retain the intestinal binding activity, are referred to herein as "C-tail. " The C-tail of the human BAL molecule has many O-linked
- the amino acid sequence of the native human C-tail contains sixteen repeating proline-rich units of eleven amino acid residues each, most having the consensus sequence of PVPPTGDSGAP (Sequence ID No. 6) (Baba et al. , Biochemistry 30:500-510 (1991)).
- the native C-tail was determined to be O-glycosylated primarily at threonine and, to a small degree, if any, at one serine residue. It is believed that the serine residue, which has an adjacent aspartic acid, is not favorable for the O-glycosylation (Elhammer et al, J.Biol.Chem. 268: 10029-10038 (1993)).
- a peptide prepared by cyanogen bromide digestion of the C-tail was found to contain most of the carbohydrate of the native BAL (Baba et al. (1991)).
- T-BAL truncated versions of BAL which lack the C-tail binding portion
- T-BAL truncated versions of BAL which lack the C-tail binding portion
- C-tail alone can bind to the intestinal surface, and in fact, can compete with native BAL for this binding, but cannot transfer cholesterol since the catalytic unit is either not functional or not present.
- C-tail proteins are ineffective in transferring cholesterol into intestinal cells since the enzyme is not bound to the intestinal surface.
- a proline-rich unit refers to any of the repeated eleven amino acid groups present in any naturally occurring form of BAL, or derivatives thereof which, when combined with two or more other proline-rich units, results in a protein which binds to intestinal endothelium cells and/or inhibits the binding of native BAL.
- Preferred proline-rich units have the consensus sequence PVPPTGDSGA-P
- C-tail protein refers to any protein containing three or more proline-rich units as defined above,
- C-tail protein is a form of C-tail protein.
- a C-tail protein should include at least three of the repeating proline-rich units of eleven amino acid residues each.
- the rat pancreatic esterase C-tail which has only four repeating units still binds to rat intestine surface.
- Preferred C-tail proteins have at least 10, and most preferably at least 16, proline-rich units. It is expected that C-tail proteins with fewer proline-rich units will bind to intestinal surface with a lower affinity.
- C-tail proteins can be constructed by combining three or more proline-rich units, where the proline-rich units have the native amino acid sequence of a proline-rich unit from any BAL, have the consensus amino acid sequence of the human proline-rich unit, or derivatives of these amino acid sequences, such that the C-tail protein retains the ability to bind to intestinal endothelium cells and/or inhibit the binding of native BAL. Amino acid sequence variants.
- the C-tail protein may be O-glycosylated to different extents with respect to the number of threonine and serine residues, and can include amino acid deletions, substitutions, or additions which do not significantly impair binding to the intestinal surface.
- the substitutions, deletions, or additions to C-tail proteins, which do not alter binding are readily determined by a screening assay, in which the protein is allowed to bind to intestinal surface, then removed by washing with buffer with increasing concentrations of salt.
- An example of a BAL which contains a deletion not affecting binding of me C-tail is a BAL lacking the heparin binding site, which is postulated to be present between amino acid residues 56 and 62 (Baba et al. (1991)).
- Amino acid sequence variants of C-tail protein fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Fusions include hybrids of mature BAL and the C-tail protein with polypeptides that are homologous with BAL, for example, in the case of human BAL, secretory leaders from other secreted human proteins. Fusions also include hybrids of BAL and the C-tail protein with polypeptides homologous to the host cell but not to BAL, as well as, polypeptides heterologous to both the host cell and BAL.
- Preferred fusions are amino terminal fusions with either prokaryotic peptides or signal peptides of prokaryotic, yeast, viral or host cell signal sequences. It is not essential that the signal sequence be devoid of any residual mature sequence from the protein whose secretion it ordinarily directs but this is preferable in order to avoid the secretion of a C-tail protein fusion. Insertions can also be introduced within the coding sequence of the proline-rich unit repeat region of the C-tail protein. Such insertions can include the addition of unrelated amino acids or the insertion of one or more additional proline-rich units. In the context of inserted amino acids, "unrelated" amino acids refer to amino acid sequences that are unrelated to the sequence of the proline-rich units of BAL.
- the inserted units can be heterologous units from non-human BAL, units having the consensus sequence (Sequence ID No. 6), or additional repeats of individual human proline-rich units.
- sequence ID No. 6 consensus sequence
- additional repeats of individual human proline-rich units In the case of insertion of unrelated amino acids, however, the insertion will ordinarily consist of smaller insertions than those of amino or carboxyl terminal fusions, or than those of proline-rich units, on the order of 1 to 4 residues.
- Insertional amino acid sequence variants of C-tail proteins are those in which one or more amino acid residues are introduced into a predetermined site in the target C-tail protein. Most commonly, insertional variants are fusions of heterologous proteins or polypeptides to
- SK -li ⁇ the amino or carboxyl terminus of the C-tail protein.
- these heterologous polypeptides are heterologous forms of the proline-rich units present in human BAL.
- Immunogenic C-tail protein derivatives are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion.
- Such immunogenic polypeptides can be bacterial polypeptides such as trpLE, beta- galactosidase and the like.
- Deletions are characterized by the removal of one or more amino acid residues from the C-tail protein sequence. It is preferred that deletions involve deletions of entire prolinerich units. If individual amino acids within the proline-rich units are deleted, no more than about from 2 to 6 residues are deleted at any one site within the C-tail protein molecule.
- These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the C-tail protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. However, variant C-tail protein fragments having up to about 100 to 150 residues may be conveniently prepared by in vitro synthesis.
- the variants typically exhibit the same qualitative biological activity as the naturally-occurring analogue, that is, specific intestinal binding, although variants also are selected in order to modify the characteristics of the C-tail protein as will be more fully described below.
- the site for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined.
- random mutagenesis may be conducted at the target colon or region and the expressed C-tail protein variants screened for the optimal combination of desired properties.
- Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis.
- Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues, or entire proline-rich units; and deletions will range about from 1 to 30 residues, or entire proline-rich units. Deletions or insertions preferably are made in adjacent pairs, that is a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. Obviously, me mutations that will be made in me DNA encoding the variant BAL or C-tail protein must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (EP 75,444A).
- substitutional variants are those in which at least one residue in the C-tail protein has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table I when is it is desired to finely modulate the characteristics of C-tail protein or BAL.
- Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table I, that is, selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain.
- substitutions which in general are expected to produce the greatest changes in BAL or C-tail protein properties will be those in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
- H SltL£26 substituted for (or by) any other residue;
- a residue having an electropositive side chain for example, lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or
- a residue having a bulky side chain for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.
- Substitutional or deletional mutagenesis can be employed to eliminate N- or O-linked glycosylation sites (for example by deletion or substitution of asparaginyl residues in Asn-X-Thr glycosylation sites), improve expression of BAL or C-tail protein or alter the half life of the protein.
- unglycosylated BAL or C-tail protein can be produced in recombinant-prokaryotic cell culture. Such unglycosylated forms are expected to lack, or have reduced, intestinal binding activity.
- Deletions of cysteine or other labile residues also may be desirable, for example in increasing the oxidative stability or selecting the preferred disulfide bond arrangement of BAL.
- Deletions or substitutions of potential proteolysis sites for example, Arg is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
- Full length BAL which has an inactivated catalytic site can also be used for competitive inhibition of binding of cholesterol. Since the catalytic site is inactivated, the BAL cannot facilitate uptake of cholesterol.
- the catalytic site can be inactivated by amino acid deletion or substitution in the active site of the recombinant BAL.
- the catalytic site can also be inactivated by chemical modification of BAL at or near the active site, for example, by proteolysis or other enzymic reactions, by binding of an irreversible enzyme inhibitor to the catalytic site, or by means which disrupt the three dimensional conformation of the catalytic unit of BAL (since the binding of the C-tail to intestine is not dependent on its conformation).
- disruptive means include detergents and heat.
- BAL derivatives that do not hydrolyze cholesterol esters or bind heparin include polypeptides that may or may not be substantially homologous with BAL. These BAL derivatives are produced by the recombinant or organic synthetic preparation of BAL fragments or by introducing amino acid sequence variations into intact BAL so that it no longer demonstrates cholesterol ester hydrolysis and/or heparin binding activity as defined above.
- BAL derivatives that do not hydrolyze cholesterol esters or bind heparin as described above are useful as immunogens for raising antibodies to active BAL.
- BAL derivatives referred to as "BAL protein antagonists” may be used to neutralize the cholesterol uptake- mediating activity or BAL.
- BAL protein antagonist may bind to the intestinal lining thereby blocking binding of native BAL.
- BAL protein antagonists are useful in the therapy of various cholesterol disorders for example, hypercholesterolemia, especially hypercholesterolemia exacerbated by dietary cholesterol intake.
- BAL, BAL derivatives, and C-tail protein molecules may also be covalently modified. Such modifications are made by reacting targeted amino acid residues of the recovered or synthesized protein with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Alternately, post-translational modification in selected recombinant host cells may be used to modify the protein.
- the resulting covalent derivatives are useful as immunogens or to identify residues important for biological activity as well as for altering pharmacological characteristics of the molecule, such as half life, binding affinity and the like, as would be known to the ordinarily skilled artisan.
- Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be used. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (Creighton, Proteins: Structure and Molecular Properties pages 79-86 (W. H. Freeman & Co., San Francisco, 1983)), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl. Production of C-tail proteins.
