EP1257592A1 - Utilisation de la conformation d'une proteine pour la protection et la liberation de composes chimiques - Google Patents

Utilisation de la conformation d'une proteine pour la protection et la liberation de composes chimiques

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Publication number
EP1257592A1
EP1257592A1 EP00916076A EP00916076A EP1257592A1 EP 1257592 A1 EP1257592 A1 EP 1257592A1 EP 00916076 A EP00916076 A EP 00916076A EP 00916076 A EP00916076 A EP 00916076A EP 1257592 A1 EP1257592 A1 EP 1257592A1
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European Patent Office
Prior art keywords
polypeptide
glu
composition
polymer
nca
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EP00916076A
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German (de)
English (en)
Inventor
Keith R. Latham
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Innovative Technologies LLC
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Innovative Technologies LLC
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/20Pills, tablets, discs, rods
    • A61K9/2004Excipients; Inactive ingredients
    • A61K9/2022Organic macromolecular compounds
    • A61K9/2031Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyethylene oxide, poloxamers
    • A61K9/2045Polyamides; Polyaminoacids, e.g. polylysine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/14Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16Anti-Parkinson drugs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10Alpha-amino-carboxylic acids

Definitions

  • the present invention is directed to the preparation of synthetic amino acid polymers and is directed to the use of these polymers to protect chemical compounds, especially drug substances, from degradation, and to release the compounds under specific conditions.
  • proteins polymer chains of peptide-linked amino acids
  • Natural proteins are peptide-linked polymers containing 20 different amino acids, each with a different side-chain. The details of the folding into higher order structures are dependent on the type, frequency and primary sequence of the amino acids in the protein. Since each position in the polymer chain can be occupied by 20 different amino acids, the thermodynamic rules that describe the details of protein folding are complex.
  • a highly preferred conformation found in many natural proteins is the 3.613 alpha-helix.
  • This right-handed helix contains 3.6 amino acids per turn and is stabilized by hydrogen bonding (about 3 kcal/mol) involving the amide hydrogen and a carbonyl oxygen, separated by 13 atoms along the backbone of the polymer chain. Since the amino acid side chains in the alpha-helix point away from and perpendicular to the helix axis, any of the amino acids (except Pro) can participate in the helix. Other structures can also appear in higher order protein conformations, including the 3 helix, and the important left-handed, three residue helix found in collagen and pleated sheets.
  • Tyrosine is frequently found internalized, with its 4- hydroxy hydrogen, hydrogen-bonded to another amino acid or potential ligand/enzyme substrate. Thus, Tyr can be utilized to produce hydrophobic pockets with a potential for hydrogen bonding.
  • Proline Pro
  • Pro has been found to be sufficient, but not always necessary, for a sharp turn in the peptide chain, allowing for cooperative interactions of different sections of the same polymer. At higher polymer concentrations, Pro can also disrupt helical structure, producing a "less organized" protein.
  • Cystine can be utilized to stabilize higher order structures by linking polymer chains through high energy (about 50 kcal/mol) disulfide bonds.
  • block polymers comprised of a limited sub-set of amino acids, would exhibit intrinsic conformational structures that could be predicted, based on an analysis of statistical distributions and ratios of the amino acids in the polymer. Moreover, it was also not realized that these synthetic proteins would have utility in protecting and releasing sensitive chemical compounds.
  • the present invention describes a use of a limited but sufficient sub-set of amino acids, to produce prototype synthetic proteins that reproduce certain conformational aspects of natural proteins.
  • the present invention describes the use and combination of only seven amino acids, each with a specific function in the resulting synthetic polypeptide: Glutamic Acid (Glu), Lysine (Lys), Phenylalanine (Phe), Proline (Pro), Tryptophan (Trp), Tyrosine (Tyr) and Cysteine (Cys). Cys is used herein as the disulfide Bis-dimer (CysS-SCys), referred to as cystine by convention. Block polymers of this amino acid subset are used to produce synthetic proteins with predictable conformations and utility.
  • the subject of this invention is the utilization of the ability of amino acid polymers (polypeptides) to form higher order structures. These structures can bind to and protect chemical entities (e.g. drugs) from chemical and enzymatic degradation and provide a mechanism for controlled release of such entities.
  • Synthetic polypeptides are described that are composed of carefully selected combinations and ratios of amino acids, including a hydrophilic/polar component (like Glu or Lys), a hydrophobic component (like Tyr, Phe or Benzyl Glu), and are designed to promote the formation of internalized domains, to accommodate chemical entities like drugs.
