WO2001012230A1 - The nasal transmucosal delivery of peptides conjugated with biocompatible polymers - Google Patents

The nasal transmucosal delivery of peptides conjugated with biocompatible polymers Download PDF

Info

Publication number
WO2001012230A1
WO2001012230A1 PCT/KR2000/000868 KR0000868W WO0112230A1 WO 2001012230 A1 WO2001012230 A1 WO 2001012230A1 KR 0000868 W KR0000868 W KR 0000868W WO 0112230 A1 WO0112230 A1 WO 0112230A1
Authority
WO
WIPO (PCT)
Prior art keywords
peg
pharmaceutical composition
transmucosal delivery
peptide
nasal
Prior art date
Application number
PCT/KR2000/000868
Other languages
French (fr)
Inventor
Myung-Ok Park
Kang Choon Lee
Original Assignee
Park Myung Ok
Kang Choon Lee
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Park Myung Ok, Kang Choon Lee filed Critical Park Myung Ok
Priority to AU64784/00A priority Critical patent/AU6478400A/en
Priority to EP00952020A priority patent/EP1204427A4/en
Priority to JP2001516573A priority patent/JP2003507344A/en
Publication of WO2001012230A1 publication Critical patent/WO2001012230A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/09—Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/23—Calcitonins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/25—Growth hormone-releasing factor [GH-RF] (Somatoliberin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • 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 relates to a pharmaceutical composition for a nasal transmucosal delivery, comprising a biologically active peptide, which is sparingly soluble in water, con ugated with an active biocompatible polymer.
  • the present invention relates to the pharmaceutical composition containing the biocompatible polymer-biologically active peptide conjugate suitable for use in the nasal transmucosal delivery, which is highly improved m water solubility and protected from being degraded by protease.
  • the pharmaceutical composition comprising of the peptide-polymer conjugate for the nasal transmucosal delivery of the present invention allows drug activity to be expressed in a short period of time and improves a bioavailability .
  • peptides or proteins are very low in body absorption efficiency because they are easily hydrolyzed or degraded by enzymes within a short period of time after being taken into the body. Further, when such peptide medicines are repetitively administered, immune reactions are frequently induced to produce antibodies which may cause so serious hypersensitivity as to menace the life of the administee, acting as a neutralizing role against the physiological activity of the medicines. In addition, the clearance attributable to the reticuloendothelial system (RES) is increased. Therefore, most of peptide medicines have been administered by injection, thus far. Injection administration, however, gives patients pain, accompanying dangers. Particularly, patients who need to be treated for a long period of time may not be able to treat themselves by injection. Thus, there remains a need to develop other routes for peptide administration .
  • RES reticuloendothelial system
  • An adult's nasal cavity is coated with a mucosa at a thickness of 2.0-4.0 mm (Mugind, Nasal Allergy, Blackwell Scientific, Oxford, 1979) and has a volume
  • the nasal cavity allows drug activity to be expressed in a short period of time because of its being abundant in fine villus and large in absorption surface area. Accordingly, extensive research has been made on the transmucosal delivery of drugs.
  • Factors which have influence on the absorption of drugs through mucosae include physical and chemical properties of drugs, such as drug' s inherent transmittance, ion strength, flow distribution coefficient and molecular weight, carrier transportation, protease degradation, and physiological conditions of nasal mucosae.
  • the nasal mucosa is a direct absorption route through which drugs can circumvent the liver metabolism, which is a great hindrance to the utilization of drugs in the body upon oral administration.
  • the nasal transmucosal route has an advantage over the oral route in that the body utilization of drugs can be significantly improved.
  • the nasal transmucosal delivery of peptides or proteins of large molecular weights are lower m absorption efficiency than intravenous injection because the peptides or proteins cannot well pass through nasal mucosae.
  • Absorption promoters have been suggested to improve the absorption of peptides. Examples of suggested absorption promoters include surfactants (Hirai et al . , In t . J. Pharm .
  • European Pat. Nos. 23,359 and 122,023 open the possibility that a powder formulation of peptide drugs is delivered through nasal mucosae.
  • U. S. Pat. No. 4,250,163 discloses a mucosa-adsorptive substance which is admixed with a powder form of peptides drugs to enhance the nasal transmucosal delivery of the drugs.
  • European Pat. No. 123,831 is directed to the administration of steroids through nasal mucosae. Germany Pat. No. 2,620,446 describes a body absorption enhancer which is effective for the nasal transmucosal delivery of insulin.
  • PCT/GB/86/00721 discloses a formulation technique of drugs into microspheres which can be delivered through nasal mucosae. However, this formulation technique can be applied only to particular drugs.
  • Japanese Pat. No. Sho . 58-189118 cyclodextnn is utilized for the nasal transmucosal delivery of peptides.
  • Japanese Pat. No. Sho. 59-89619 discloses ethereal surfactants, for example, polyoxyethylene lauryl ether, as neutral absorption enhancers for nasal transmucosal delivery.
  • these surfactants are not suitable for clinical use because they cause damage to nasal mucosae.
  • Japanese Pat. No. Sho. 61-118325 describes alkaline or neutral amino acids for use m the nasal transmucosal delivery of calcitomn.
  • sucrose fatty acid ester is used as an absorption promoter for the nasal transmucosal delivery of drugs.
  • these absorption promoters are also toxic to mucosae.
  • the above-mentioned reference patents most of which are based on the sustained release of peptide drugs, enable drugs to be released continuously, but cannot solve the problem m that peptides or protein drugs administered through mucosae are degraded m a short period of time. These conventional techniques find difficulty m being applied for the nasal transmucosal delivery of peptide drugs.
  • Conjunction of pharmaceutically active proteins or molecules to synthetic macromolecules may afford great advantages when they are applied m vivo and in vi tro .
  • physiologically active molecules When being covalently bonded to macromolecules, physiologically active molecules may be changed in surface properties and solubility. For example, an increase may be brought into their solubility m water or organic solvents.
  • macromolecules may make the conjugated peptides more stable m vivo as well as reduce the clearance attributed to the intestinal system, the kidney, the spleen and/or the liver.
  • conjunction of polymers to peptides can bring about a great improvement m the stability of the peptides in solutions and effectively protect the intrinsic surface properties of peptides to prevent non-specific protein adsorption.
  • U. S. Pat. No. 4,179,337 discloses conjugates between peptides or polypeptides and polyethylene glycol (hereinafter, referred to as "PEG") with a molecular weight of 500-20,000 or water-soluble polymers, which are reduced in antigenicity and anti- lmmunity while maintaining the biological activity of the peptides or polypeptides. It is described in U. S, Pat. No. 4,301,144 that hemoglobin is increased in oxygen molecule-carrying potential when being associated with PEG or water-soluble polymers.
  • PEG polyethylene glycol
  • the conjugation of PEG to polypeptides is achieved by reacting activated PEG to amino residues of polypeptides. Suitable for use in this purpose are a lysme residue and N-termini .
  • PEG activation one of the hydroxy groups of PEG is substituted with a methyl ether group while the other hydroxy group is bonded to an electrophile functional group (Abuchowski, A. and Davis, F. F. (1981), in Enzymes as Drugs (Holsenberg, J. and Roberts, J. , eds . ) ) .
  • activated polymers include PEG-N-hydroxysuccmeimide-activated esters, which contain amide bonds, PEG-epoxides and PEG- tresylate, which contain alkyl bonds, PEG-carbonyl lmidazole and PEG-nitrophenyl carbonates, which contain urethane bonds, PEG-aldehyde, which contains a Schiff' s base at the N-terminus .
  • PEG derivatives able to specifically react to cystein groups of polypeptides include PEG- vi ⁇ yl sulphone, PEG-iodoacetamide, PEG-maleimide, and PEG-orthopyridyl disulfide with most preference to male mide-containing PEG.