- C-tail protein There are several ways to produce a C-tail protein. For example, one may obtain natural or recombinant BAL and cleave off the native C- tail using proteases, such as trypsin, chymotrypsin, pepsin and others, or chemical methods such as cyanogen bromide cleavage at Met-X bonds. Alternatively, one can express in an eukaryotic host the gene or cDNA encoding the sequence including the C-tail region of BAL, or any C-tail protein as described above, either by itself or as fusion to other non-BAL DNA sequences which may facilitate either the expression or the purification of recombinant C-tail protein.
- proteases such as trypsin, chymotrypsin, pepsin and others
- chemical methods such as cyanogen bromide cleavage at Met-X bonds.
- BAL or C-tail protein fusions can be subjected to cleavages and purification to obtain purified C-tail.
- BAL can be purified from natural sources, such as the milk of human and certain species or animal intestines or pancreatic juice, although this is impractical on a large scale. It is preferably produced by genetic engineering using standard recombinant DNA technology and eukaryotic host cells, such as yeast or cultured mammalian or insect cells, so the C-tail can be properly glycosylated, or in the milk of transgenic non-human animals.
- EET ILE 26 from blood and/or tissues by extraction and purification.
- Other materials include infectious organisms such as, for example, the causative agent of acquired deficiency syndrome (AIDS).
- BAL and C-tail protein produced by the method of the instant invention is greater than or equal to 95% purity.
- sequence ID No. 3 encoding human milk bile salt activated lipase (Sequence ID No. 2) set out in U.S. Patent No. 5,200,183 to Tang and Wang; Baba et al. (1991); or Nilsson et al., Eur. J. Biochem. 192:543-550 (1990), all incorporated herein by reference.
- This sequence can be readily adapted for expression of any C-tail-protein as described above, either by deletion, addition, or substitution of the sequence encoding native BAL C-tail, as described above.
- a DNA isolate is understood to mean chemically synthesized DNA, cDNA or genomic DNA with or without the 3' and/or 5' flanking regions.
- DNA encoding BAL can be obtained from other sources than human by a) obtaining a cDNA library from the liver, breast, pancreas, or other tissues containing BAL mRNA of the particular animal, b) conducting hybridization analysis with labelled DNA encoding human BAL or fragments thereof (usually, greater than 100 bp) in order to detect clones in the cDNA library containing homologous sequences, and c) analyzing the clones by restriction enzyme analysis and nucleic acid sequencing to identify full-length clones.
- men appropriate fragments may be recovered from the various clones using nucleic acid sequence disclosed herein and ligated at restriction sites common to the clones to assemble a full-length clone encoding BAL.
- Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. Unless indicated otherwise, me method used herein for transformation of the host cells is the method of Graham and van der Eb, Virology 52:456-457 (1973). However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, the preferred method of transfection is calcium treatment using calcium chloride as described by Cohen et al, Proc. Natl. Acad. Sci. USA 69:2110 (1972).
- plasmids containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and relegated in the form desired to form the plasmids required. Since glycosylation of the C-tail is essential, prokaryotic cells are not useful as expression hosts for C-tail which binds to receptor. However, in general, prokaryotes are used for cloning of DNA sequences in constructing the vectors useful for expression. For example, E. coli W3110 (F, a prototrophic, ATTC No.
- bacilli such as Bacillus subtilus
- enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans
- various pseudomonas species can be used.
- plasmid vectors containing promoters and control sequences which are derived from species compatible with the host cell are used with these hosts.
- the vector ordinarily carries a replication site as well as one or more marker sequences which are capable of providing phenotypic selection in transformed cells.
- E. coli is typically transformed using a derivative of pBR322 which is a plasmid derived from an E. coli species (Boliver et al, Gene 2:95 (1977)).
- pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
- the pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters and omer control elements commonly used in recombinant DNA construction.
- Promoters suitable for use with prokaryotic hosts illustratively include the
- the ligation mixtures are used to transform E. coli K12 strain 294 (ATCC 31446) and successful transformants selected by ampicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction and/or sequenced by me method of Messing et al, Nucleic Acids Res. 9:309 (1981) or by the method of Maxam et al, Methods in Enzymology 65:499 (1980).
- Host cells can be transformed with the expression vectors and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes.
- the culture conditions such as temperature and pH, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
- Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO 4 and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell. In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.
- UBSI ⁇ oTt SHEET (BUIE 26) -21- of another DNA fragment at the restriction site. Procedures and reagents for dephosphorylation are conventional (Maniatis et al, Molecular Cloning, 133-134 (Cold Spring Harbor, 1982). Reactions using BAP are carried out in 50 mM Tris at 68 °C to suppress the activity of any exonucleases which may be present in the enzyme preparations.
- Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis et al, Id. at 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
- ligase T4 DNA ligase
- “Filling” or “blunting” refers to the procedures by which the single stranded and in the cohesive terminus of a restriction enzyme- cleared nucleic acid is converted to a double strand. This eliminates the cohesive terminus and forms a blunt end. This process is a versatile tool for converting a restriction cut end that may be cohesive with the ends created by only one or a few other restriction enzymes into a terminus compatible with any blunt-cutting restriction endonuclease or other filled cohesive terminus.
- blunting is accomplished by incubating 2 to 15 ⁇ g of the target DNA in 10 mM MgCl 2 , ImM dithiothreitol, 50 mM NaCl, 10 mM Tris (pH 7.5) buffer at about 37 °C in the presence of a 8 units of the Klenow fragment of DNA polymerase I and 250 ⁇ M of each of the four deoxynucleoside triphosphates.
- the incubation generally is terminated after 30 minutes by phenol and chloroform extraction and ethanol precipitation.
- Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis et al, Id. at 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
- ligase T4 DNA ligase
- “Filling” or “blunting” refers to the procedures by which the single stranded and in the cohesive terminus of a restriction enzyme- cleared nucleic acid is converted to a double strand. This eliminates the cohesive terminus and forms a blunt end. This process is a versatile tool for converting a restriction cut end that may be cohesive with the ends created by only one or a few other restriction enzymes into a terminus compatible with any blunt-cutting restriction endonuclease or other filled cohesive terminus.
- blunting is accomplished by incubating 2 to 15 ⁇ g of the target DNA in 10 mM MgCl 2 , ImM dithiothreitol, 50 mM NaCl, 10 mM Tris (pH 7.5) buffer at about 37°C in the presence of a 8 units of the Klenow fragment of DNA polymerase I and 250 ⁇ M of each of the four deoxynucleoside triphosphates.
- the incubation generally is terminated after 30 minutes by phenol and chloroform extraction and ethanol precipitation.
- both human BAL cDNA and human BAL genomic DNA can be used to direct the synthesis of recombinant human BAL protein(s) in native or modified forms.
- Human BAL gene(s) are expected to contain in their untranslated region sequences which regulate the expression of the enzyme. These regulatory sequences may even be directly used in transgenic animal expression.
- Recombinant BAL protein can be produced from human BAL cDNA or genes by many different methods. These include the expression of BAL in hosts such as E. coli, Bacillus, yeast, fungi, insect cells, mammalian cells, and transgenic animals. Expression of T-BAL (truncated form of BAL without C-tail) in E. coli has been described by Downs et al, Biochemistry 33:7980-7985 (1994). Since prokaryotic hosts cannot excise mammalian introns from mRNA, it is preferable to express the cDNA, with appropriate modifications, in procaryotic systems, ramer than the gene.
- prokaryotes cannot glycosylate BAL
- eukaryotic systems for expression of BAL.
- eukaryotic cells either human BAL genes or cDNA can be used to direct the synthesis of the enzyme.
- There can also be glycosylation on BAL provided ttiat a 'leader' or 'signal' sequence is present to direct newly synthesized BAL to the inside of the rough endoplasmic reticulum. This is important for the C-tail to reduce cholesterol uptake since the interaction of intestinal surface is through the oligosaccharides on the C-tail.
- the human BAL cDNA or gene can be inserted into appropriate expression vectors containing expression regulatory elements, such as transcription initiation signals, translation initiation signals, starting codon, termination codon, transcription terminating signals, polyadenylation signals, and others. Suitable vectors are commercially available from a variety of companies. After the recombinant vectors containing BAL cDNA or gene is transfected into the host cells, they may remain as extrachromosomal DNA or they may be integrated into the host
- SUESTiTiJTE SI1 ⁇ E ⁇ (RULE 26) genome. In either case, they may direct the synthesis of recombinant BAL in the host cells.
- Some examples for me expression of heterologous genes are described in Methods in Enzymology, Vol. 153, Chapters 23 to 34 (Editors, Wu and Grossman, Academic Press, 1987).
- Large scale culture of the BAL synthesizing host cells and the purification of the enzyme may form a cost effective commercial means of production of BAL or the C-tail. Methods are well known to those skilled in the art for the large scale production of enzymes.
- yeast and Fungi as host The principles for the expression of recombinant BAL in the yeast are similar to that for E. coli expression. Examples are provided by Bitter et al. Methods in Enzymology 153:516- 544 (1987). Like E. coli, yeast host cells may express a foreign gene either in the cytosol or, preferably in this case, as secreted protein.
- the secreted expression in yeast is capable of glycosylation.
- yeast expression of secretion proteins There are many vectors, promoters, and leaders available for yeast expression of secretion proteins.
- An excellent example is the system based on the control by alcohol oxidase promoter in methylotrophic yeast, Pichia pastoris.
- Vectors pHIL-Sl and pPIC9 are commercially available (Invitrogen) and can make large quantities of secretory eukaryotic recombinant proteins (1-4 mg per L of human serum albumin, Barr et al, Pharmaceutical Engineering 12:48-51 (1992); 12 mg per L of tetanus toxin fragment C, Clare et al, Bio/Technology 9:455-460 (1991).