  • the method relates to the protection of a chemical compound from degradation comprising combining the chemical compound with a synthetic protein which may be a homo-polymer, containing for example Glu or Lys, or may be a co-polymer with an amino acid having hydrophobic character, contributes a hydrogen bonding capacity, or stabilizes higher order structures.
  • a synthetic protein which may be a homo-polymer, containing for example Glu or Lys, or may be a co-polymer with an amino acid having hydrophobic character, contributes a hydrogen bonding capacity, or stabilizes higher order structures.
  • the invention in another embodiment, relates to cell culture media comprising a synthetic polypeptide containing Gin that is co-polymerized with an amino acid, like Glu.
  • the polymer provides a chemically stable nutritional source of Gin in the culture.
  • a related embodiment utilizes a Gin containing synthetic protein as a nutritional source of Gin in humans.
  • the invention in another embodiment, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an active ingredient that has been combined with a synthetic amino acid polymer.
  • the synthetic protein may be a homo-polymer of Glu or Lys, for example, or may be a co-polymer containing Glu or Lys and Tyr, Phe or Benzyl Glu.
  • the active ingredient of such pharmaceutical compositions is L-DOPA, aspirin, hydrocortisone, or estrogen.
  • the protein/active ingredient combination may also be combined with other pharmaceutically acceptable excipients to aid in tablet formation and properties, for example.
  • the invention relates to a method of controlling the release of a chemical compound based on response to changes in pH.
  • This embodiment is comprised of manipulating the higher order structure of a synthetic protein by choice of amino acid composition and combining said chemical compound with the protein.
  • the invention relates to the release of chemical compounds by regulating the rate of proteolytic digestion through the manipulation of higher order structures of a synthetic protein by choice of amino acid composition, and combining said chemical compounds with the protein.
  • the invention relates to the release of chemical compounds by regulating thermal diffusion of said compounds from a synthetic protein. Regulation of diffusion rate occurs by manipulating the higher order structure of the synthetic protein, by choice of amino acid composition, and combining said chemical with the protein.
  • a parameter that is qualified by “about” may vary ⁇ 10 % from the stated value. That is, “about 50 °C” means 45-55 °C. Further, unless otherwise indicated, all amino acids are in the L-form.
  • triphosgene is a preferred reagent for amino acid NCA formation. Additional advantages to the Fuchs-Farthing chemistry are that the NCA's are generally easy to purify in crystalline form, negligible racemization occurs at the alpha carbon and the polymerization reaction yields only the protein polymer and non-toxic carbon dioxide.
  • the polymerization reaction normally contains an amino acid NCA (for homopolymers) or mixture of NCA's, for the synthesis of co- or heteropolymers, together with a polymerization initiator, all dissolved in a compatible solvent system. Highly preferred for the polymerization is a non-protic organic solvent that has high solubility for the NCA and the polymer.
  • the preferred solvents include ethyl acetate, THF, benzene, dichloromethane, DMF and dioxane. Most preferred solvents include THF dioxane and DMF.
  • solubility of the polymer in the polymerization solvent is important, since early precipitates of polymer, prior to complete use of all the NCA monomers, may favor the appearance of one amino acid over another, in the first precipitates.
  • the initiator of the polymerization reaction can be water, a base (organic or inorganic) or a preformed amino acid polymer.
  • the average number of amino acid residues in the final polymer product is a direct result of the molar ratio of the monomer NCA's to the initiator. Since initiation may not behave ideally, through, for example, partitioning of the initiator into non-polymerizing compartments in the reaction, care must also be taken to use an initiator that is highly soluble in the reaction solvent.
  • a tertiary amine like triethyamine or tert-butylamine is used, since primary and secondary amines may stay covalently attached to the polymer, forming stable end-labeled polymer products.
  • the preferred average number of residues (N) in the polymer chains is between 5 and 400.
  • N The preferred average number of residues (N) in the polymer chains is between 5 and 400.
  • an average N between 10 and 400 is most preferred.
  • a useful embodiment of the invention is the formulation of tablets or liquids intended for oral delivery of an active drug substance.
  • Standard formulations of drugs may have limited shelf-life (e.g. from oxidation) or be inactivated by the acidic conditions of the stomach. In this case, a drug delivery mechanism that circumvents the stomach would be desirable. Alternatively, rapid release of a drug substance in the stomach may be preferred.
  • chemical compounds may be formulated as defined herein. "Macroformulation”: blending of a powdered chemical compound and a powdered synthetic polypeptide prior to formulation and tableting.