  • PEG-vmyl sulfone is best m view of the stability in water solutions while PEG- orthopyridyl disulfide can be reversibly degraded in vivo because of the presence of disulfide bonds.
  • Peptides taking advantage of these derivatives can be exemplified by mterleu ⁇ n-3 and ⁇ nterleuk ⁇ n-2.
  • PEG derivatives reactive specifically to oligo sugars of polypeptides may be exemplified by PEG- hydrizide, which is able to react with aldehyde- containing compounds to form relatively stable hydrozone bonds. Advantage is taken of the specific bonding of PEG-hydrizides to sugar moieties of glycoproteins .
  • PEG-isocyanates react specifically with hydroxy groups of polypeptides.
  • PEG derivatives containing phenylglyoxal which is highly reactive to the guanidmo group.
  • the nasal transmucosal delivery of peptides alone is significantly improved absorption efficiency compared with the oral administration because the peptides are not subjected to liver metabolism, but poor in the bioavailability of the peptides because they are degraded by endogenous enzymes.
  • a pharmaceutical composition for the nasal transmucosal delivery comprising a sparingly soluble, biologically active polypeptide conjugated with an activated biocompatible polymer.
  • the present invention have confirmed that, when the polymer- peptide conjugate is administered through the nasal cavity, it is improved in water solubility and protected from being degraded by proteases, whereby the medicinal activity of the pharmaceutical composition can be sustained for an extended period of
  • the present invention provides a pharmaceutical composition for the nasal transmucosal delivery, comprising a sparingly soluble, biologically active polypeptide conjugated with an activated biocompatible polymer.
  • FIG. 1 represents a result of size exclusion chromatography which isolates sCT and PEG-sCT, where
  • FIG. 2 shows a pH effect to the production of mono-PEG-sCT that PEG is conjugated with N-termmus of calcitonin, Lys 18 or Lys 11 , where I ; mono-PEG-sCT (N-terminal conjugate) ,
  • FIG. 3 shows a molar ratio effect of calcitonm : sCT to the production of mono-PEG-sCT that PEG is conjugated with N-termmus of calcitonm, Lys 18 or Lys 11 , where ⁇ ; mono-PEG-sCT (N-termmal conjugate),
  • FIG. 4 shows a reaction time effect of calcitonm : sCT to the production of mono-PEG-sCT that PEG is conjugated with N-termmus of calcitonm, Lys 18 or Lys 11 , where
  • FIG. 5 represents a result of size exclusion chromatography which isolates hPTH and PEG-hPTH ( 1-34 ) , where
  • FIG. 6 shows a reduction effect of calcitonm concentration m blood when sCT and PEG-sCT conjugate are administered through the nasal cavity, where A ; sCT,
  • FIG. 7 shows a calcium reduction effect of isolated mono-PEG-sCT isomers
  • mono-PEG-sCT (Lys n -conjugate, M3), ⁇ ; sCT, D ; mono-PEG-sCT not isolated its isomers.
  • biocompatible polymers as used herein means naturally occurring or synthetic compounds which are dissolved in water.
  • the biocompatible polymers include polyethylene glycol, polypropylene glycol (PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and its derivatives, polyacrylic acid and their derivatives, polyamino acid, polyvinyl alcohols, polyurethane, polyphosphazene, poly(L- lysine) , polyalkylene oxide (PAO), and water-soluble polymers such as polysaccharide, dextran, and non- immumogenic polymers such as polyvinyl alcohol and polyacryl amide.
  • the present invention provides a peptide-polymer conjugate for a nasal transmucosal delivery, which can be prepared by bonding an activated polymer to a biologically active peptide.
  • the bond between the peptides and the polymers may be a covalent bond or a non-covalent bond such as a lipophilic bond or a hydrophobic bond.
  • the molar ratio of polypeptides to activated polymers are in the range of about 1:1 to 1:10 and preferably in the range of 1:1 to 1:7.
  • one to three activated polymers may be conjugated to one polypeptide molecule.
  • Peptide-mono polymer conjugates exert the most effective pharmaceutical activity.
  • PEG can be conjugated to the N- termmus of calcitonm and/or Lys 18 and/or Lys 11 .
  • the calcitonm- (mono) PEG conjugate in which one PEG molecule is conjugated to one calcitonm molecule exhibits the highest calcium reduction effect.
  • a conjugate isomer m which PEG is conjugated to the N- termmus or the Lys 18 shows more sustained and effective calcium reduction activity than does a conjugate isomer in which PEG is conjugated to Lys 11 .
  • pH 5, 6 or 7 of the reaction solution more than 80 % of PEG is conjugated to the N-termmus of calcitonm.
  • pH 8 or higher PEG is increasingly conjugated to Lys 11 and Lys 18 . In contrast, changes in reaction time and molar ratio cannot affect the proportion of isomers.
  • the binding reaction of the peptide-activated polymer in the present invention is performed m 0.1 M phosphate buffer ranged, m pH, from 6 to 9, at 0 to 25C° of reaction temperature for several minutes to 12 hours.
  • a method of polymer activation consists of the following steps of; preparing the polymer into polyalkylene oxide (hereinafter, referred to as "PAO") such as monoethoxy-poly (ethylene glycol, mPEG) ; and changing the other part of PAO into a reaction group having reactivity.
  • PAO polyalkylene oxide
  • the activated polymer forms the prptide-polymer conjugate by reacting with ⁇ -amme group of lysme.
  • the peptide can be used as a conjugated moiety.
  • the present inventors have measured the blood concentration of peptide according to time after the peptide-polymer conjugate is administered to rats through the nasal cavity. Thus, the present inventors have confirmed that the peptide by the nasal transmucosal delivery has a better stability living body and sustains its biological activity for a long time .
  • the peptide of the present invention is not limited to the specific therapeutic agents but applied to the all substances having biological activity, particularly, it is desirable to use calcitonin, parathyroid hormone (hereinafter, referred to as "PTH”).
  • PTH parathyroid hormone
  • insuline synthetic enkephalin
  • GHRP growth hormone releasing peptide
  • LHRH leutenizing hormone releasing hormone
  • CGRP calcitonin gene related peptide
  • THF thyroid stimulating hormone and thymic humoral factor
  • Calcitonin is a single chain peptide composed of 32 amino acids, forms a ring at N-terminus and has a proline amide group at C-terminus. Calcitonin inhibits a bone absorption by acting directly to osteoclast and is used for cure of hypercalciumia, Pajet's disease, pain from the bone absorption and osteporosis. Calcitonin is produced in salmon, eel, human, pig, etc., and salmon and eel calcitonin have the most effect.
  • Parathyroid hormone (PTH) is a peptide hormone composed of 84 ammo acids and secreted from parathyroid.
  • parathyroid cell Since parathyroid cell has a recognition site for calcium concentration, PTH secretion increases when calcitonm is lower, and PTH secretion decreases when calciton higher. PTH increases a calcium absorption taken from foods at the small intestine, transfers calcium from bone to blood, and , finally, increases a blood calcium concentration.
  • the major active site of PTH is adrenal cortex. PTH binds to the membrane of adrenal cortex to increase the production of cAMP, IP 3 (mos ⁇ tol triphosphate) and DAG(d ⁇ acyl glycerol) .
  • Insulin is a peptide composed of A chain and B chain.
  • a chain composed of 21 ammo acids and B chain composed of 30 ammo acids are connected by 1 pair of disulfide bond. Disulfide bond exists between 6 th ammo acid of A chain and 11 th ammo acid of B chain.
  • Disulfide bond exists between 6 th ammo acid of A chain and 11 th ammo acid of B chain.
  • Enkephalm is a pentapeptide representing a similar action to opium. Enkephalm is devided into two groups of methionme enkephalm and leucme enkephalm according to C-termmus bounded to Try-Gly- Gly-Phe. Enkephalm inhibits pain from transferring to bram by preventing a release of substance P from the end of analgesic nerve fiber.
  • Growth hormone releasing peptide presents m the form of hepta and hexa and affects a release of growth hormone.
  • the growth hormone releasing effect of GHRP is related to a dose, increases until aldolescence and decreases after then.
  • GHRP-6 is a hexapeptide having a structure of H ⁇ s-Asp-Try-Ala-Try-Asp-Lys-NH 2 , and is very stable in acetate buffer ranged, in pH, from 5.5 to 6.0. In case of oral administration, bioavailability of GHRP-6 is 0.3%, an absorption half life is 15 minutes, and an elimination half life is 60 minutes. GHRP-6 secrets selectively growth hormone through a receptor in hypothalamus and pituitary gland.