- the plasmid YRp7 for example, (Stinchcomb et al, Nature 282:39 (1979); Kingsman et al, Gene 7:141 (1979); Techemper et al, Gene 10:157 (1980)) is commonly used.
- This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC no. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)).
- the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective means of selection by growth in the absence of tryptophan.
- Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerata kinase (Hitzeman et al, J. Adv. Enzyme Reg. 7:149 (1968); and Holland, Biochemistry 17:4900 (1978)), such, as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
- 3-phosphoglycerata kinase such, as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinas
- yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
- Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al. , European Patent Publication No. 73,657A.
- Yeast enhancers also are advantageously used with yeast promoters.
- Insect cells as host Baculovirus expression vectors for me synthesis of foreign genes in insect cells have been successfully used to express many mammalian and viral proteins as well as described for rat
- Mammalian cells as host Expression of many heterologous genes in mammalian cells has been successfully accomplished for commercial purposes. The commercial production of recombinant human tissue plasminogen activator is an example. Most of these expression vectors contains either mammalian promoter or viral promoters, polyadenylation signals, and appropriate regulatory elements for E. coli cloning, including antibiotic resistance genes. After the insertion of DNA encoding BAL or a C-tail protein downstream from the promoter, the vector can be first cloned in E. coli, isolated and transfected into mammalian cells. Neomycin or similar resistant selection markers can be either cotransfected into mammalian cells.
- Neomycin or similar resistant selection markers can be either cotransfected in another vector or in the same vector.
- a gene amplification system is advantageous.
- the transformant clones secreting BAL or C-tail protein can be identified by enzyme assays or by western blots.
- Successful examples of this approach with other mammalian proteins include the synthesis of glycosylated recombinant prorenin (Poorman et al, Proteins 1:139-145 (1986)) and human immune interferon (Scahill et al, Proc. Natl. Acad. Sc , U.S.A 80:4654-4658 (1983)).
- Preferred promoters for controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polymoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably
- viruses such as: polymoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably
- the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al, Nature 273:113 (1978)).
- the immediate early promoter of the human- cytomegalovirus is conveniently obtained as a HindTJI E restriction fragment (Greenaway et al, Gene 18:355-360 (1982)).
- promoters from the host cell or related species also are useful herein.
- Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp, that act on a promoter to increase its transcription initiation capability. Enhancers are relatively orientation and position independent having been found 5' (Laimins et al, Proc. Natl. Acad. Sci. USA 78:993 (1981)) and 3' (Lusky et al, Mol. Cell Bio.
- enhancer sequences are known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein and insulin). Typically, however, one will use an enhancer from an eukaryotic cell virus.
- Examples include the SV40 enhancer on the late side of the replication origin (nucleotides 100 to 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
- Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding BAL or C- tail protein. The 3' untranslated regions also include transcription termination sites.
- Expression vectors may contain a selection gene, also termed a selectable marker.
- selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase or neomycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure.
- DHFR dihydrofolate reductase
- thymidine kinase thymidine kinase
- neomycin neomycin.
- the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, Southern and Berg, Molec. Appl. Genet. 1:327 (1982), mycophenolic acid, Mulligan, and Berg, Science 209:1422 (1980) or hygromycin, Sugden et al, Mol. Cell. Biol. 5:410-413 (1985). The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.
- Amplification refers to the increase or replication of an isolated region within a cell's chromosomal DNA. Amplification is achieved using a selection agent, for example, methotraxate (MTX) which inactivates DHFR. Amplification or the making of successive copies of the DHFR gene results in greater amounts of DHFR being produced in the face of greater amounts of MTX. Amplification pressure is applied notwithstanding the presence of endogenous, DHFR, by adding ever greater amounts of MTX to the media. Amplification of a desired gene can be achieved by contransfecting a mammalian host cell with a plasmid having a DNA encoding a desired protein and the DHFR or amplification gene permitting cointegration.
- MTX methotraxate
- Preferred suitable host cells for expressing the disclosed vectors encoding BAL or C-tail protein in higher eukaryotes include: monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293, Graham et al, J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol Reprod.
- monkey kidney cells CVI ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); and, TRI cells (Mather et al, Annals N. Y. Acad. Sci. 383:44-68 (1982)).
- the cloned vector is then microinjected into a newly fertilized egg of cow or sheep and the egg transferred to a 'foster mother' for the fetal development and birth.
- the transgenic offsprings are analyzed for gene transfer by Southern blots and for the production of BAL or the C-tail protein in the milk. It is important to note that cows and sheep do not produce BAL in their milks.
- the transgenic animals can be interbred in order to produce a high yielding strain.
- the C-tail protein secreted in the milk would be fully glycosylated. Since cow and sheep do not make milk BAL, the recombinant product can be easily differentiated from the host milk proteins.
- Sugar or sugar analogue containing compounds can also be synthesized by chemical reactions to mimic the C-tail structures. These can be used to bind the intestinal surface and compete with endogenous BAL in the same manner as the C-tail itself.
- the following examples demonstrate that galactose and fucose can elute endogenous bound BAL from rat intestinal surface, indicating tiiat synthetic mimics of C-tail containing these sugars or their structural analogues can be used to affect the binding to intestinal surface. Since the C-tail contains repeating sequences and many glycosylation sites, the synthetic mimics can contain many sugar containing sites.
- the chemical linkages of sugars can be modeled based on the oligosaccharide structures of the C-tail, or the structural analogues of these oligosaccharide structures may contain essential features for effective binding to the intestinal BAL receptors.
- the sugars can be chemically attached to a polymer to create repeating units. Examples of suitable polymers include polypeptides, polyethylene glycol, dextran like sugar polymers and other synthetic polymers with appropriate functional groups for chemical linkage to sugars.
- BAL binds through the oligosaccharides of its C-tail to lectin-like receptors on intestinal surface. This binding is an essential step in mediating cholesterol uptake by the intestine. Further, it has been discovered that the isolated C-tail of BAL can compete with BAL for binding to the receptors on the intestinal surface. It has further been shown that C-tail can competitively inhibit the intestinal uptake of cholesterol by reducing the intestinal bound BAL. The reduction of cholesterol uptake by C-tail is specific, since BAL is the only enzyme in the intestine that can mediate cholesterol uptake. BAL also hydrolyses other fatty acid esters, and there fore can facilitate the uptake of other fats.
- C-tail proteins can be administered as a therapeutic agent to individuals in need of specific reduction of cholesterol uptake, and/or more general reduction in uptake of other fats, and thereby to treat hyperlipoproteinemia, hypercholesterolemia, and diseases associated with atherosclerosis.
- the C-tail protein is administered orally in an amount effective to reduce cholesterol intake from food as measured by a reduction in cholesterol levels in the blood.
- the dosage will vary depending on the formulation, me rate of excretion, individual variations such as the number of receptors on the intestinal surface, the cholesterol levels to be decreased, and me frequency of administration, as well as other factors routinely optimized by physicians.
- the cholesterol uptake from diet by the intestine is about 200 mg/day/person.
- Cholesterol synthesized by the body is about 500 mg/day/person.
- the pancreas secrets cholesterol at about 500 mg/day/person, which is reabsorbed through the intestine.
- the C- tail competition at the intestinal surface must be effective to reduce the amount of cholesterol from an uptake of about 700 mg cholesterol/day /person. If the reduction is to a level less than 500 mg/day, the body reduces body cholesterol.
- compositions containing C-tail protein designed to improve the pharmaceutical activity of the C-tail protein when administered to a patient in an amount effective to reduce cholesterol uptake in the intestine and thereby decrease blood cholesterol levels, can be prepared in combination with appropriate pharmaceutical stabilization compounds, delivery vehicles, carriers, inert diluents, and/or other additives appropriate for enteral (oral) administration according to methods well known in the art.
- the formulation usually provides for release within the stomach or the intestine.
- the C-tail protein can be formulated into a liquid, paste, suspension, gel, powders, tablets, capsules, food additives or other standard forms.
- Pharmaceutically compatible binding agents and/or adjuvant materials can be included as part of the composition.
- Examples include a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, PrimogenTM, or corn starch; a lubricant such as magnesium stearate or sterotes; aglidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and/or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
- a binder such as microcrystalline cellulose, gum tragacanth, or gelatin
- an excipient such as starch or lactose
- a disintegrating agent such as alginic acid, PrimogenTM, or corn starch
- a lubricant such as magnesium stearate or sterotes
- aglidant such as colloidal silicon dioxide
- a sweetening agent such as sucrose or saccharin
- a flavoring agent such as peppermint,
- the C-tail protein can be administered as a component of a fluid such as an elixir, suspension, beverage, liquid dietary supplement or substitute, or syrup; or of a solid such as a wafer or candy.
- the C-tail protein can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as other blood lipid-lowering pharmaceutical compositions.
- C-tail protein is encapsulated within carriers that effect release in the small intestine, such as microparticles, microcapsules, or microspheres prepared from synthetic or natural polymers such as proteins, polyhydroxy acids, or polysaccharides. Appropriate systems are known to those skilled in the art.
- microsphere formulations have been proposed as a means for oral drug delivery.
- Enteric coated formulations have been widely used for many years to protect drugs administered orally, as well as to delay release, other formulations designed to deliver compounds into the blood stream, as well as to protect the encapsulated drug, are formed of a hydrophobic protein, such as zein, as described in PCT/US90/06430 and PCT/US90/06433; "proteinoids", as described in U.S. Patent No. 4,976,968 to Steiner; or synthetic polymers, as described in European Patent application 0 333 523 by me UAB Research Foundation and Southern Research Institute.