  • Microformulation incorporation of a chemical compound into a synthetic polypeptide, for example by inclusion into hydrophobic pockets, prior to formulation and tableting.
  • Covalent formulation incorporation of a chemical compound by peptide linkages into a synthetic polypeptide prior to formulation and tableting.
  • a focus of the present invention makes use of a dramatic effect of pH on secondary and tertiary structures of synthetic protein polymers containing an ionizable R group (e.g. Glu, Asp, Lys, Arg).
  • an ionizable R group e.g. Glu, Asp, Lys, Arg.
  • the ionizable portion makes a transition from uncharged to highly charged.
  • higher order polymeric structures like alpha helices, are rapidly converted to "random coils.
  • Random coils are highly flexible and dynamic; this form promotes drug release and enhanced proteolytic cleavage by digestive enzymes.
  • PGlu polyglutamic acid
  • the pH of the intestinal contents increases to about 6.5 and the carboxyl groups become de-protonated and highly charged.
  • the closely spaced, highly charged carboxyl groups repel each other strongly enough to overcome intrachain bonds (e.g. hydrogen bonds) responsible for higher order structure of the PGlu.
  • the drug then diffuses from the loose, random coils of the polymer.
  • the enhanced digestability of the random coil structure also aids drug release. Through this mechanism, the drug is released preferentially in the small intestine.
  • a drug intended for oral delivery is "macro-formulated" with polymeric lysine (PLys) by blending and tableting (e.g. by direct compression).
  • PLAs polymeric lysine
  • the omega amino group of Lys has a pK / of about 10.0. Once entering the stomach, the omega amino group becomes fully protonated and highly charged. The closely spaced amino groups repel each other, releasing the drug substance by diffusion and enhanced digestability of the random coil structure.
  • a further enhancement of drug stability and controlled release properties, especially in the digestive system, can be realized by incorporation of a hydrophobic amino acid, like Phe, to form a synthetic co-polymer.
  • a synthetic co-polymer containing Glu and Phe, in a preferred ratio is, "macro- formulated" with a hydrophobic drug substance. Stability of the drug in the compressed tablet is again enhanced by sequestration from water and oxygen. Release of the drug in stomach is slow. However, once in the small intestine, the PGlu/Phe becomes less organized due to pH/charge effects and there is an initial release of drug substance accompanied by re-partitioning of the drug into hydrophobic domains in the polymer. Finally, terminal digestive proteolysis releases the entire store of drug.
  • PLys/Phe can be used for release of a drug substance in the stomach, except that drug release and digestion of the polymer are enhanced in the stomach, and the drug release profile is blunted by successive re-partitioning of the drug into the hydrophobic domain of the polymer. Finally, digestive proteolysis destroys even the hydrophobic pockets, releasing all the drug.
  • a further enhancement of drug stability is accomplished by microformulation involving inclusion of the drug, into the internal matrix of the synthetic protein prior to tableting and oral administration.
  • a hydrophobic drug substance is combined in solution with a co-polymer of glutamic acid and phenylalanine (PGlu/Phe) at a pH that favors the random coil form of the polymer (pH > 4).
  • PGlu/Phe co-polymer of glutamic acid and phenylalanine
  • the solution is slowly acidified to promote the formation of higher order structures in the polymer, with attendant formation of internalized hydrophobic domains containing the "dissolved" hydrophobic drug substance.
  • the PGlu/Phe - Drug Substance combination precipitates at lower pH; precipitation can be enhanced by the addition of an organic solvent like acetone.
  • the vacuum or freeze dried product is especially stable since the drug substance is partitioned into anhydrous, hydrophobic domains inside the protein structure.
  • a similar process of drag inclusion applies to PLys/Phe except that higher order structures, like alpha helices, occur above pH 10 for this polymer.
  • the polymer/drug combination is adjusted to pH > l l in solution and freeze dried. Again, the product is especially stable due to partitioning of the drug into hydrophobic domains inside the protein.
  • the polymer/drug complexes can be formulated with other excipients that may facilitate tableting.
  • Gin is an essential amino acid for most mammalian cells and is therefore an important nutritional component, for example in cell culture.
  • monomeric Gin is chemically unstable, degrading to ammonia and pyrrolidonecarboxylic acid, under physiologic conditions. Gin is chemically stabilized as a co-polymer with Glu by incorporation into the structure of the polymer.
  • Gin release is regulated by normal proteolytic digestion of the polymer.