  • LH-RH is a hypothalamus peptide and stimulates the relases of LH and follicle stimulating hormone.
  • LH-RH regulates a function of brain and many peripheral organs by binding to receptors on the targeted cell surface.
  • LH-RH has a decapeptide structure of Pyro-Glu-His-Trp-Ser-Tyr-Gly- Leu-Arg-Pro-Gly-NH 2 , and is degraded in tubular of the kidney.
  • LH-RH derivatives include nafarelin, busecilin, zilidexin, etc.
  • composition for the nasal transmucosal delivery including the peptide-polymer conjugate of the present invention can be formulated into the suitable form, and mainly administered by spray as a medicine for external use.
  • the peptide-polymer conjugate is dissolved in a solvent, or a suspended, medicinal solution is filled in a container having a specific spraying device (valve) with a low viscous spraying agent.
  • a specific spraying device valve
  • the medicinal solution is sprayed in the type of smog using pressure.
  • the material quality of container uses a metal such as a tinned iron and aluminum.
  • the inside of container is coated with moth-proof painting.
  • the container made of glass or synthetic resin is possible.
  • the spraying agent uses generally a compressed air and an incombustible liquefied gas, freon(freon 11, CC 13 F; freon 12, CC 1£ F; freon 114, C 2 C 12 F 4 ).
  • a dose of the pharmaceutical compositions containing the peptide-polymer conjugate of the present invention is 0.1 ug - 10 mg/kg/day, and can be widly altered according to the kind of peptide and patient's condition.
  • sCT calcitonin-phosphate buffered saline
  • PBS pH 7.4
  • PEG (MW 12000) was activated with succinimidyl succinate, and PEG12000-sCT was prepared by using 9.2 mg of the activated SS-PEG. All procedures were performed as indicated in the Example ⁇ 1-1>.
  • PEG (MW 2000) was activated with succinimidyl succinate, and PEG2000-sCT was prepared by using 2 mg of the activated SS-PEG. All procedures were performed as indicated in the Example ⁇ 1-1>.
  • Example 2 Isolation of PEG-sCT PEG-sCT conjugates prepared in the Examples ⁇ 1-1> to ⁇ l-3> were isolated with the aid of size exclusion column (Super HR 12/30, PharmaciaLKB, Sweden) using PBS (pH 7.0) as eluent at 0.4 ml/min of flow rate (FIG. 1).
  • FIG. 1 represented a fluorescence intensity of each peak measured by fluorescence spectrometer wherein each peak was eluted from the size-exclusion column.
  • tri-PEG-sCT which was conjugated 3 molecules of PEG with 1 molecule of calcitonin and had a biggest molecular size, was firstly eluted from the column and collected within 30 minutes after sample injection into the column. After the tri-PEG-sCT, di-PEG-sCT and mono-PEG-sCT were isolated in succession. In addition, since calsitonin molecule conjugated with PEG had the smallest molecular size, it was eluted last from the column and time for elution took more 40 minutes. That time, the fluorescence intensity of each peak eluted from the column had measured by the fluorescence spectrometer directly connected to the column, and each peak was collected.
  • Example 3 Preparation of PEG-sCT According to Various pHs , Molar Ratios and Reaction Times
  • PEG-sCT conjugates prepared in Examples ⁇ 3-l> to ⁇ 3-3> were separated into tri-PEG-sCT, di-PEG-sCT and mono-PEG- sCT . This separation was conducted in a similar manner to that of Example 2.
  • the mono-PEG-sCT obtained was divided into three isomers by reverse phase high performance liquid chromatography (HPLC) .
  • HPLC high performance liquid chromatography
  • the gradient condition was changed with 36-42 % solvent B (0.1 % PFPA added acetonitrile) and 64-58 % solvent A (0.1% PFPA added distilled water).
  • Quantitative measurements were made using a UV absorption meter (215 mm) or a fluorescent absorption meter (excitation 280 nm, emissm 315 nm) .
  • 0.1 M PBS, pH 7.0 was added with 50 ⁇ l of a PTH solution (2.4 mg/0.15 ml, 0.1 M PBS, pH 7.0) to give a mixture containing a molar ratio of 5:1 of SS- PEG5000:PTH.
  • the mixture was allowed to react for 30 mm at ambient temperature with shaking. After 30 mm of the reaction, an excess amount of a 0.1 M glycme solution was added to stop the reaction. Unreacted parathyroid gland hormone and PEG were removed by dialysis using PBS to obtain PEG5000-PTH (1-34 ) .
  • the PEG 5000-PTH (1-34) conjugate thus prepared was purified by size-exclusion chromatography using an HPLC system and superdex.
  • PBS pH 7.0
  • Eluted fractions were stored at 4C° until they were measured for UV absorbance. UV absorbance was measured at 215 nm while the excitation and emission waves of fluorescence were fixed in the range of 280 to 315 nm, as shown in Fig. 5.
  • PEG2000-PTH (1-3 ) conjugates were prepared and isolated in a similar manner to that of Example ⁇ 5-l> except that SS-PEG2000 was used at an amount of 2 mg .
  • GHRP-6 prepared PEG-GHRP-6, and isolated and purified as indicated in the Example ⁇ 5-l>. Isolated mono-PEG- GHRP-6 was immediately frozen-dried by freeze dryer.
  • PEG2000 GHRP-6 conjugate as indicated in the Example ⁇ 6-l>.
  • SS-PEG5000 (23 mg/0.20 ml, MW 5,000, 0.1 M PBS, pH 7.0) was added with 50 ⁇ l of a LHRH solution (0.5 mg/0.1 ml, 0.1 M PBS, pH 7.0) to give a mixture containing a molar ratio of 1:5 of LHRH: SS-PEG, prepared PEG-LHRH, and isolated and purified as indicated in the Example ⁇ 5-l>.
  • PEG2000-LHRH conjugate was prepared, isolated and purified as indicated in the Example ⁇ 7-l>.
  • Example 8 Preparation and Isolation of PEG5000- triptorelin Except for use of triptorelm, LHRH derivative, instead of LHRH, PEG5000-tr ⁇ ptorelm conjugate was prepared, isolated and purified as indicated m the Example ⁇ 7-l>.
  • LHRH agonist instead of LHRH, PEG5000-ornt ⁇ de conjugate was prepared, isolated and purified as indicated in the Example ⁇ 7-l>,
  • SD (Spraque Dawley) male rat (220 - 300 g, Charles River Japan, Atsugi, Japan) was put under anesthesia by administrating 45 mg/kg of peptobarbital into terperitoneal .
  • Bronchus and esophagus were cannulated with PE-250 tube and thigh arteria with SP- 45 tube.
  • 20 ul of a placebo and experimental drugs were administered into the nasal cavity of rats with Hamilton syringe.
  • the placebo used physiological saline and the experimental drug used PEG5000-SCT, PEG12000-sCT, PEG2000-sCT and sCT prepared from the Example 2.
  • Salmon calcitonm was administered at dose of 0.05 to 4.5 IU per rat, and rats were grouped into 4.
  • AUC area-under the curve
  • ⁇ D ( % ) ( AUCca-plac-AUC C a-sC ⁇ ) / AUC C a-plac X 100
  • AUC Ca -piac was AUC from placebo administration to 960 minutes
  • AUC Ca -sc ⁇ was AUC from calcitonm and experimental drugs administration to 960 minutes .
  • the calcium concentration of calcitonm conjugated without any PEG was decreased for 1 hour after the nasal transmucasal administration, but restored at the original concentration after 2 hours.
  • the calcitonm reduction effect represented for 4 hours to 8 hours after the nasal transmucasal administration.
  • the calcium reduction effect was significantly altered according to the molecular size of PEG.
  • the nasal transmocosal delivery using the PEG conjugates of the present invention could reduce a used amount of calcitonm and side effects of drugs .
  • Example 7 The calcium reduction effect of the mono-PEG-sCT isomers prepared from the Example 4 was measured as indicated in the Example 1 (FIG. 7) .
  • isomers conjugated w th PEG at N-termmus and Lys 18 represented a better sustaining and effective calcium reduction effect than isomers conjugated with PEG at Lys 11 .
  • Lys 18 isomers having a lower calcium reduction effect than N-termmus isomers represented a considerable reduction effect to sCT or Lys 11 isomers.
  • PEG (MW 2000) was activated with succinimidyl succinate, and PEG2000-GLP-1 was prepared by using 2 mg of the activated SS-PEG. All procedures were performed as indicated m the Example ⁇ 1-1>. In addition, PEG2000-GLP-1 was isolated and purified as indicated in the Example ⁇ 5-l>.
  • a pharmaceutical composition comprising a peptide-polymer conjugate for a nasal transmucosal delivery of the present invention increases the water solubility of peptide, which is sparingly soluble m water, improves a stability by protecting from being degraded by protease, and, consequently, reduces an administration number of drug to decrease side-effects induced by drug abuse.
  • the pharmaceutical composition comprising a peptide-polymer conjugate for the nasal transmucosal delivery of the present invention is delivered through the nasal cavity, it allows drug activity to be expressed in a short period of time and improves a bioavailability .