- EPA 0 333 523 described microparticles of less than ten microns in diameter that contain antigens, for use in oral administration of vaccines. Larger sizes are preferred for the uses described herein to avoid uptake into the blood and lymph systems of the encapsulated C-tail protein.
- the microparticles can be formed of rapidly bioerodible polymers such as poly[lactide-co-glycolide], poly anhydrides, and polyorthoesters, whose carboxylic groups are exposed on the external surface as their smooth surface erodes; natural polymers such as proteins, like zein, modified zein, casein, gelatin, gluten, serum albumin, or collagen, and polysaccharides, like cellulose, dextrans, polyhyaluronic acid, polymers of acrylic and methacrylic esters and alginic acid; synthetic polymers such as polyphosphazines, poly (vinyl alcohols), polyamides, polycarbonates, polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and cop
- Representative polymers include methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly (methyl methacrylate), poly(ethyl methacrylate), poly (butyl methacrylate), poly ⁇ sobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly (methyl acrylate), poly(isopropyl aery late), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate),
- bioerodible polymers include polylactides, polyglycolides and copolymers thereof, poly(ethylene terephthalate), poly(butyric acid), poly (valeric acid), poly(lactide-co-caprolactone), poly[lactide-coglycolide], polyanhydrides, polyorthoesters, blends, and copolymers thereof.
- polymers can be obtained from sources such as Sigma Chemical Co., St. Louis, MO., Polysciences, Warrenton, PA, Aldrich, Milwaukee, WI, Fluka, Ronkonkoma, NY, and BioRad, Richmond, CA. or else synthesized from monomers obtained from these suppliers using standard techniques.
- compositions containing C-tail protein must be stable under the conditions of manufacture and storage and may be preserved against contamination by microorganisms, such as bacteria and fungi, through the use of antioxidants such as Vitamin E and ethoxyquin
- SUBST ⁇ UTESHEET (WILE26) and bacteriostatic agents, which are on the list of compounds approved for use by the Food and Drug Administration.
- inactivation and excretion rates of the C-tail protein will affect the amount of drug which is administered to a patient.
- the determinative criteria is whether the amount is effective to reduce the blood cholesterol. Blood cholesterol levels are assayed by standard techniques used by clinical laboratories.
- the C-tail protein can be used to reduce the uptake of cholesterol by the intestine and thereby for the treatment of hyperlipidemias including hypercholesterolemia, hypertriglyseridemia and associated disease states such as atherosclerosis, cardiovascular disease, and pancreatitis.
- the C- tail protein can also be used in normal subjects as a preventative measure to prevent the occurrence of these disorders. It is preferable that human serum cholesterol levels be maintained below 200 mg/dl, with values of 240 mg being considered clinically high and values of 160 mg being considered to be low.
- the formulation is administered as a single daily dose or divided daily doses, most preferably three doses given before, during, or after meals.
- Patients can be maintained on C-tail protein indefinitely to reduce the uptake of cholesterol by the intestine.
- Conditions to be considered in selecting dosage level, frequency, and duration primarily include the severity of the patient's disorder, the patient's serum cholesterol level, adverse side effects such as gastric distress, and the patient's need for preventive therapy, as well as the therapeutic efficacy.
- C-tail protein should be enough to cover 10 meters in length of the intestine for blocking the BAL binding sites.
- a five-fold excess (100 mg) should be more than sufficient for preventing cholesterol absorption by the intestinal cells. Further adjustment of the dosage will be based on the monitoring of the dosage response of patients to C-tail protein in lowering blood cholesterol.
- Example 1 Proline-rich Domain and Glycosylation are not Essential for the Enzymatic Activity of Bile Salt-activated Lipase.
- T- BAL truncated human milk bile salt-activated lipase
- PAN A p- nitrophenyl acetate
- PANB /J-nitrophenyl butyrate
- SDS sodium dodecyl sulfate
- T-BAL truncated form of bile salt-activated lipase
- PCR polymerase chain reaction
- TN 50 mM Tris-HCl, pH 8.0, and 100 mM NaCl
- EGTA ethylene glycol-bis( ⁇ -aminoethyl ether)N,N,N',N'- tetraacetic acid
- FPLC fast protein liquid chromatography
- BSA bovine serum albumin. Materials.
- the purification of BAL from human milk was performed as described in Baba et al. (1991), incorporated herein by reference.
- the ⁇ gtlO cDNA library from human lactating breast tissue was purchased from Clontech (Palo Alto, CA).
- Glycerol tri[9,10- 3 H]oleate was obtained from Amersham (Arlington Heights, II.).
- the oligonucleotides Primer I (Sequence ID No. 4) and Primer II (Sequence ID No. 5) were prepared by Dr. K. Jackson in me Molecular Biology Resource Facility, Oklahoma University Health Science Center. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Molecular Biology Methods.
- Double stranded DNA templates were first denatured as described by Kraft et al, BioTechniques 6:544-547 (1988). Then 2.5 ⁇ g of DNA, 15 ng of primer and 2 ⁇ l of reaction buffer were combined in a total volume of 10 ⁇ l and heated to 65 °C for 2 min, then cooled to 25 °C over a period 30 min to anneal the template. Labeling mixture was diluted 5-fold with water and Sequenase version 2. 0 was diluted 8-fold with water. 1 ⁇ l of DTT (0.1 M), 2 ⁇ l of diluted labeling mix, 5 ⁇ Ci of [cc 32 ?] dATP and 2 ⁇ l of diluted Sequenase were added to the annealed template-primer.
- PCR was performed as described by Innis and Gelfand, in PCR Protocols. A Guide to Methods and Application 3-12 (Innis et al. (Eds.), Academic Press, New York, N.Y. (1990)), incorporated herein by reference.
- the PCR generated DNA was subcloned into PCPJI using the TA cloning kit from Invitrogen (San Diego, Ca.) as described below. Ligation with the PCRII vector were set up as 1:1 and 1:3 molar ratio of vector:PCR product.
- the transformed cells were spread onto LB plates containing 50 ⁇ g/ml kanamycin and 1 mg of X-Gal. The plates were then inverted and incubated at 37 °C overnight for the selection of the transformed clones. Plasmid isolation was performed using the Magic MiniprepTM system supplied by Promega (Madison, WI). Three ml of overnight culture was centrifuged at 4000 rpm for 5 minutes to pellet the cells. The pellet was suspended in 200 ⁇ l of Cell Resuspension Solution. 200 ⁇ l of cell lysis solution was added and mixed by inverting the tubes until the suspension cleared. 200 ⁇ l of Neutralization Solution was added and mixed by inverting the tubes several times.
- a Minicolumn was attached to a syringe barrel and the assembly was inserted into a vacuum manifold.
- the resin/DNA mixture was transferred to the syringe barrel and poured into the column by applying vacuum.
- the column was washed with 2 ml of Column Wash Solution and the resin was dried.
- the minicolumn was centrifuged at 14000 rpm for 20 sec. to remove the remaining wash solution.
- the DNA was eluted from the column by applying 50 ⁇ l of 70°C water to the column then centrifuge the column after 1 minute for 20 seconds. Construction of Vector.
- the unique Smal restriction site of the overlapping clones G10-2 and G10-5 was utilized for construction of the cDNA G10-6 to encompass the entire coding sequence region of BAL.
- PCR was used for preparing T-BAL cDNA with the use of Primers I and II for expression of the truncated form of BAL.
- the T-BAL cDNA was ligated to the pETlla cloning vector (Invitrogen, San Diego, CA.) at the Ndel/BamHl cloning site for the subsequent expression of T-BAL using e T7-expression system in E. coli.
- T-BAL Using pETlla Vector.
- the general approach and methodology for using T7 polymerase to direct the expression of the cloned genes are described by Studier et al , Methods Enzymol. 185:60-89 (1990), incorporated herein by reference.
- the cDNA of T-BAL was ligated to the Ndel/BamHI cloning sites of pETlla (Novagen, Madison, WI.).
- the vector was used to transform the E. coli BL21 (DE3) for the expression of T-BAL.
- the cells harboring the vector were cultured overnight in ZB medium (Studier et al. (1990)) with ampicillin.
- the refolded T-BAL was then mixed with 60 ml of saturated ammonium sulfate (adjusted to pH 8.0 with NH 4 OH), which caused the precipitation of T-BAL.
- the T-BAL was collected from the precipitate after centrifugation at 18,000 rpm for 1 hour.
- the collected precipitate was solubilized with 40 ml of the refolding buffer and stirred at 40°C for 30 minutes.
- the supernatant fraction was then concentrated to 3 to 5 ml in an Amicon CentriprepTM-10 concentrator (Beverly, MA). After further ultracentrifugation at 105,000 x g for 30 minutes, the supernatant fraction (1 ml) was subjected to molecular sieving fractionation by fast protein liquid chromatography
- FPLC Fluorescence Chroxade
- a buffer solution containing 1 M urea, 0.15 M NaCl, 0.1 mM / 8-mercaptoethanol, and 50 mM Tris-HCl, pH 8.0.
- the column was eluted with a flow rate of 0.5 ml/minutes.
- the eluate was collected in 1 ml fractions and monitored by measuring absorbance at 280 ran and by assaying esterase activity with PANA as substrate.
- the enzyme assays were performed with 1 mM PANA as substrate and 2 mM taurocholate as activator.
- One unit of enzyme Activity was defined as one ⁇ mole of the product released per minute.