  • Gin is released for metabolic use by cultured cells by slow extracellular hydrolysis of the synthetic protein in the culture media or by pinocytotic mechanisms in which the synthetic protein is internalized by the cultured cells and digested by lysozomes to become a metabolic source of Gin.
  • hydrophobic amino acids find special use when co-polymerized with a hydrophilic component like Glu or Lys.
  • Tyrosine Tyrosine
  • Trp Tryptophan
  • Trp-NCA synthesis is also facilitated since the secondary nitrogen does not need protected.
  • Proline is used in the present invention to provide obligatory turns in structures like helices, providing for enhanced intrachain interactions and promoting the formation of more globular synthetic proteins, when preferred.
  • Pro destabilizes higher order protein structures since it cannot participate in helical structures and can be used in a co-polymer to enhance diffusion of a drug substance from hydrophobic domains internalized in a synthetic protein.
  • D-amino acids also inhibit formation of helical structures of L-amino acids but are less desirable due to their possible unwanted metabolic effects as an unnatural amino acid.
  • Cystine is used in the present invention to stabilize higher order structures via intra- and inter-chain disulfide linkages. This is accomplished in the present invention using the bifunctional Bis-disulfide NCA, since the disulfide linkage serves as its own thiol protecting group.
  • An additional object of the present invention is the synthesis of a synthetic protein for use in the preparation of a synthetic serum.
  • a globular protein that is metabolically stable, non-immunogenic and non-allergenic is highly desired.
  • an artificial serum component a heteropolymer containing Glu, Pro, Tyr, and Cys has been prepared with the desired properties.
  • polypeptides in the current application are also referred to as synthetic proteins or synthetic amino acid polymers.
  • Polypeptides of the invention have two or more amino acids linked by a peptide bond. In a preferred embodiment, polypeptides have five or more peptide linked amino acids.
  • L-Glutamic acid 200 gm is dissolved in 2.5 L hot water (T > 95 C). The hot solution is added slowly to 2.5 L of rapidly stirred, cold (T ⁇ 10 C) acetone to form a thick slurry. After cooling, the precipitated solid is separated by filtration, washed with 200 ml of acetone and the filter cake compressed to remove excess solvent. The white filter cake is dried in vacuo at 80 C for 4 hours and is suitable for use in Example 2. Yield: 189 gm (94 %). "Microcrystalline" means that the crystalline nature is not obvious by macroscopic inspection; i.e. , the resulting L- Glutamic acid is amorphous.
  • Microcrystalline, dry L-Glutamic Acid (73.6 gm, 0.5 mol) from Example 1 is suspended in 2.0 L of anhydrous THF containing triphosgene (98 gm, 1 Eq.) and heated with stirring to 50 °C for 4 hours or until the reaction is homogeneous.
  • the reaction is then heated to a gentle reflux for about 1 hour, using a condenser protected with a drying tube. "About 1 hour” means 1 hour + 20 min.
  • the solution is then decanted or filtered from any remaining solids and evaporated under oil vacuum using a water bath less than 40 °C, until a precipitate forms or until a thick oil remains, with no additional solvent evaporation.
  • the product is dissolved in 360 ml of dry ethyl acetate, any insoluble material ( ⁇ 5 gm) is filtered off, and the crude product is precipitated by the rapid addition of 360 ml of hexanes with active stirring. After 20 min. , an additional 200 ml of hexanes is added to fully precipitate the crude Glu-NCA. The precipitate is collected by filtration under a dry carbon dioxide curtain, wash the filter cake with hexanes (100 ml) and compact under pressure to remove excess solvent.
  • the hexane-damp filter cake is dissolved in a combination of 350 ml anhydrous THF and 350 ml of anhydrous ethyl acetate. Any insoluble material is filtered off and is precipitated with rapid addition of 700 ml of hexanes with stirring. Once precipitation has commenced, an additional 350 ml hexanes is added to complete the precipitation. After 30 min, the precipitate is isolated by filtration under a dry carbon dioxide curtain. The product is compacted with pressure to remove excess solvent, and it is then washed with hexanes (100 ml) and dried in vacuo (T ⁇ 30 °C). The product is stable when stored under dry carbon dioxide, in the cold (temperature is less than about 10 °C, where "about 10 °C" means ⁇ 5 °C).