Abstract

The present invention relates to a pharmaceutical composition for a nasal transmucosal delivery, comprising a biocompatible polymer-biologically active peptide conjugate. The pharmaceutical composition of the present invention increases the water solubility of peptide, which is sparingly soluble in water, improves a stability by protecting from being degraded by protease, and, consequently, reduces an administration number of drug to decrease side-effects induced by drug abuse. In addition, since the pharmaceutical composition of the present invention is delivered through the nasal cavity, it allows drug activity to be expressed in a short period of time and improves a bioavailability.

Description

THE NASAL TRANSMUCOSAL DELIVERY OF PEPTIDES CONJUGATED WITH BIOCOMPATIBLE POLYMERS
FIELD OF THE INVENTION
The present invention relates to a pharmaceutical composition for a nasal transmucosal delivery, comprising a biologically active peptide, which is sparingly soluble in water, con ugated with an active biocompatible polymer.
More particularly, the present invention relates to the pharmaceutical composition containing the biocompatible polymer-biologically active peptide conjugate suitable for use in the nasal transmucosal delivery, which is highly improved m water solubility and protected from being degraded by protease.
The pharmaceutical composition comprising of the peptide-polymer conjugate for the nasal transmucosal delivery of the present invention allows drug activity to be expressed in a short period of time and improves a bioavailability .
BACKGROUND
In the body, various peptides play important roles, existing as various forms such as hormones and cytokines. With recent great advances in genetic engineering, various peptides have been able to be synthesized on a mass scale and be used as medicines.
Use of peptides or proteins as medicines, however, suffers from many problems. First, peptides or proteins are very low in body absorption efficiency because they are easily hydrolyzed or degraded by enzymes within a short period of time after being taken into the body. Further, when such peptide medicines are repetitively administered, immune reactions are frequently induced to produce antibodies which may cause so serious hypersensitivity as to menace the life of the administee, acting as a neutralizing role against the physiological activity of the medicines. In addition, the clearance attributable to the reticuloendothelial system (RES) is increased. Therefore, most of peptide medicines have been administered by injection, thus far. Injection administration, however, gives patients pain, accompanying dangers. Particularly, patients who need to be treated for a long period of time may not be able to treat themselves by injection. Thus, there remains a need to develop other routes for peptide administration .
An adult's nasal cavity is coated with a mucosa at a thickness of 2.0-4.0 mm (Mugind, Nasal Allergy, Blackwell Scientific, Oxford, 1979) and has a volume
? of about 20 ml. It is thought that the nasal cavity allows drug activity to be expressed in a short period of time because of its being abundant in fine villus and large in absorption surface area. Accordingly, extensive research has been made on the transmucosal delivery of drugs. Factors which have influence on the absorption of drugs through mucosae include physical and chemical properties of drugs, such as drug' s inherent transmittance, ion strength, flow distribution coefficient and molecular weight, carrier transportation, protease degradation, and physiological conditions of nasal mucosae. In fact, the nasal mucosa is a direct absorption route through which drugs can circumvent the liver metabolism, which is a great hindrance to the utilization of drugs in the body upon oral administration. Thus, the nasal transmucosal route has an advantage over the oral route in that the body utilization of drugs can be significantly improved. The nasal transmucosal delivery of peptides or proteins of large molecular weights are lower m absorption efficiency than intravenous injection because the peptides or proteins cannot well pass through nasal mucosae. Absorption promoters have been suggested to improve the absorption of peptides. Examples of suggested absorption promoters include surfactants (Hirai et al . , In t . J. Pharm . 9, 165-169, 1981), acylcarnitine, cholinester, alpha-cyclodextrin, and chelating agents (Lee, In: Del ivery Systems for Peptide Drug, Plenum, New York, pp 87-104, 1986). These absorption promoters, however, are difficult to apply in practice because they give rise to a decrease in the stability of the drugs upon formulation, or irritate nasal mucosae.
In regard to the nasal transmucosal delivery of drugs, many research results are disclosed in patents. European Pat. Nos. 23,359 and 122,023 open the possibility that a powder formulation of peptide drugs is delivered through nasal mucosae. U. S. Pat. No. 4,250,163 discloses a mucosa-adsorptive substance which is admixed with a powder form of peptides drugs to enhance the nasal transmucosal delivery of the drugs. European Pat. No. 123,831 is directed to the administration of steroids through nasal mucosae. Germany Pat. No. 2,620,446 describes a body absorption enhancer which is effective for the nasal transmucosal delivery of insulin.
PCT/GB/86/00721 discloses a formulation technique of drugs into microspheres which can be delivered through nasal mucosae. However, this formulation technique can be applied only to particular drugs.
In Japanese Pat. No. Sho . 58-189118, cyclodextnn is utilized for the nasal transmucosal delivery of peptides. Japanese Pat. No. Sho. 59-89619 discloses ethereal surfactants, for example, polyoxyethylene lauryl ether, as neutral absorption enhancers for nasal transmucosal delivery. However, these surfactants are not suitable for clinical use because they cause damage to nasal mucosae.
Japanese Pat. No. Sho. 61-118325 describes alkaline or neutral amino acids for use m the nasal transmucosal delivery of calcitomn. In Japanese Pat. No. Sho. 63-39822, sucrose fatty acid ester is used as an absorption promoter for the nasal transmucosal delivery of drugs. However, these absorption promoters are also toxic to mucosae. The above-mentioned reference patents, most of which are based on the sustained release of peptide drugs, enable drugs to be released continuously, but cannot solve the problem m that peptides or protein drugs administered through mucosae are degraded m a short period of time. These conventional techniques find difficulty m being applied for the nasal transmucosal delivery of peptide drugs.
Conjunction of pharmaceutically active proteins or molecules to synthetic macromolecules may afford great advantages when they are applied m vivo and in vi tro . When being covalently bonded to macromolecules, physiologically active molecules may be changed in surface properties and solubility. For example, an increase may be brought into their solubility m water or organic solvents. Further, the presence of macromolecules may make the conjugated peptides more stable m vivo as well as reduce the clearance attributed to the intestinal system, the kidney, the spleen and/or the liver. Hence, conjunction of polymers to peptides can bring about a great improvement m the stability of the peptides in solutions and effectively protect the intrinsic surface properties of peptides to prevent non-specific protein adsorption.
U. S. Pat. No. 4,179,337 discloses conjugates between peptides or polypeptides and polyethylene glycol (hereinafter, referred to as "PEG") with a molecular weight of 500-20,000 or water-soluble polymers, which are reduced in antigenicity and anti- lmmunity while maintaining the biological activity of the peptides or polypeptides. It is described in U. S, Pat. No. 4,301,144 that hemoglobin is increased in oxygen molecule-carrying potential when being associated with PEG or water-soluble polymers.
Various proteins are reported to show extended half-life spans and reduced immunogenicity in plasma when being conjugated with PEG (Abuchowski et al . , Cancer Biochem . Biophys . , 1 , 175-186, 1984). Uricase- PEG conjugates are demonstrated to be increased in m - vivo half life span and show reduced side-effects during the metabolism of uric acid (Davis et al . , Lancet , 2, 281-283, 1981). As apparent from the preceding patents, the conjugation of PEG allows biologically active peptides or proteins to be increased m-vivo half life span and solubility and to be reduced in immune reactions.
Most frequently, the conjugation of PEG to polypeptides is achieved by reacting activated PEG to amino residues of polypeptides. Suitable for use in this purpose are a lysme residue and N-termini . As for PEG activation, one of the hydroxy groups of PEG is substituted with a methyl ether group while the other hydroxy group is bonded to an electrophile functional group (Abuchowski, A. and Davis, F. F. (1981), in Enzymes as Drugs (Holsenberg, J. and Roberts, J. , eds . ) ) . Examples of activated polymers include PEG-N-hydroxysuccmeimide-activated esters, which contain amide bonds, PEG-epoxides and PEG- tresylate, which contain alkyl bonds, PEG-carbonyl lmidazole and PEG-nitrophenyl carbonates, which contain urethane bonds, PEG-aldehyde, which contains a Schiff' s base at the N-terminus .
On a polypeptide sequence, lysme residues are randomly located, so that PEG is non-specifically bonded to the polypeptide. In order to obtain uniform PEG-peptide conjugates, there have been made attempts of bonding PEG to targeted sites such as cystein residues, oligo sugars, hydroxy groups, argmine groups.
Examples of PEG derivatives able to specifically react to cystein groups of polypeptides include PEG- viπyl sulphone, PEG-iodoacetamide, PEG-maleimide, and PEG-orthopyridyl disulfide with most preference to male mide-containing PEG. PEG-vmyl sulfone is best m view of the stability in water solutions while PEG- orthopyridyl disulfide can be reversibly degraded in vivo because of the presence of disulfide bonds. Peptides taking advantage of these derivatives can be exemplified by mterleu ιn-3 and ιnterleukιn-2.
PEG derivatives reactive specifically to oligo sugars of polypeptides may be exemplified by PEG- hydrizide, which is able to react with aldehyde- containing compounds to form relatively stable hydrozone bonds. Advantage is taken of the specific bonding of PEG-hydrizides to sugar moieties of glycoproteins .
PEG-isocyanates react specifically with hydroxy groups of polypeptides. In order to conjugate PEG to argmine residues of polypeptides, there is used PEG derivatives containing phenylglyoxal, which is highly reactive to the guanidmo group. As mentioned above, the nasal transmucosal delivery of peptides alone is significantly improved absorption efficiency compared with the oral administration because the peptides are not subjected to liver metabolism, but poor in the bioavailability of the peptides because they are degraded by endogenous enzymes.
To overcome the foregoing and other disadvantages, we, the inventors of the present invention, have developed a pharmaceutical composition for the nasal transmucosal delivery, comprising a sparingly soluble, biologically active polypeptide conjugated with an activated biocompatible polymer. The present invention have confirmed that, when the polymer- peptide conjugate is administered through the nasal cavity, it is improved in water solubility and protected from being degraded by proteases, whereby the medicinal activity of the pharmaceutical composition can be sustained for an extended period of
Figure imgf000010_0001
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a biologically active peptide-polymer conjugate suitable for use in a nasal transmucosal delivery.