- T-BAL Thermostability of T-BAL.
- 0.1 mg/ml each of T-BAL and native BAL in sodium phosphate (0.15 M, pH 7.4) were incubated for 10 minutes in a water bath at 30°C, 40°C, 45 °C and 50°C for 10 minutes. After incubation, the solutions were cooled in ice-water. These treated samples were then assayed for remaining activity with PANA as substrate.
- the lipase assay of BAL was performed according to a modification of the method of Nilsson-Ehle and Schotz, J. Lipid Res. 17:536-541 (1976), incorporated herein by reference, using glycerol tri[9,10- 3 H]oleate as substrate.
- the two-fold concentrated stock substrate solution was prepared by emulsifying 28 ⁇ mol of trioleoylglycerol (specific activity 1.4 ⁇ Ci/ ⁇ mol) and 2.8 ⁇ mol of dioleoylphosphatidylcholine in 10 ml of 50 mM NH 4 OH-HCl buffer, pH 8.5.
- the mixture was emulsified using a W-380 sonicator (Heat Systems- Ultrasonics, Inc., Farmingdale, NY) at a setting of 5 (50% maximum output) for 30 seconds in an ice bath. After cooling, the mixture was further sonicated for an additional 30 seconds.
- the assay mixture with final volume of 100 ⁇ l contained 50 mM NH 4 OH-HCl buffer, pH 8.5, 1.4 mM trioleoylglycerol, 0.14 mM dioleoylphosphatidylglycerol, taurocholate, and 10 ⁇ l of the enzyme solution.
- the SDS-polyacrylamide gel electrophoresis was performed using the LKB-Pharmacia Phast system (Piscataway, NJ) and with a 8-25% polyacrylamide gel slab manufactured by Pharmacia (Piscataway, NJ). The samples were treated with 10 mM 3-mercaptoethanol and 2% SDS at 100°C for 6 minutes prior to electrophoresis. Protein Assay. The protein content of the enzyme preparation was determined by a modification (Wang and Smith, Anal. Biochem. 63:414-417 (1975),
- Fluorescence Measurement of the Interaction of T-BAL with Taurocholate Fluorescence measurements of the interaction of T-BAL with taurocholate were made at 25 °C with the aminco-Bowman Series 2 Fluorescence Spectrometer (Urbana, IL). The T-BAL tryptophanyl fluorescence was used for studying the interaction of T-BAL with taurocholate. Fluorescence was recorded at 340 nm with excitation wave length at 280 nm. The band-width of excitation and emission were both set a 2 nm. The sensitivity of the instrument was set at 1,000 volts of the detector high voltage.
- the amino acid sequence of native human milk BAL cDNA (Baba et al. (1991); Hui and Kissel, FEBS Lett. 276:131-134 (1990); Nilsson et al, Eur. J. Biochem. 192:543-550 (1990)) encodes for a 722-residue mature enzyme and is shown in Sequence ID No. 3.
- the cDNA structure is identical to that of the pancreatic BAL (Reue et al, J. Lipid Res. 32:267-27G (1991)), supporting the concept that the enzyme from mammary gland and from pancreas are expressed from the same gene.
- the T-BAL synthesized in E. coli is inactive and is insoluble in aqueous buffer.
- Active T-BAL was obtained by refolding. This procedure included the solubilization of the inclusion bodies with urea and 0-mercaptoethanol at a high pH (12.5), followed by dialysis against a low concentration of urea at lower pH (8.0). Based on the enzyme activity measurement, the yield of the active enzyme was about 10 mg per 4 liters of the cultured LB-medium. SDS-polyacrylamide gel electrophoresis gave rise to a major protein (greater than 90%) in the urea-soluble fraction corresponding to a molecular weight of 60,000, which approximated closely the expected molecular weight of T-BAL (59,270).
- N-terminal sequence analysis of this protein fraction gave the expected BAL N-terminal sequence of Ala-Lys-Leu-Gly-Ala-Val-Tyr-Thr- (Amino acids 1 to 8 of Sequence ID No. 1), indicating the successful synthesis of T-BAL and the removal of initiation methionine.
- the specific activity of T-BAL after the initial step of me refolding was about 5 to 10 units/mg, which is only 10 to 20% of that of the native BAL.
- the recombinant T-BAL expressed in E. coli is not glycosylated, indicating further that the highly glycosylated C-terminal region of BAL is not essential for catalytic function.
- T-BAL Purification of T-BAL was achieved by molecular sieving with FPLC after prior partial purification of the refolded T-BAL with ammonium sulfate precipitation. Two major peaks were found in the column fractions. The first peak, eluted at the void volume (17 ml), represents the major protein peak and contains mainly the aggregate form of the inactive T-BAL. The second peak (eluted at 28 ml) contains BAL activity. SDS-polyacrylamide gel patterns of fractions 27-29 indicate that T-BAL eluted in this peak was homogeneous. From four individual batches, an average specific activity of 64 2 units/mg for the purified enzyme (fraction 28) was obtained.
- T-BAL With me availability of a sufficient amount of purified recombinant T-BAL, the specificity and kinetics of T-BAL and the native BAL were compared and otiier characteristics were noted.
- T-BAL Thermostability of T-BAL. To compare the stability of T-BAL with the native enzyme, these two enzyme forms were treated at temperatures ranging for 30°C to 50°C. The heat inactivation patterns for T-BAL and native BAL were similar, with both showing a loss of about 90% of activity with treatment at 50°C for 10 minutes. This further suggests that the folding of T-BAL is similar to that of the catalytic domain of the native enzyme.
- the dissociation constants K A of T-BAL and native BAL are similar.
- native BAL upon binding with the bile salt showed about 20% decrease of me protein tryptophanyl fluorescence at a saturating concentration of taurocholate (Wang and Kloer (1983)), which probably resulted from a conformational change of BAL upon ligand
- T-BAL Similar to the native BAL, T-BAL was found to contain basal activity when assayed in the absence of bile salts with the esterase substrates. Therefore, taurocholate can also be considered as a non- essential activator of T-BAL. From the Lineweaver-Burke plots, the kinetic parameters K s , k ⁇ (for basal enzyme) and ⁇ K,, and /Sk a ,, (taurocholate-activated enzyme) for T-BAL and PANA and PANB as substrates were obtained.
- T-BAL a slightly higher specific activity of T-BAL (64 units/mg) than that of native enzyme (52 units/mg) (Wang and Johnson (1983)) was obtained, the derived of T- BAL was about 2 to 8 fold lower than that of the native enzyme.
- the deduced K, and ⁇ lC, of T-BAL were only slightly higher than that of me native enzyme.
- the presence of the proline-rich sequence plays a role mainly in enhancing the turnover rate of the enzyme, but has only a minor effect on the substrate binding affinity.
- there is a change of the preferential reactivity of the enzyme Previously it was reported that among the short chain acyl-esters of p— nitrophenol, native BAL has the higher with PANB.
- T-BAL has a higher with PANA than with PANB, when assayed in the presence of taurocholate.
- T-BAL also has higher substrate specificity constants of T-BAL with PANB. This is similar to what is found for the native enzyme.
- the activation effect of taurocholate on BAL-catalyzed hydrolytic reaction with PANA as substrate demonstrates that the proline-rich domain of BAL does not represent the bile salt-binding site of the enzyme. Lipase Activity of T-BAL.
- serum albumin is not a fatty acid acceptor for BAL catalysis, it is also a poor fatty acid acceptor in the BAL-catalyzed reaction; thus only taurocholate, and not BSA, was included in the lipase mixture as the fatty acid acceptor.
- T-BAL truncated form of recombinant BAL
- the iodination of human milk BAL and recombinant T-BAL was performed using iodo beads (Pierce, Rockford, IL). Iodine 121 was obtained from Amersham (Arlington Heights, IL). The iodination was performed by adding 6 ⁇ l of 125 I to 94 ⁇ l of the enzyme solution (2 mg/ml in sodium phosphate buffer, pH 6.5). One washed bead was placed in the solution and let stand at room temperature for 15 minutes. The mixture was then passed dirough a Bio-spinTM 6 column (Bio Rad, (Hercules, CA) and centrifuged at 2275 rpm for 4 minutes at 4°C and me eluate collected.
- Bio-spinTM 6 column Bio Rad, (Hercules, CA)
- the eluate was diluted wim 400 ⁇ l of 0.1 M sodium phosphate buffer, pH 6.5.
- the eluted fractions were combined and 1 ⁇ l of the eluate was examined for radioactivity.
- the lipase assay of BAL was performed as described in Example 1 using glycerol tri[9,10- 3 H]oleate as substrate.
- the two-fold concentrated stock substrate solution was prepared by emulsifying 28 ⁇ mol of trioleoylglycerol (specific activity 1.4 ⁇ Ci/ ⁇ mol) and 2.8 ⁇ mol of dioleoylphosphatidylcholine in 10 ml of 50 mM NH 4 OH-
- the assay mixture with final volume of 100 ⁇ l contained 50 mM NH 4 OH-HCl buffer, pH 8.5, 1.4 mM trioleoylglycerol, 0.14 mM dioleoylphosphatidylglycerol, 30 mM taurocholate, and 10 ⁇ l of the enzyme solution. Following a one hour incubation at 37 °C with agitation, the reaction was terminated by the addition of 3.2 ml of chloroform-heptanemethanol (5:4:5.6, vol/vol/vol) and 1 ml of 0.2 M
- the duodenum and jejunum were removed from mouse (about 20 grams each) small intestine and cut into 12-cm segments.