  • Example 3 Synthesis of Polymeric Glutamic Acid (PGlu)
  • Example 2 The dry Glu-NCA product (17.3 gm, 100 mmol) of Example 2 is dissolved in anhydrous THF (86 ml) and polymerization is initiated by the addition of 86 ml of anhydrous ethyl acetate containing 0.1 gm triethylamine. The reaction is warmed to reflux for 15 min then allowed to cool and react at 25 °C for 24 hours with continuous stirring. The precipitate is isolated by filtration, washed with anhydrous diethyl ether and dried in vacuo at 60 °C for 2 hours, to yield a white powder. Yield: 10.2 gm (79%).
  • Polymeric L-Lysine is prepared as previously described (Sela et. al. , Biopolymers 1,517, 1963).
  • the dry polymer is converted to the alpha helical form, and contaminant bromine is removed by dissolving in water as a 10 % solution and titrating the pH to 12 by the addition on 1.0 N NaOH.
  • the spontaneous precipitate is further precipitated by the addition of acetone and collected by filtration and vacuum dried to obtain a white powder. Yield: 78 % from the starting polymeric Lysine.
  • the helical form is confirmed by measuring the optical rotation [alpha] D of the pH 12 solution (minimal negative value of -40 degrees optical rotation, compared to -130 degrees optical rotation for the random coil).
  • Example 3 The conditions of Example 3 are repeated except that Glu-NCA (12.46 gm, 72 mmol) and Phe-NCA (5.35 gm, 28 mmol) are co-dissolved in the THF.
  • Crude Phe-NCA is prepared by the method of Poche (Tet. Letters 29:5859-5862, 1988).
  • the crude Phe-NCA is further purified prior to polymerization by dissolving 50 gm in a mixture of 100 ml of THF and 100 ml of ethyl acetate followed by precipitation with 600 ml of hexanes.
  • the fluffy, white product is isolated by filtration, washed with hexanes and dried in vacuo to yield 36 gm (72%).
  • Example 6 Synthesis of Lysine/Phenylalanine Co-Polymer (PLys/Phe)
  • Example 4 is repeated except that Phe-NCA is included in the polymerization to for the co-polymer in a final Lys/Phe ratio of 3.6.
  • the polymer is converted to the helical form as described in Example 4.
  • Example 3 The polymer of Example 3 is converted to the Glu/ Gin co-polymer.
  • the partial ammonium salt is then heated to 80 °C in a vacuum oven for conversion to the Glu/Gln co-polymer. Yield: 18.6 gm. Elemental analysis for N shows this preparation to contain 8% Gin. Titrations to higher pH with ammonia yield higher % Gin in the final co-polymer.
  • Example 3 is repeated except that 4 separate amino acid NCA's: Glu-NCA ( 10.71 gm, 62 mmol), Pro-NCA (1.42 gm, 10 mmol), Tyr-NCA (3.54 gm, 17 mmol) and Cys-NCA (1.14 gm, 4 mmol) are dissolved in 86 ml THF prior to polymerization.
  • Glu-NCA is used a synthesized in Example 2.
  • Pro-NCA, Tyr- NCA and Cys-NCA are synthesized as summarized by Blacklock, Hirschmann and Veber (77te Peptides 9:39-95, 1987).
  • Cys-NCA is prepared and used as the Bis- Cysteine- NCA (NCA-Cys-S-S-Cys-NCA).
  • Pro-NCA is prepared just prior to use but the remaining NCA's are stable when stored as described in Example 2. Yield: 12.2 gm (76 %).
  • a uniform blend of PGlu (19.0 gm) and Trp (1.0 gm) is prepared in a ball mill and 100 mg tablets are prepared as described in Example 13. Tablets are subjected to a standard dissolution test under two pH conditions (pH 1.0 and 6.5) in order to mimic conditions in the stomach and small intestine.
  • a tablet is placed in a jacketed (37 °C) glass beaker containing 100 ml of the test solution, and stirred at 100 RPM.
  • Samples (0.1 ml) of the solution are taken at 30, 60 and 120 min, diluted 1/10 in water, and the OD measured at a wavelength of 280 nanometers and compared to the control samples containing 5 mg of Trp dissolved in 100 ml of solution. Release of Trp is expressed as % of Control (100%).
  • Example 9 is repeated except that tablets were formulated using the helical form of PLys from Example 4.
  • Tryptophan is included in hydrophobic sites in PGlu/Phe from Example 5 by dissolving 10 gm of the polymer in a solution composed of 50% ethanol and 0.05 M sodium phosphate buffer at pH 7.2.