Further objects and advantages of the present invention will appear hereinafter.
The present invention provides a pharmaceutical composition for the nasal transmucosal delivery, comprising a sparingly soluble, biologically active polypeptide conjugated with an activated biocompatible polymer.
Further features of the present invention will appear hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 represents a result of size exclusion chromatography which isolates sCT and PEG-sCT, where
Figure imgf000011_0001
C ; mono-PEG-sCT,
D ; sCT.
FIG. 2 shows a pH effect to the production of mono-PEG-sCT that PEG is conjugated with N-termmus of calcitonin, Lys18 or Lys11, where I ; mono-PEG-sCT (N-terminal conjugate) ,
D ; mono-PEG-sCT (Lys18-conjugate) ,
Δ ; mono-PEG-sCT (Lysu-conj ugate ) . FIG. 3 shows a molar ratio effect of calcitonm : sCT to the production of mono-PEG-sCT that PEG is conjugated with N-termmus of calcitonm, Lys18 or Lys11, where Φ ; mono-PEG-sCT (N-termmal conjugate),
D ; mono-PEG-sCT (Lys18-conjugate) ,
Δ ; mono-PEG-sCT (Lys -conj ugate) .
FIG. 4 shows a reaction time effect of calcitonm : sCT to the production of mono-PEG-sCT that PEG is conjugated with N-termmus of calcitonm, Lys18 or Lys11, where
Φ ; mono-PEG-sCT (N-termmal conjugate),
D ; mono-PEG-sCT (Lys18-conjugate) ,
Δ ; mono-PEG-sCT (Lys -conjugate) . FIG. 5 represents a result of size exclusion chromatography which isolates hPTH and PEG-hPTH ( 1-34 ) , where
A ; tπ-PEG-hPTH(l-34) ,
B ; di -PEG-hPTH ( 1 - 34 ) , C ; mono-PEG-hPTH ( l - 34 ) ,
D ; hPTH ( l - 34 ) .
FIG. 6 shows a reduction effect of calcitonm concentration m blood when sCT and PEG-sCT conjugate are administered through the nasal cavity, where A ; sCT,
B ; mono-PEG5000-sCTs,
C ; mono-PEG2000-sCTs, D ; mono-PEG12000-sCTs.
FIG. 7 shows a calcium reduction effect of isolated mono-PEG-sCT isomers, where
Φ ; mono-PEG-sCT (N-terminal conjugate, Ml), ■ ; mono-PEG-sCT (Lys18-conjugate, M2 ) ,
♦ ; mono-PEG-sCT (Lysn-conjugate, M3), Δ ; sCT, D ; mono-PEG-sCT not isolated its isomers.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The term "biocompatible polymers" as used herein means naturally occurring or synthetic compounds which are dissolved in water. By way of example, not limitation, the biocompatible polymers include polyethylene glycol, polypropylene glycol (PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and its derivatives, polyacrylic acid and their derivatives, polyamino acid, polyvinyl alcohols, polyurethane, polyphosphazene, poly(L- lysine) , polyalkylene oxide (PAO), and water-soluble polymers such as polysaccharide, dextran, and non- immumogenic polymers such as polyvinyl alcohol and polyacryl amide. Available in the present invention are the polymers ranging, in molecular weight, from about 200 to 20,000 and preferably from 500 to 12,000. The present invention provides a peptide-polymer conjugate for a nasal transmucosal delivery, which can be prepared by bonding an activated polymer to a biologically active peptide. In this regard, the bond between the peptides and the polymers may be a covalent bond or a non-covalent bond such as a lipophilic bond or a hydrophobic bond.
In preparing the peptide-polymer conjugates of the present invention, the molar ratio of polypeptides to activated polymers are in the range of about 1:1 to 1:10 and preferably in the range of 1:1 to 1:7. In addition, one to three activated polymers may be conjugated to one polypeptide molecule. Peptide-mono polymer conjugates exert the most effective pharmaceutical activity. In the case of calcitonin- PEG conjugates, PEG can be conjugated to the N- termmus of calcitonm and/or Lys18 and/or Lys11. Of the resulting calcitonm-PEG conjugates, the calcitonm- (mono) PEG conjugate in which one PEG molecule is conjugated to one calcitonm molecule exhibits the highest calcium reduction effect. A conjugate isomer m which PEG is conjugated to the N- termmus or the Lys18 shows more sustained and effective calcium reduction activity than does a conjugate isomer in which PEG is conjugated to Lys11. At pH 5, 6 or 7 of the reaction solution, more than 80 % of PEG is conjugated to the N-termmus of calcitonm. On the other hand, at pH 8 or higher, PEG is increasingly conjugated to Lys11 and Lys18. In contrast, changes in reaction time and molar ratio cannot affect the proportion of isomers.
The binding reaction of the peptide-activated polymer in the present invention is performed m 0.1 M phosphate buffer ranged, m pH, from 6 to 9, at 0 to 25C° of reaction temperature for several minutes to 12 hours.
A method of polymer activation consists of the following steps of; preparing the polymer into polyalkylene oxide (hereinafter, referred to as "PAO") such as monoethoxy-poly (ethylene glycol, mPEG) ; and changing the other part of PAO into a reaction group having reactivity. The activated polymer forms the prptide-polymer conjugate by reacting with ε-amme group of lysme. Besides the amme group of lysme, carboxy group, activated carbonyl group, oxidized sugar and mercapto group m the peptide can be used as a conjugated moiety.
The present inventors have measured the blood concentration of peptide according to time after the peptide-polymer conjugate is administered to rats through the nasal cavity. Thus, the present inventors have confirmed that the peptide by the nasal transmucosal delivery has a better stability living body and sustains its biological activity for a long time .
The peptide of the present invention is not limited to the specific therapeutic agents but applied to the all substances having biological activity, particularly, it is desirable to use calcitonin, parathyroid hormone (hereinafter, referred to as "PTH"). insuline, synthetic enkephalin, growth hormone releasing peptide (hereinafter, referred to as "GHRP"), leutenizing hormone releasing hormone (hereinafter, referred to as "LHRH") and its derivatives, secretory components of hypothalamus, calcitonin gene related peptide (hereinafter, referred to as "CGRP") and thyroid stimulating hormone and thymic humoral factor (hereinafter, referred to as "THF").
Calcitonin is a single chain peptide composed of 32 amino acids, forms a ring at N-terminus and has a proline amide group at C-terminus. Calcitonin inhibits a bone absorption by acting directly to osteoclast and is used for cure of hypercalciumia, Pajet's disease, pain from the bone absorption and osteporosis. Calcitonin is produced in salmon, eel, human, pig, etc., and salmon and eel calcitonin have the most effect. Parathyroid hormone (PTH) is a peptide hormone composed of 84 ammo acids and secreted from parathyroid. Since parathyroid cell has a recognition site for calcium concentration, PTH secretion increases when calcitonm is lower, and PTH secretion decreases when calciton higher. PTH increases a calcium absorption taken from foods at the small intestine, transfers calcium from bone to blood, and , finally, increases a blood calcium concentration. The major active site of PTH is adrenal cortex. PTH binds to the membrane of adrenal cortex to increase the production of cAMP, IP3(mosιtol triphosphate) and DAG(dιacyl glycerol) .
Insulin is a peptide composed of A chain and B chain. A chain composed of 21 ammo acids and B chain composed of 30 ammo acids are connected by 1 pair of disulfide bond. Disulfide bond exists between 6th ammo acid of A chain and 11th ammo acid of B chain. When blood sugar level is increased, insulin is immediately excreted from pancreas. Sugar is, then, stored n the form of glycogen by insulin or used as energy source for fat or protein synthesis. In addition, insulin plays an important role in homeostasis of calcium. When insulin presents at a high concentration, calcium uptake is increased from cell exterior to cell interior and hypercalciumia is induced.
Enkephalm is a pentapeptide representing a similar action to opium. Enkephalm is devided into two groups of methionme enkephalm and leucme enkephalm according to C-termmus bounded to Try-Gly- Gly-Phe. Enkephalm inhibits pain from transferring to bram by preventing a release of substance P from the end of analgesic nerve fiber.
Growth hormone releasing peptide (GHRP) presents m the form of hepta and hexa and affects a release of growth hormone. The growth hormone releasing effect of GHRP is related to a dose, increases until aldolescence and decreases after then.
GHRP-6 is a hexapeptide having a structure of Hιs-Asp-Try-Ala-Try-Asp-Lys-NH2, and is very stable in acetate buffer ranged, in pH, from 5.5 to 6.0. In case of oral administration, bioavailability of GHRP-6 is 0.3%, an absorption half life is 15 minutes, and an elimination half life is 60 minutes. GHRP-6 secrets selectively growth hormone through a receptor in hypothalamus and pituitary gland.
Leutemizmg hormone releasing hormone (LH-RH) is a hypothalamus peptide and stimulates the relases of LH and follicle stimulating hormone. LH-RH regulates a function of brain and many peripheral organs by binding to receptors on the targeted cell surface. . LH-RH has a decapeptide structure of Pyro-Glu-His-Trp-Ser-Tyr-Gly- Leu-Arg-Pro-Gly-NH2, and is degraded in tubular of the kidney. By way of examples, LH-RH derivatives include nafarelin, busecilin, zilidexin, etc.
Pharmaceutical composition for the nasal transmucosal delivery including the peptide-polymer conjugate of the present invention can be formulated into the suitable form, and mainly administered by spray as a medicine for external use.
For preparation of spray, the peptide-polymer conjugate is dissolved in a solvent, or a suspended, medicinal solution is filled in a container having a specific spraying device (valve) with a low viscous spraying agent. For this, the medicinal solution is sprayed in the type of smog using pressure. The material quality of container uses a metal such as a tinned iron and aluminum.
If necessary, the inside of container is coated with moth-proof painting. When a internal volume is lower than 100 ml, the container made of glass or synthetic resin is possible. The spraying agent uses generally a compressed air and an incombustible liquefied gas, freon(freon 11, CC13F; freon 12, CC1£F; freon 114, C2C12F4). A dose of the pharmaceutical compositions containing the peptide-polymer conjugate of the present invention is 0.1 ug - 10 mg/kg/day, and can be widly altered according to the kind of peptide and patient's condition.
EXAMPLES
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1 : Preparation of PEG-sCT
<!-!> Preparation of PEG5000-sCT
According to the method of Abuchowski (Abuchowski et al , Cancer Biochem . Bi ophys . , 1 , 175-86, 1984), monomethoxy-poly (ethylene glycol) was prepared from PEG (MW 5000) so that one hydroxyl group of PEG was protected. Phosgene and N-hydroxysuccmimide was added to it to activate in the form of succmyl-N- hydroxysuccimide ester (hereinafter, referred to as "SS-PEG"). 4.38 mg of the activated SS-PEG was dissolved in phosphate buffer (pH 8.0), added with 0.2 ml of salmon calcitonin (hereinafter, referred to as
"sCT") (Novabiochem, LA Jolla, CA, USA) (5mg/ml, 0.1 M phosphate buffer, pH 8.0), and stirred for 30 minutes at ambient temperature. 0.1 M glycine was added to stop the reaction. Unreacted calcitonin and PEG were removed by analysis using phosphate buffered saline
(hereinafter, referred to as "PBS", pH 7.4) to obtain PEG5000-SCT.
<l-2> Preparation of PEG12000-SCT
PEG (MW 12000) was activated with succinimidyl succinate, and PEG12000-sCT was prepared by using 9.2 mg of the activated SS-PEG. All procedures were performed as indicated in the Example <1-1>.
<l-3> Preparation of PEG2000-sCT
PEG (MW 2000) was activated with succinimidyl succinate, and PEG2000-sCT was prepared by using 2 mg of the activated SS-PEG. All procedures were performed as indicated in the Example <1-1>.