- Three experiments were performed in which T-BAL, without the C-tail, was used in parallel experiments as a control.
- the segments were washed once with 0.15 M NaCl and twice wim 0.1 M sodium phosphate buffer, pH 6.5.
- Twenty ⁇ l of the labeled samples were first mixed with 20 ⁇ l of 8 M urea and then diluted wim 720 ⁇ l distilled water and 240 ⁇ l 20% albumin with a final volume of 1 ml.
- Five hundred ⁇ l of the solution were then injected into each intestinal segment wim both ends ligated.
- the segments were incubated at room temperature for two hours. After incubation, the intestine was washed diree times with 0.1 M sodium phosphate buffer, pH 6.5, and 2 cm of the intestinal pieces were cut for counting the radioactivity.
- native BAL was retained by the intestinal mucosa in amounts nearly twelve times greater than T BAL, as shown in Table II and Table III. Since the native BAL and T-BAL differ only in C-tail, these results indicate that the attachment to intestinal surface is mediated by the glycosylated C-tail, possibly to a receptor on the intestinal surface.
- pancreatic BAL attaches to the surface of the intestine in an adult animal
- BAL enzymatic activity was measured in mouse intestine after the segments were thoroughly washed with physiological saline. The results demonstrated that me intestine possessed high BAL activity.
- Mouse pancreatic BAL has a C-terminal region containing four repeating motifs similar to that of the human enzyme. The results above confirm a similarity in the structure of the oligosaccharides, since human
- BAL binds to mouse intestine.
- Example 3 BAL elution from intestine.
- rat intestine was used to demonstrate the elution of BAL by various compounds.
- the initial experiment was to demonstrate that, like the observation done with mouse intestine, the rat endogeneous BAL can also be similarly eluted with galactose and fucose. Since calcium ion is required for the ligand binding of C-type lectin, EGTA elution was included for a test of calcium requirement for the BAL binding to intestinal surface.
- Table V shows the results of three experiments which indicate that bound rat intestinal BAL was eluted by galactose (0.1 M), fucose (0.1 M) and EGTA (1 mM).
- a cholesterol (oleate)ester emulsion solution was prepared as follows.
- a two-fold concentrated cholesterol oleate stock solution was prepared by emulsifying 6 ⁇ mol of [ 3 H] cholesterol oleate (1.6 ⁇ Ci/ ⁇ mol) (Amersham, Arlington Heights, IL) with 0.6 ⁇ mol of dioleoylphosphatidylcholine in 15 ml of isotonic phosphate buffer, pH 7.4.
- the mixture was emulsified using a W380 sonicator (Heat System- Ultrasonics, Inc., Farmingdale, NY) at a setting of 5 (50% maximum output) for 30 seconds in an ice bath. After cooling, the mixture was further sonicated for an additional 30 seconds.
- W380 sonicator Heat System- Ultrasonics, Inc., Farmingdale, NY
- experimental segments received me same isotonic buffer containing 1 mM EGTA.
- the intestinal segments were placed on a polystyrene weighing dish (14 cm 2 ) containing 5 ml of the same isotonic buffer and gently shaken for 30 minutes.
- the experimental segments were then washed once wim isotonic phosphate-EGTA solution and twice wim the isotonic buffer.
- the control intestine was washed three times with the isotonic buffer.
- s ⁇ s ⁇ nESHEEr nuu segment was removed and further cut into four segments of about 2.5 cm, each of which was placed into scintillation vials containing 1 ml of 2% sodium dodecylsulfate plus 8 M urea for the solubilization of the tissue. After the solubilization process, 10 ml of HydrocountTM, (J.T. Baker, Inc., Phillipsburg, NJ) was added to each vial, and the amount of radioactivity from 3 H-cholesterol in the intestinal segments was counted using a Beckman Scintillation Counter (Fullerton, CA) .
- SUBST ⁇ UTE SHEET 8ULE 26 0.6 ⁇ mol of dioleoylphosphatidylcholine in 15 ml of isotonic phosphate buffer, pH 7.4.
- the mixture was emulsified using a W-380 sonicator (Heat-System-Ultrasonics, Inc) at a setting of 5 (50% maximum output, for 30 s. in an ice bath. After cooling, the mixture was further sonicated for an additional 30 sec.
- Table VIII shows that the control (column- 1 presence of taurocholate and absence of heparin), the mean uptake is 20.65 nmol/g/h. In the presence of heparin (column 2) is 2.62 nmol/g/h. The difference is significant (p ⁇ .05). The value from heparin inhibition is near that of the background uptake in the absence of taurocholate.
- Heparin (ICN Chemicals, Costa Mesa, CA) concentration in the uptake solution was 10 mg/ml.
- N-BAL Native human milk bile salt-activated lipase
- a two-fold concentrated cholesterol oleate stock solution was prepared by emulsifying 6 ⁇ mol of 3 H-cholesterol (1.6 ⁇ Ci/ ⁇ mol) and 0.6 ⁇ mol of dioleoyl-phosphatidylcholine 4 in 15 ml of isotonic sodium
- SUBSTITUTE SKEH (RULE 26) phosphate buffer, pH 7.4.
- the mixture was emulsified using a W-380 sonicator (Heat-system Ultrasonics, Inc., Farmingdale, NY) at a setting of 5 (50% maximum output) for 30 seconds in an ice bath. After cooling, the mixture was further sonicated for an additional 30 seconds. Methods.
- the intestinal segments were washed once with the EGTA-containing isotonic buffer, and twice with isotonic buffer containing 0.2 mM calcium chloride.
- One of the segments was then injected with I ml of the purified N-BAL, and the second segment was injected with 1 ml of T-BAL, with the average enzyme concentration of 0.8 mg/ml, in isotonic phosphate buffer and in the presence of 0.2 mM Ca +2 .
- JN-OAL is native BA puntie ⁇ trom numan m ⁇ ; I -BAL IS recombinant truncated BAL (without C-tail).
- Example 4 Addition of C-tail to intestinal content releases bound endogenous BAL.
- C-tail can compete for binding to intestinal surface CT-receptors resulting in the displacement of bound endogenous BAL.
- (a) Preparation of C-tail A procedure has been devised to prepare pure C-tail of BAL from BAL. It should be noted that the purification of C-tail is very effective, so the starting BAL need not be completely homogeneous.
- BAL used in these C-tail isolation experiments was enriched by Heparin-SepharoseTM column (bed volume 14 x 1.5 cm) chromatography as described previously (Wang and Johnson (1983)).
- the fractions was assayed for me esterase activity of BAL using p- nitrophenyl acetate as substrate.
- HEET RULE 26 activity was pooled and dialyzed against distilled water and lyophilized. The yield was about 100 to 150 mg of dried materials from each batch.
- C-tail purification a pooling from three batches of the partially purified BAL (about 350 mg) was first treated with 8 M urea (10 mg/ml) for 2 h at room temperature and dialyzed against 50 mM Tris-HCl overnight.
- the denatured BAL was digested with trypsin and chymotrypsin (substrate: protease ratio of 50:1, w/w) for 4 h at 37°C.
- N-terminal sequence and amino acid composition analyses of the material from the carbohydrate containing FPLC fractions indicated tiiat the material corresponded to human BAL region containing residue of 528-712.
- Table X the amino acid composition of the purified C-tail and the composition based on me known sequence are very similar. From the / 3-elimination experiment using alkali for the release of the O-linked oligosaccharide and in the further amino acid composition analysis of the sample, it was determined tiiat between 8 to 10 residues of
- Table X The amino acid composition of isolated of me C-tail of human milk BAL and after treatment with alkali(0.1 N NaOH).
- C-tail causes the release of endogeneous BAL from rat intestine.
- SiiBS TE SHEET (RULE 26) Table XII: Release of endogeneous BAL from Rat intestine by EGTA and various doses of purified C-tail.
- Example 5 Competitive Inhibition of Cholesterol Uptake by the C-Tail of BAL.
- C-tail can elute the endogenously bound BAL from the rat intestinal surface.
- the following study demonstrates that the isolated C-tail can competitively inhibit cholesterol uptake in the rat intestine. a) In vitro Inhibition of cholesterol uptake by the C-tail of BAL.
- intestine segments were washed with isotonic phosphate buffer, pH 7.4.
- One of the intestine segment was incubated with the substrate [ 3 H] cholesterol oleate (0.2 mM) alone as control.
- the control intestine segment should have a higher rate of cholesterol uptake because the endogenous BAL on me intestinal surface is available for the transport of cholesterol.
- the second intestine segment was incubated with the same [ 3 H] cholesterol oleate and EGTA (1 mM). EGTA is known to elute endogenous BAL from the intestinal surface.
- the third intestine segment was incubated wim the same [ 3 H] cholesterol oleate and the isolated C-tail (0.2 mg/ml).
- BAL The scheme of BAL's physiological function in cholesterol uptake by intestine, shown in Figure 1, has been constructed based on data generated from in vitro experiments.
- BAL is first bound to the intestinal surface by putative receptors.
- the cholesterol or cholesterol esters is then transferred from hydrophobic food vesicles to the active site of the enzyme.
- Cholesterol ester is hydrolyzed in the BAL active site.
- the free cholesterol in the active site is transferred to the intestinal cells.
- This last step is assisted by d e cell surface heparin, which possibly serves as an orientational factor.
- the results described above show that BAL not bound to intestinal surface is ineffective in mediating me uptake of cholesterol.
- the scheme predicts that the feeding of isolated C-tail to live animals will compete for the putative receptor on the intestinal surface thus competitively inhibit cholesterol uptake. The following results confirm this prediction.