  • the solution is bubbled with nitrogen to remove dissolved oxygen. Tryptophan (1.0 gm) is then added and the pH slowly adjusted to 3.0 by titration with 1.0 N HCl. The precipitate formed is collected by centrifugation and washed by re-suspension in water and the product is freeze dried. Solution of the product in 0.05 M sodium phosphate and measuring the OD at 280 nanometers is used to measure the Trp included in the polymer.
  • Example 11 is repeated except that L-DOPA is substituted for tryptophan.
  • Decomposition of DOPA is measured by formation of colored quinone oxidation products, when compared to DOPA alone.
  • DOPA loss is measured by reverse phase HPLC as previously described. Gerlach et al., J. Chromat. 380:379-385, 1986.
  • Oral dosage forms of drugs combined with synthetic polypeptides can be prepared by direct compression of the polypeptide drug combination.
  • the synthetic polypeptide/drug combination can be combined with other excipients to enhance tablet properties, as described for 5 mg hydrocortisone in a 200 mg tablet.
  • a uniform blend of equal masses of PGlu and aspirin is prepared in a shell blender and 200 mg tablets containing 100 mg aspirin each are compressed as described in Example 13 to a hardness of 7 kg.
  • Example 11 is repeated except that hydrocortisone (17- hydroxycorticosterone) is substituted for Trp and tableted with other excipients as described in Example 13. Hydrocortisone in the tablets is determined by quantitative reverse phase HPLC of solutions containing dissolved tablets as previously described. Waters Corporation, Symmetry Applications Notebook II, August, 1994, p. 19.
  • Stability of Gin in the PGlu/Gln co-polymer of Example 7 is measured by the production of free ammonia in 0.2 M phosphate buffer, as described previously (Gilbert et al. , J. Biol. Chem. 180:209, 1949).
  • the Glu/Gln co-polymer yields no detectable ammonia in this assay, while the control sample, containing free Gin is almost completely de-amidated.
  • Example 7 The co-polymer of Example 7 is dissolved as an 0.8% solution (0.8 gm/100 ml) in a standard media, devoid of monomer ic glutamine (alpha-MEM, without glutamine) and combined with insulin (5 ug/ul) and transferrin (holo, 5 ⁇ g/ ⁇ l). The solution is adjusted to pH 7.2 with 1.0 N NaOH and filtered through a 0.2 micron sterilization membrane. This combined media is found to support growth of cultured human amniocytes, through multiple passages, with no addition of monomer ic L- glutamine.
  • Example 18 Treatment of Gin Deficiency in Humans with Oral Dosage of PGlu/Gln
  • the Glu/Gln co-polymer of Example 7 is formed into tablets by direct compression of 125 mg of the co-polymer, as described in Example 13. This oral dose will suffice as an oral preparation to deliver about 10 mg L-glutamine, in order to treat a deficiency of this essential amino acid in humans.
  • the heteropolymer product of example 8 is dissolved in phosphate buffered saline (PBS) and adjusted to pH 7.2 with 1.0 N NaOH, prior to sterile filtration.
  • PBS phosphate buffered saline
  • This sterile solution is intended as a synthetic serum replacement, to supplement serum volume in humans and other mammals.
  • Example 14 is tableted by direct compression, as in Example 13, to contain about 50 mg of aspirin per tablet. This preparation is intended as an oral treatment for inflammation in mammals, especially humans.
  • Example 15 The product of Example 15 is combined with other excipients to formulate an oral dosage for the treatment of adrenal insufficiency or inflammation in mammals, especially humans.
  • Example 22 Treatment for Parkinson 's Disease
  • Example 12 is tableted by direct compression, as in example 13, to contain 50 mg of L-DOPA per tablet, as oral dosage form for the treatment of Parkinsons disease in humans.

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Abstract

L'invention concerne des procédés utilisés pour combiner des composés chimiques avec des polymères d'acides aminés synthétiques conçus pour lutter contre la dégradation et assurer la libération contrôlée des composés. L'invention porte également sur un procédé de libération sélective de substances médicamenteuses à partir d'un polymère synthétique d'acides aminés dans l'estomac ou l'intestin grêle, qui utilise des changements dépendant du pH dans une structure d'ordre supérieure. L'invention concerne, en outre, une composition pharmaceutique contenant une substance médicamenteuse qui a été combinée avec un polymère d'acides aminés et une composition d'excipients pharmaceutiquement acceptable.
EP00916076A 1999-03-05 2000-03-06 Utilisation de la conformation d'une proteine pour la protection et la liberation de composes chimiques Withdrawn EP1257592A1 (fr)

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