Example 2: Isolation of PEG-sCT PEG-sCT conjugates prepared in the Examples <1-1> to <l-3> were isolated with the aid of size exclusion column (Super HR 12/30, PharmaciaLKB, Sweden) using PBS (pH 7.0) as eluent at 0.4 ml/min of flow rate (FIG. 1). The peptides isolated from the column as a time difference according to the molecular size was isolated into each peak using fluorescence spectrometer (Hitach, Japan) . Each isolated fraction was concentrated using Centricon-10 (Amicon, USA) and stored at refrigerator. FIG. 1 represented a fluorescence intensity of each peak measured by fluorescence spectrometer wherein each peak was eluted from the size-exclusion column.
As illustrated in the FIG. 1, tri-PEG-sCT which was conjugated 3 molecules of PEG with 1 molecule of calcitonin and had a biggest molecular size, was firstly eluted from the column and collected within 30 minutes after sample injection into the column. After the tri-PEG-sCT, di-PEG-sCT and mono-PEG-sCT were isolated in succession. In addition, since calsitonin molecule conjugated with PEG had the smallest molecular size, it was eluted last from the column and time for elution took more 40 minutes. That time, the fluorescence intensity of each peak eluted from the column had measured by the fluorescence spectrometer directly connected to the column, and each peak was collected. Example 3 : Preparation of PEG-sCT According to Various pHs , Molar Ratios and Reaction Times
<3-l> Preparation of PEG-sCT According to pH
To 88.2 μl of each of 0.1 M phosphate buffered solutions ranging, in pH, from 5 to 9, 6.8 μl of calcitonin (10 mg/ml) and 5 μl of SS-PEG (60 mg/ml) were added and stirred for 30 min at ambient temperature. Addition of 5 μl of 1 M glycine ceased the reaction between calcitonin and SS-PEG. Unreacted calcitonin and PEG were removed by dialysis using PBS (pH 7.4) .
<3-2> Preparation of PEG-sCT According to Molar Ratio of SS-PEG/Calcitonin
To 6.8 μl of calcitonin (10 mg/ml), 10 μl of SS- PEG solutions were added to give mixtures of calcitonin and SS-PEG at molar ratios of 1:1, 1:2, 1:3, 1:5 and 1:10 and these five mixtures were added with 78.2 μl of a PBS (pH 8.0). From them, PEG-sCT was prepared in the same procedure as in Example <3-l>.
<3-3> Preparation of PEG-sCT According to Reaction Time 6.8 μl of calcitonin (10 mg/ml) was added with 10 μl of an SS-PEG solution to give a mixture of calcitonin and SS-PEG in a molar ratio of 1:3, and then, five copies of reaction solutions were prepared by adding to the mixture with 78.2 μl of PBS (pH 8.0). Five copies of this solution were allowed to react at room temperature for 5, 10, 20, 30 and 60 min with stirring to prepare PEG-sCT as indicated in Example <3-l>.
Example 4 : Isolation of Mono-PEG-Calcitonin Isomer
With the aid of size-exclusion columns, PEG-sCT conjugates prepared in Examples <3-l> to <3-3> were separated into tri-PEG-sCT, di-PEG-sCT and mono-PEG- sCT . This separation was conducted in a similar manner to that of Example 2.
The mono-PEG-sCT obtained was divided into three isomers by reverse phase high performance liquid chromatography (HPLC) . In this regard, 100RP-8
(4.0X125 MM, 5μM, Merck) used as a column while a linear gradient of pentafluoropropionic acid (PFPA)- containing acetonitrile was used as a mobile phase.
The gradient condition was changed with 36-42 % solvent B (0.1 % PFPA added acetonitrile) and 64-58 % solvent A (0.1% PFPA added distilled water).
Quantitative measurements were made using a UV absorption meter (215 mm) or a fluorescent absorption meter (excitation 280 nm, emissm 315 nm) .
Ammo acid analysis showed that the three mono- PEG-sCT isomers thus separated were identified to contain PEG conjugated to the N-termmus, Lys18 and Lys11 of calcitonm, respectively. In addition, it was found that when the reaction solution was 5, 6 or 7 m pH, more than 80 % of PEG was selectively conjugated to the N-termmus. At pH 8 or higher, PEG was increasingly conjugated to the Lys11 and the Lys18, as shown m Fig. 2. However, there were no differences in the proportion of isomers when the reaction time and the molar ratio were changed, as shown in Figs. 3 and 4.
Example 5: Preparation and Isolation of PEG-PTH (1-34)
<5-l> Preparation and Isolation of PEG5000-PTH (1-34)
50 μl of SS-PEG5000 (23 mg/0.20 ml, MW 5,000,
0.1 M PBS, pH 7.0) was added with 50 μl of a PTH solution (2.4 mg/0.15 ml, 0.1 M PBS, pH 7.0) to give a mixture containing a molar ratio of 5:1 of SS- PEG5000:PTH. The mixture was allowed to react for 30 mm at ambient temperature with shaking. After 30 mm of the reaction, an excess amount of a 0.1 M glycme solution was added to stop the reaction. Unreacted parathyroid gland hormone and PEG were removed by dialysis using PBS to obtain PEG5000-PTH (1-34 ) .
The PEG 5000-PTH (1-34) conjugate thus prepared was purified by size-exclusion chromatography using an HPLC system and superdex. For this, PBS (pH 7.0) was used as a developing buffer at a flow rate of 0.8 ml/min. Eluted fractions were stored at 4C° until they were measured for UV absorbance. UV absorbance was measured at 215 nm while the excitation and emission waves of fluorescence were fixed in the range of 280 to 315 nm, as shown in Fig. 5.
<5-2> Preparation and Isolation of PEG2000-PTH (1-34)
PEG2000-PTH (1-3 ) conjugates were prepared and isolated in a similar manner to that of Example <5-l> except that SS-PEG2000 was used at an amount of 2 mg .
Example 6: Preparation and Isolation of PEG-GHRP
<6-l> Preparation and Isolation of PEG5000-GHRP-6
50 μl of SS-PEG5000 (23 mg/0.20 ml, MW 5,000,
0.1 M PBS, pH 7.0) was added with 50 μl of a GHRP-6 solution (0.6 mg/0.15 ml, 0.1 M PBS, pH 7.0) to give a mixture containing a molar ratio of 5:1 of SS-PEG:
GHRP-6, prepared PEG-GHRP-6, and isolated and purified as indicated in the Example <5-l>. Isolated mono-PEG- GHRP-6 was immediately frozen-dried by freeze dryer.
<6-2> Preparation and Isolation of PEG2000-GHRP-6
Except for use of PEG2000, PEG2000 : GHRP-6 conjugate as indicated in the Example <6-l>.
Example 7: Preparation and Isolation of PEG-LHRH
<7-l> Preparation and Isolation of PEG5000-LHRH
50 μl of SS-PEG5000 (23 mg/0.20 ml, MW 5,000, 0.1 M PBS, pH 7.0) was added with 50 μl of a LHRH solution (0.5 mg/0.1 ml, 0.1 M PBS, pH 7.0) to give a mixture containing a molar ratio of 1:5 of LHRH: SS-PEG, prepared PEG-LHRH, and isolated and purified as indicated in the Example <5-l>.
<7-2> Preparation and Isolation of PEG2000-LHRH
Except for use of PEG2000, PEG2000-LHRH conjugate was prepared, isolated and purified as indicated in the Example <7-l>.
Example 8: Preparation and Isolation of PEG5000- triptorelin Except for use of triptorelm, LHRH derivative, instead of LHRH, PEG5000-trιptorelm conjugate was prepared, isolated and purified as indicated m the Example <7-l>.
Example 9: Preparation and Isolation of PEG5000- orntide
Except for use of orntide, LHRH agonist, instead of LHRH, PEG5000-orntιde conjugate was prepared, isolated and purified as indicated in the Example <7-l>,
Experimental Example 1 : Comparison of Calcium Reduction Effect of sCT and PEG-sCT
SD (Spraque Dawley) male rat (220 - 300 g, Charles River Japan, Atsugi, Japan) was put under anesthesia by administrating 45 mg/kg of peptobarbital into terperitoneal . Bronchus and esophagus were cannulated with PE-250 tube and thigh arteria with SP- 45 tube. 20 ul of a placebo and experimental drugs were administered into the nasal cavity of rats with Hamilton syringe. The placebo used physiological saline and the experimental drug used PEG5000-SCT, PEG12000-sCT, PEG2000-sCT and sCT prepared from the Example 2. Salmon calcitonm was administered at dose of 0.05 to 4.5 IU per rat, and rats were grouped into 4. 200 ul of blood was taken at before, 5, 10, 30, 60, 120, 240, 480 and 360 minutes after the placebo and the experimental drugs were administered, and immediately centrifuged to obtain 100 ul of plasma. The calcium concentration m plasma was measured using a calcium assay kit (Sigma, USA), and calcium remaining efficiency was calculated as the calcium concentration before administration was 100. The unit of the measured calcium concentration in plasma was mg/dl, and the remaining efficiency was calculated as the ratio (%) to the primary value.
After the remaining coefficient was calculated according to each time, area-under the curve (AUC) was calculated about the average value of remaining coefficient at each time per experimental group. The reduction ratio of calcium concentration was calculated by the mathematical formula 1, and the results were represented in the table 1(FIG. 6) .
Mathematical formula 1>
Δ D ( % ) = ( AUCca-plac-AUCCa-sCτ ) / AUCCa-plac X 100
where, AUCCa-piac was AUC from placebo administration to 960 minutes, and AUCCa-scτ was AUC from calcitonm and experimental drugs administration to 960 minutes .
<Table 1> The reduction ratio of calcitonm concentration in plasma at each time (n=4)
Figure imgf000030_0001
As illustrated in the table 1, the calcium concentration of calcitonm conjugated without any PEG was decreased for 1 hour after the nasal transmucasal administration, but restored at the original concentration after 2 hours. However, when calcitonm conjugated with PEG was administered, the calcitonm reduction effect represented for 4 hours to 8 hours after the nasal transmucasal administration. In addition, the calcium reduction effect was significantly altered according to the molecular size of PEG.
Namely, in case of lower 5,000 of molecular weight, the calcium reduction effect was similar sustained, but, in case of 12,000, the calcium reduction effect was not sufficient. This result was because that the more the molecular weight of PEG was increased, the more the degree of absorption into body through the nasal transmucosal was decreased. Consequently, calciton conjugated with the lower molecular weight of PEG had a better calcitonm reduction effect than calciton conjugated without any PEG, and its calcium reduction effect was sustained for a long time.
Therefore, the nasal transmocosal delivery using the PEG conjugates of the present invention could reduce a used amount of calcitonm and side effects of drugs .
Experimental Example 2 : Comparison of Calcium Reduction Effect of Mono-PEG-sCT Isomers
The calcium reduction effect of the mono-PEG-sCT isomers prepared from the Example 4 was measured as indicated in the Example 1 (FIG. 7) . As illustrated in the FIG. 7, isomers conjugated w th PEG at N-termmus and Lys18 represented a better sustaining and effective calcium reduction effect than isomers conjugated with PEG at Lys11. In addition, in case of mono-PEG-sCT not isolated, Lys18 isomers having a lower calcium reduction effect than N-termmus isomers represented a considerable reduction effect to sCT or Lys11 isomers.
Example 10: Preparation and Isolation of PEG2000-GLP- 1 (glucagons like peptide)
PEG (MW 2000) was activated with succinimidyl succinate, and PEG2000-GLP-1 was prepared by using 2 mg of the activated SS-PEG. All procedures were performed as indicated m the Example <1-1>. In addition, PEG2000-GLP-1 was isolated and purified as indicated in the Example <5-l>.
INDUSTRIAL APPLICABILITY
A pharmaceutical composition comprising a peptide-polymer conjugate for a nasal transmucosal delivery of the present invention increases the water solubility of peptide, which is sparingly soluble m water, improves a stability by protecting from being degraded by protease, and, consequently, reduces an administration number of drug to decrease side-effects induced by drug abuse.
In addition, since the pharmaceutical composition comprising a peptide-polymer conjugate for the nasal transmucosal delivery of the present invention is delivered through the nasal cavity, it allows drug activity to be expressed in a short period of time and improves a bioavailability .
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