- both animals were tube fed with 1 ml of emulsified mixture containing 10 ⁇ Ci [ 3 H)-cholesterol oleate plus cold cholesterol oleate with a final concentration of 0.2 mM, 4 mg of isolated C-tail, 0.2 mM triolein and 0.02 mM dioleoylphosphatidylcholine.
- the emulsification procedure was performed as described in section e) of Example 3 above. Again, the control animal received the same mixture but without C-tail. Blood samples were taken at 1 hour intervals for 5 hours in short term studies.
- Figure 5 shows the results from a typical experiment of a single experimental rat and a control rat. Results have been obtained from a total of 6 control rats and 6 experimental rats.
- Table XIV shows the data from all 6 experiments and Figure 6 shows the average cholesterol uptake of control group and experiment group receiving C-tail. The inhibition of cholesterol uptake by the administration of C-tail was 35%, 48.8%, 55.7%, 62.2% and 55.5% respectively for measurements taken at 1, 2, 3, 4 and 5 hours after die administration of radiolabeled cholesterol.
- Table XTV the data between die control and experimental groups at 2, 3, 4 and 5 hour are statistically highly significant(p ⁇ 0.01).
- Table XIV Appearance of Orally Fed Radiolabelled, Cholesterol in the Blood of Rats wim and without Administration of C-tail Fragments.
- Example 6 BAL mediates uptake of triglycerides but not taurocholate by isolated rat intestinal tissue.
- BSTmirtSHEET(RULE26) used in place of cholesterol.
- the emulsions of [ 3 H] -trioleoylglycerol (Amersham, Arlington Heights, IL) were prepared similarly as described previously (Downs et al. (1994).
- the 2-fold- concentrated substrate solution was prepared by emulsifying 15 ⁇ mol dioleoylphosphatidylcholine in 7.5 ml of isotonic phosphate buffer, pH 7.4.
- the mixture was emulsified using a W-380 sonicator (Heat Systems- Ultrasonics, Inc.) at a setting of 5 (50% maximum output) for 30 s in an ice bath. After cooling the mixture was further sonicated for an additional 30 s. A volume of 1 ml of this emulsified solution was placed inside of isolated rat intestines for the measurement of uptake as described previously.
- a stock solution of taurocholate (6 mM) was made by including 2 ⁇ Ci of tauro(carbonyl- 14 )cholate and 30 ⁇ mol of taurocholate (Sigma) in 5 ml of isotonic phosphate buffer. Results.
- Table XVI Taurocholate Uptake by Rat Intestine.
- compositions including all or a portion of the carboxy terminal (C tail) region of bile salt-activated lipase (BAL), or functional equivalents thereof, (C-tail peptides) are described, which, in the intestine, compete with native BAL in binding to the intestinal surface, and which are conjugated to a biologically active composition.
- BAL C-tail molecules are attached to a substance to be delivered thus enabling the substance to be delivered specifically to the intestine upon oral administration of the conjugate.
- these compositions bind to the intestinal surface resulting in delivery and/or long-term presence of the therapeutic compound at the intestinal lining.
- the C tails can be modified by covalent attachment of a bioactive agent to a carboxylic group or amino group on the C tail.
- the C tails can be modified using any of a number of different coupling chemistries that covalently attach ligands to C tails.
- One useful protocol involves the "activation" of hydroxyl groups on the C tail carbonyldiimidazole (CDI) in aprotic solvents such as DMSO, acetone, or THF.
- CDI forms an imidazolyl carbamate complex with the hydroxyl group which may be displaced by binding the free amino group of a bioactive ligand such as a protein.
- the reaction is an N-nucleophilic substitution and results in a stable N-alkylcarbamate linkage of the ligand to the C tail.
- the resulting ligand-C tail complex is stable and resists hydrolysis for extended periods of time.
- Another coupling method involves the use of l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (ED AC) or "water-soluble CDI" in conjunction with N-hydroxylsulfosuccinimide (sulfo NHS) to couple the exposed carboxylic groups of the C tail to the free amino groups of bioactive ligands.
- ED AC and sulfo-NHS form an activated ester witii the carboxylic acid groups of the C tail which react witii the amine end of a ligand to form a C tail bond.
- the resulting peptide bond is resistant to hydrolysis.
- a useful coupling procedure for attaching ligands with free hydroxyl and carboxyl groups to the C tail involves the use of the cross- linking agent, divinylsulfone. This method is useful for attaching sugars or other hydroxylic compounds to hydroxyl groups on the C tail.
- the activation involves the reaction of divinylsulfone witii the hydroxyl groups of the C tail to a vinylsulfonyl ethyl ether.
- the vinyl groups will couple to alcohols, phenols and amines.
- Activation and coupling take place at pH 11.
- the linkage is stable in the pH range from 1-8 and is suitable for transit through the intestine.
- the therapeutic compound can be covalently coupled to C-tail protein either directly or indirectly using a linker molecule.
- Linker molecules will typically be used when additional flexibility or space is needed between the C-tail protein and the therapeutic compound.
- Any suitable molecule that can be coupled to both C-tail protein and a therapeutic compound can be used as a linked.
- Exemplary linkers are peptides or molecules with straight carbon chains. Because the C-tail composition will be used in the intestine, the bond or linker coupling the C-tail protein and the therapeutic compound must be stable in the intestinal environment.
- bioactive agent can be attached to the C-tail using standard techniques.
- the resulting conjugate of the C-tail and the bioactive agent is referred to herein as a C-tail composition or a C-tail- drug conjugate.
- the C-tail fragments may be attached to any biologically active agent.
- biologically active material refers to a protein, carbohydrate, nucleic acid, lipid, organic compound such as a drug, or a combinations thereof, that causes a biological effect when administered in vivo to an animal including humans.
- Nonlimiting examples are antigens, enzymes, hormones, receptors, peptides, proteins, polysaccarides, nucleic acids, nucleosides, nucleotides, liposomes, vitamins, minerals, inorganic compounds and viruses.
- the C- tail also can be used to deliver procaryotic and eucaryotic cells, e.g., bacteria, yeast, and mammalian cells, including human cells, and components thereof, such as cell walls, and conjugates of cellular components.
- useful proteins include hormones such as insulin, growth hormones including somatometins, transforming growth factors,
- RULE26 and other growth factors, antigens for oral vaccines, enzymes such as lactase or lipases, and digestive aids such as pancreatin.
- useful drugs include ulcer treatments such as CarafateTM from Marion Pharmaceuticals, neurotransmitters such as L- DOPA, antihypertensives or saluretics such as Metolazone from Searle Pharmaceuticals, carbonic anhydrase inhibitors such as Acetazolamide from Lederle Pharmaceuticals, insulin like drugs such as glyburide, a blood glucose lowering drug of the sulfonylurea class, synthetic hormones such as Android F from Brown Pharmaceuticals and Testred (metiiyltestosterone) from ICN Pharmaceuticals, and antiparasitics such as mebendzole (VermoxTM, Jannsen Pharmaceutical).
- ulcer treatments such as CarafateTM from Marion Pharmaceuticals
- neurotransmitters such as L- DOPA
- antihypertensives or saluretics such as Metolazone from Searle Pharmaceuticals
- carbonic anhydrase inhibitors such as Acetazolamide from Lederle Pharmaceuticals
- insulin like drugs such as glyburide
- the C-tail drug conjugates are especially useful for treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.
- ulcerative colitis inflammation is restricted to the colon
- Crohn's disease inflammatory lesions may be found throughout the gastrointestinal tract, from the mouth to the rectum.
- Sulfasalazine is one of the drugs that is used for treatment of the above diseases.
- Sulfasalazine is cleaved by bacteria within the colon to sulfapyridine, an antibiotic, and to 5-amino salicylic acid, an anti- infiammatory agent.
- the 5-amino salicylic acid is the active drug and it ⁇ is needed locally.
- Direct administration of the degradation product (5- amino salicylic acid) may be more beneficial.
- a protein-drug delivery system could improve the therapy by retaining the drug for a prolonged time in the intestinal tract.
- retention of 5- aminosalicylic acid in the upper intestine is of great importance, since bacteria cleave the sulfasalazin in the colon, the only way to treat inflammations in the upper intestine is by local administration of 5- aminosalicylic acid.
- Antigens can be attached to the peptide to provide a vaccine.
- the vaccines can be produced to have different retention times in the gastrointestinal tract depending on the strength of the covalent bond binding the peptide to me vaccine.
- the different retention times can stimulate production of more than one type (IgG, IgM, IgA, IgE, etc.) of antibody.
- antigen includes any chemical structure that stimulates the formation of antibody or elicits a cell-mediated response, including but not limited to protein, polysaccharide, nucleoprotein, lipoprotein, synthetic polypeptide, or a small molecule linked to a protein.
- Specific antigens that can be attached to the peptide include attenuated or killed viruses, toxoids, polysaccharides, cell wall and surface or coat proteins of viruses and bacteria. These can also be used in combination with conjugates, adjuvants, or other antigens.
- Hemophilus influenzae in the form of purified capsular polysaccharide (Hib) can be used alone or as conjugate with diphtheria toxoid.
- organisms from which these antigens are derived include poliovirus, rotavirus, hepatitis A, B, and C, influenza, rabies, HIV, measles, mumps, rubella, Bordetella pertussus, Streptococcus pneumoniae, Diphtheria,
- Tetanus Cholera, Salmonella, Neisseria, Shigella, and Enterotoxigenic E. coli.