What is Claimed is
1. A pharmaceutical composition for a nasal transmucosal delivery which is conjugated a biologically active peptide with an active biocompatible polymer.
2. The pharmaceutical composition for the nasal transmucosal delivery according to claim 1, wherein the peptide is selected from the group of calcitonm, parathyroid hormone (PTH), insulin, synthetic enkephalm, growth hormone releasing peptide (GHRP) , lutemizmg hormone releasing hormone (LHRH) and its derivatives, secretory components of hypothalamus, calcitonm gene related peptide (CGRP) , thyroid stimulating hormone and thymic humoral factor (THF) .
3. The pharmaceutical composition for the nasal transmucosal delivery according to claim 1, wherein the biocompatible polymer has 200-20,000 of the molecular weight.
4. The pharmaceutical composition for the nasal transmucosal delivery according to claim 1, wherein the biocompatible polymer has 500-12,000 of the molecular weight.
"> ->
5. The pharmaceutical composition for the nasal transmucosal delivery according to claim 1, wherein the biocompatible polymer is selected one from the group of polyethylene glycol, polypropylene glycol (PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and its derivatives, polyacrylic acid and their derivatives, polyammo acid, polyvinyl alcohols, polyurethane, polyphosphazene, poly (L-lysme) , polyalkylene oxide (PAO), and water-soluble polymers such as polysacchaπde, dextran, and non-immumogenic polymers such as polyvinyl alcohol and polyacryl amide.
6. The pharmaceutical composition for the nasal transmucosal delivery according to claim 1, wherein a molar ratio of the peptide and the active biocompatible polymer is 1:1 to 1:7.
7. The pharmaceutical composition for the nasal transmucosal delivery according to claim 1, wherein a reaction pH of the peptide and the active biocompatible polymer is 5.0 to 8.0.
8. The pharmaceutical composition for the nasal transmucosal delivery according to claim 1, which is conjugated 1 to 3 molecules of the active biocompatible polymer with 1 molecule of the peptide .
9. The pharmaceutical composition for the nasal transmucosal delivery according to claim 8, which is conjugated 1 to 3 molecules of PEG with 1 molecule of calciton .
10. The pharmaceutical composition for the nasal transmucosal delivery according to claim 9, wherein PEG conjugated at N-termmus, Lys18 or Lys11 of calciton .
33
PCT/KR2000/000868 1999-08-17 2000-08-07 The nasal transmucosal delivery of peptides conjugated with biocompatible polymers WO2001012230A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU64784/00A AU6478400A (en) 1999-08-17 2000-08-07 The nasal transmucosal delivery of peptides conjugated with biocompatible polymers
EP00952020A EP1204427A4 (en) 1999-08-17 2000-08-07 The nasal transmucosal delivery of peptides conjugated with biocompatible polymers
JP2001516573A JP2003507344A (en) 1999-08-17 2000-08-07 Nasal mucosal delivery of peptides conjugated with biocompatible polymers