- C tail can also be used to deliver water soluble or water insoluble drugs such as nonsteroidal antiinflammatory compounds, anesthetics, chemotiierapeutic agents, immunosuppressive agents, steroids, antibiotics, antivirals, antifungals, steroidal antiinflammatories, and anticoagulants.
- Imaging agents also may be attached to C-tail, including metals, radioactive isotopes, radioopaque agents, fluorescent dyes, and radiolucent agents. Radioisotopes and radioopaque agents include gallium, technetium, indium, strontium, iodine, barium, and phosphorus.
- the therapeutic compound (i.e., the biologically active agent) attached to C-tail can be any compound that will have a useful effect when delivered to the intestinal lining.
- the therapeutic compound can act to either reduce or enhance the uptake of a compound ingested by die individual, or to break down harmful compounds.
- the therapeutic compound can be an enzyme, non-enzymatic binding molecule, or a ligand. An enzyme that catabolizes an undesirable
- 1BSTITUTESHEET(R1JI£26) compound ingested by the individual, or an antibody or receptor specific for such a compound, would be useful a the therapeutic compound.
- the therapeutic compound must be stable an active in the intestinal environment.
- Preferred therapeutic compounds are catabolic enzymes, catalyzing the breakdown of specific molecules, especially enzymes with activities not normally present in the intestines.
- a preferred therapeutic compound of this type is lactase.
- a C-tail/lactase composition can be used to deliver lactase to the intestinal lining of individuals who lack lactase or have a diminished lactase activity.
- the C-tail-drug conjugate is administered orally in an amount effective for a particular therapeutic application.
- the dosage will vary depending on the formulation, the rate of excretion, individual variations such as the number of receptors on the intestinal surface, the type of therapy, and the frequency of admin- istration, as well as other factors routinely optimized by physicians.
- the BAL C-tail composition is administered orally in an amount effective to reduce or enhance uptake of certain compounds from food, or lower the intestinal concentration of an undesirable compound.
- NAME Pabst, Patrea L.
- MOLECULE TYPE protein
- HYPOTHETICAL NO
- Trp lie Tyr Gly Gly Ala Phe Leu Met-Gly 100 105 110
- Ala Asn Ala Ala Asp lie Asp Tyr lie Ala Gly Thr Asn Asn Met Asp 305 310 315 320
- MOLECULE TYPE protein
- HYPOTHETICAL NO
- TTCATACATC TTTGTTGGAT TTCCTGTGTA CTTGGTCTTT GTTTTCTCCT CGATGTACAT 180
- CTGAAGGCCA AGAACTTCAA GAAGAGATGC CTGCAGGCCA CCATCACCCA GGACAGCACC 960
- CAGGAGGCCA CCCCTGTGCC CCCCACAGGG GACTCCGAGG CCACTCCCGT GCCCCCCACG 2400
- GTGCCGCCCA CGGGTGACTC CGGGGCCCCC CCCGTGCCGC CCACGGGTGA CTCCGGGGCC 2580
- MOLECULE TYPE protein
- HYPOTHETICAL NO
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Obesity (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Diabetes (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Optics & Photonics (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
- Peptides Or Proteins (AREA)
- Steroid Compounds (AREA)
Abstract
Compositions dérivées de la région intégrale ou partielle de l'extrémité carboxylique de la lipase activée par les sels biliaires humains (BAL). Lorsqu'elles sont administrées par voie orale, ces compositions concurrencent la BAL à l'état natif en se liant à la paroi de l'intestin, ce qui réduit le rôle physiologique de la BAL dans la médiation du transport de cholestérol dans les cellules intestinales, d'où une réduction de la quantité de cholestérol absorbée par l'intestin et introduite dans le courant sanguin. Les dérivés utiles de la région de l'extrémité carboxylique de la BAL, sont dérivés de la région intégrale ou partielle contenant les restes d'acide aminé en position 539 à 722 et ont une structure semblable à celle de la mucine et contiennent au moins trois des unités récurrentes riches en proline comportant chacune onze restes d'acide aminé.
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US479160 | 1983-03-28 | ||
US347718 | 1994-12-01 | ||
US08/347,718 US5696087A (en) | 1994-12-01 | 1994-12-01 | Method and compositions for reducing cholesterol absorption |
US08/482,262 US5821226A (en) | 1994-12-01 | 1995-06-07 | BAL C-tail drug delivery molecules |
US08/479,160 US5681819A (en) | 1994-12-01 | 1995-06-07 | Method and compositions for reducing cholesterol absorption |
US482262 | 1995-06-07 | ||
PCT/US1995/015647 WO1996017054A1 (fr) | 1994-12-01 | 1995-12-01 | Technique et compositions permettant de reduire l'absorption de cholesterol |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0795011A1 true EP0795011A1 (fr) | 1997-09-17 |
Family
ID=27407792
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP95943643A Withdrawn EP0795011A1 (fr) | 1994-12-01 | 1995-12-01 | Technique et compositions permettant de reduire l'absorption de cholesterol |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0795011A1 (fr) |
JP (1) | JP3007161B2 (fr) |
CA (1) | CA2206526A1 (fr) |
WO (1) | WO1996017054A1 (fr) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5821226A (en) * | 1994-12-01 | 1998-10-13 | Oklahoma Medical Research Foundation | BAL C-tail drug delivery molecules |
US5681819A (en) * | 1994-12-01 | 1997-10-28 | Oklahoma Medical Research Foundation | Method and compositions for reducing cholesterol absorption |
AU2001255501A1 (en) * | 2000-04-21 | 2001-11-07 | Monsanto Technology Llc | Blood-pressure reducing polypeptides containing vpp derived from microorganisms |
AU2001255469A1 (en) * | 2000-04-21 | 2001-11-07 | Monsanto Technology Llc | Purification of ace inhibiting polypeptides containing vpp from milk |
US7267950B2 (en) * | 2003-05-01 | 2007-09-11 | Veridex, Lcc | Rapid extraction of RNA from cells and tissues |
JP6310780B2 (ja) * | 2013-06-12 | 2018-04-11 | 第一三共ヘルスケア株式会社 | 経口用組成物 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IS4130A (is) * | 1993-03-01 | 1994-09-02 | Ab Astra | Ný fjölpeptíð |
-
1995
- 1995-12-01 JP JP8519095A patent/JP3007161B2/ja not_active Expired - Fee Related
- 1995-12-01 CA CA002206526A patent/CA2206526A1/fr not_active Abandoned
- 1995-12-01 WO PCT/US1995/015647 patent/WO1996017054A1/fr not_active Application Discontinuation
- 1995-12-01 EP EP95943643A patent/EP0795011A1/fr not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of WO9617054A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1996017054A1 (fr) | 1996-06-06 |
JP3007161B2 (ja) | 2000-02-07 |
AU707558B2 (en) | 1999-07-15 |
JPH10510166A (ja) | 1998-10-06 |
CA2206526A1 (fr) | 1996-06-06 |
AU4506496A (en) | 1996-06-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Whitcomb et al. | Human pancreatic digestive enzymes | |
JP4503708B2 (ja) | ヒト血漿ヒアルロニダーゼ | |
US7838274B2 (en) | Phospholipases and uses thereof | |
Kitabchi et al. | Degradation of insulin and proinsulin by various organ homogenates of rat | |
US6864064B2 (en) | 18891, a novel human lipase | |
JP2008546396A (ja) | 薬学的に使用するためのアミラーゼ | |
JP2008500055A (ja) | 医薬用途のための酵素 | |
Osorio et al. | Targeted modification of wheat grain protein to reduce the content of celiac causing epitopes | |
De Caro et al. | Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice | |
JP3837444B2 (ja) | 胆汁酸塩刺激リパーゼの変種、それらをコードするdna分子およびトランスジェニック非ヒト哺乳動物 | |
JPH08508399A (ja) | ホスホリパーゼ及びヒアルロニダーゼのようなスズメバチ毒酵素のクローニング及び組換え体の生産並びにそれに基づく免疫学的治療 | |
US5681819A (en) | Method and compositions for reducing cholesterol absorption | |
EP0795011A1 (fr) | Technique et compositions permettant de reduire l'absorption de cholesterol | |
US6432400B1 (en) | Specific pancreatic lipase inhibitors and their applications | |
US5696087A (en) | Method and compositions for reducing cholesterol absorption | |
AU707558C (en) | Method and compositions for reducing cholesterol absorption | |
US5821226A (en) | BAL C-tail drug delivery molecules | |
KR20170105104A (ko) | 소화 촉진을 위한 약제로서 광범위한 pH 활성 범위를 갖는 효소의 용도 | |
Dolphin | Lipolytic enzymes and the role of apolipoproteins in the regulation of their activity | |
EP1261719B1 (fr) | 27411, phosphatidyl-glycerolphosphate (pgp) synthase humaine | |
US6652851B1 (en) | Cloning and recombinant production of polistinae venom enzymes, such as phospholipase and hyaluronidase, and immunological therapies based thereon | |
Osorio | Expression of glutenases in wheat (Triticum aestivum L.) endosperm and increase in the thermostability of prolyl endopeptidase from Flavobacterium meningosepticum as an alternative therapy for celiac patients | |
Mas et al. | [20] Glycosylation of bile salt-dependent lipase (Cholesterol esterase) | |
US20070092503A1 (en) | Human alkaline sphingomyelinase and use thereof | |
Dupuis et al. | Preduodenal Lipases and Their Role in Lipid Digestion |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 19970701 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE |
|
17Q | First examination report despatched |
Effective date: 20010730 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20021001 |