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1999/33984 1999-08-17
KR1019990033984A KR100345214B1 (en) 1999-08-17 1999-08-17 The nasal transmucosal delivery of peptides conjugated with biocompatible polymers

Publications (1)

Publication Number Publication Date
WO2001012230A1 true WO2001012230A1 (en) 2001-02-22

Family

ID=19607583

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2000/000868 WO2001012230A1 (en) 1999-08-17 2000-08-07 The nasal transmucosal delivery of peptides conjugated with biocompatible polymers

Country Status (6)

Country Link
US (1) US6506730B1 (en)
EP (1) EP1204427A4 (en)
JP (1) JP2003507344A (en)
KR (1) KR100345214B1 (en)
AU (1) AU6478400A (en)
WO (1) WO2001012230A1 (en)

Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001024831A2 (en) * 1999-10-04 2001-04-12 Shearwater Corporation Polymer stabilized neuropeptides
US6815530B2 (en) 2001-06-04 2004-11-09 Nobex Corporation Substantiallymonodispersed mixtures of polymers having polyethylene glycol moieties
EP1477496A1 (en) * 2002-02-01 2004-11-17 Chugai Seiyaku Kabushiki Kaisha Peg-conjugated pth or peg-conjugated pth derivate
WO2005007714A1 (en) * 2003-07-18 2005-01-27 Japan Exlan Company Limited Polymer sustainedly releasing amino acid derivative, cosmetic and fiber construct containing the polymer, method of producing the same and method of regenerating the same
EP1569676A2 (en) * 2002-11-08 2005-09-07 Nobex Corporation Calcitonin drug-oligomer conjugates, and uses thereof
WO2006135930A2 (en) * 2005-06-13 2006-12-21 Nastech Pharmaceutical Company Inc. Transmucosal delivery of peptide derivatives
EP1779864A1 (en) * 2004-07-14 2007-05-02 Chugai Seiyaku Kabushiki Kaisha Pth-containing preparation for transmucosal administration
WO2008089426A2 (en) * 2007-01-19 2008-07-24 Hananja Ehf Methods and compositions for the delivery of a therapeutic agent
EP1973952A1 (en) * 2006-01-23 2008-10-01 Gwangju Institute of Science and Technology Conjugate comprising pharmaceutical active compound covalently bound to mucoadhesive polymer and transmucosal delivery method of pharmaceutical active compound using the same
US7786133B2 (en) 2003-12-16 2010-08-31 Nektar Therapeutics Chemically modified small molecules
US7872095B2 (en) 2004-07-19 2011-01-18 Biocon Limited Insulin-oligomer conjugates, formulations and uses thereof
CN101531756B (en) * 2009-04-16 2011-06-15 山东大学 Method for preparing water soluble polyethylene glycol/polylactic segmented polycarbonate resin
US8034618B2 (en) 2001-12-18 2011-10-11 Eldgenossische Technische Hochschule Zurich PTH containing cell growth matrix
US8067431B2 (en) 2003-12-16 2011-11-29 Nektar Therapeutics Chemically modified small molecules
US8114383B2 (en) 2003-08-06 2012-02-14 Gruenenthal Gmbh Abuse-proofed dosage form
US8226942B2 (en) 2007-12-28 2012-07-24 Kuros Biosurgery Ag PDGF fusion proteins incorporated into fibrin foams
US8293869B2 (en) 2005-12-16 2012-10-23 Nektar Therapeutics Polymer conjugates of GLP-1
US8309122B2 (en) 2001-07-06 2012-11-13 Endo Pharmaceuticals Inc. Oxymorphone controlled release formulations
US8323889B2 (en) 2004-07-01 2012-12-04 Gruenenthal Gmbh Process for the production of an abuse-proofed solid dosage form
US8329216B2 (en) 2001-07-06 2012-12-11 Endo Pharmaceuticals Inc. Oxymorphone controlled release formulations
US8633153B2 (en) 2002-04-04 2014-01-21 Amgen Inc. Transthyretin variants
US9161917B2 (en) 2008-05-09 2015-10-20 Grünenthal GmbH Process for the preparation of a solid dosage form, in particular a tablet, for pharmaceutical use and process for the preparation of a precursor for a solid dosage form, in particular a tablet
US9241908B2 (en) 2007-10-16 2016-01-26 Biocon Limited Orally administrable solid pharmaceutical composition and a process thereof
US9289432B2 (en) 2007-01-19 2016-03-22 HANANJA EHF and UNIVERSITY OF ICELAND Methods and compositions for the delivery of a therapeutic agent
US9629807B2 (en) 2003-08-06 2017-04-25 Grünenthal GmbH Abuse-proofed dosage form
US9636303B2 (en) 2010-09-02 2017-05-02 Gruenenthal Gmbh Tamper resistant dosage form comprising an anionic polymer
US9655853B2 (en) 2012-02-28 2017-05-23 Grünenthal GmbH Tamper-resistant dosage form comprising pharmacologically active compound and anionic polymer
US9675610B2 (en) 2002-06-17 2017-06-13 Grünenthal GmbH Abuse-proofed dosage form
US9737490B2 (en) 2013-05-29 2017-08-22 Grünenthal GmbH Tamper resistant dosage form with bimodal release profile
US9750701B2 (en) 2008-01-25 2017-09-05 Grünenthal GmbH Pharmaceutical dosage form
US9855263B2 (en) 2015-04-24 2018-01-02 Grünenthal GmbH Tamper-resistant dosage form with immediate release and resistance against solvent extraction
US9872835B2 (en) 2014-05-26 2018-01-23 Grünenthal GmbH Multiparticles safeguarded against ethanolic dose-dumping
US9913814B2 (en) 2014-05-12 2018-03-13 Grünenthal GmbH Tamper resistant immediate release capsule formulation comprising tapentadol
US9925146B2 (en) 2009-07-22 2018-03-27 Grünenthal GmbH Oxidation-stabilized tamper-resistant dosage form
US10058548B2 (en) 2003-08-06 2018-08-28 Grünenthal GmbH Abuse-proofed dosage form
US10064945B2 (en) 2012-05-11 2018-09-04 Gruenenthal Gmbh Thermoformed, tamper-resistant pharmaceutical dosage form containing zinc
US10080721B2 (en) 2009-07-22 2018-09-25 Gruenenthal Gmbh Hot-melt extruded pharmaceutical dosage form
US10154966B2 (en) 2013-05-29 2018-12-18 Grünenthal GmbH Tamper-resistant dosage form containing one or more particles
US10201502B2 (en) 2011-07-29 2019-02-12 Gruenenthal Gmbh Tamper-resistant tablet providing immediate drug release
US10300141B2 (en) 2010-09-02 2019-05-28 Grünenthal GmbH Tamper resistant dosage form comprising inorganic salt
US10335373B2 (en) 2012-04-18 2019-07-02 Grunenthal Gmbh Tamper resistant and dose-dumping resistant pharmaceutical dosage form
US10449547B2 (en) 2013-11-26 2019-10-22 Grünenthal GmbH Preparation of a powdery pharmaceutical composition by means of cryo-milling
US10624862B2 (en) 2013-07-12 2020-04-21 Grünenthal GmbH Tamper-resistant dosage form containing ethylene-vinyl acetate polymer
US10695297B2 (en) 2011-07-29 2020-06-30 Grünenthal GmbH Tamper-resistant tablet providing immediate drug release
US10729658B2 (en) 2005-02-04 2020-08-04 Grünenthal GmbH Process for the production of an abuse-proofed dosage form
US10842750B2 (en) 2015-09-10 2020-11-24 Grünenthal GmbH Protecting oral overdose with abuse deterrent immediate release formulations
US11844865B2 (en) 2004-07-01 2023-12-19 Grünenthal GmbH Abuse-proofed oral dosage form

Families Citing this family (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6191105B1 (en) * 1993-05-10 2001-02-20 Protein Delivery, Inc. Hydrophilic and lipophilic balanced microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents such as insulin
KR19980018262A (en) * 1996-08-01 1998-06-05 윌리엄 비.켐플러 I / O port and RAM memory addressing technology
US6638906B1 (en) * 1999-12-13 2003-10-28 Nobex Corporation Amphiphilic polymers and polypeptide conjugates comprising same
US20040096437A1 (en) * 2000-07-27 2004-05-20 Bon-Hong Min Pharmaceutical composition comprising arginine deiminasa for inhibiting angio-genesis
WO2002009741A1 (en) * 2000-07-27 2002-02-07 Angiolab, Inc. The pharmaceutical composition comprising arginine deiminase for inhibiting angiogenesis
US6867183B2 (en) * 2001-02-15 2005-03-15 Nobex Corporation Pharmaceutical compositions of insulin drug-oligomer conjugates and methods of treating diseases therewith
US7060675B2 (en) * 2001-02-15 2006-06-13 Nobex Corporation Methods of treating diabetes mellitus
US20090281023A9 (en) * 2001-06-04 2009-11-12 Nobex Corporation Mixtures Of Calcitonin Drug-Oligomer Conjugates And Methods Of Use In Pain Treatment
WO2005004792A2 (en) * 2003-06-24 2005-01-20 Nobex Corporation Mixtures of calcitonin drug-oligomer conjugates and methods of use in pain treatment
US6713452B2 (en) * 2001-06-04 2004-03-30 Nobex Corporation Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6828305B2 (en) * 2001-06-04 2004-12-07 Nobex Corporation Mixtures of growth hormone drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6828297B2 (en) * 2001-06-04 2004-12-07 Nobex Corporation Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
BR0210855A (en) * 2001-07-06 2006-10-24 Penwest Pharmaceuticals Compan Method of Manufacturing Extended Release Formulations
US7196059B2 (en) * 2001-09-07 2007-03-27 Biocon Limited Pharmaceutical compositions of insulin drug-oligomer conjugates and methods of treating diseases therewith
AU2002337686B2 (en) * 2001-09-26 2008-05-15 Penwest Pharmaceuticals Company Opioid formulations having reduced potential for abuse
BR0314619A (en) * 2002-09-18 2005-08-02 Univ Montreal Ct Hospitalier Chum Ghrh's analogs
WO2004043396A2 (en) * 2002-11-09 2004-05-27 Nobex Corporation Modified carbamate-containing prodrugs and methods of synthesizing same
EP1569683B1 (en) * 2002-11-26 2010-03-03 Biocon Limited Modified natriuretic compounds, conjugates, and uses thereof
US7648962B2 (en) * 2002-11-26 2010-01-19 Biocon Limited Natriuretic compounds, conjugates, and uses thereof
KR20060135661A (en) * 2003-12-18 2006-12-29 노보 노르디스크 에이/에스 Novel glp-1 compounds
US7115561B2 (en) * 2004-09-22 2006-10-03 Patterson James A Medicament composition and method of administration
US20060147443A1 (en) * 2004-12-22 2006-07-06 Kuros Biosurgery Ag Synthetic biomaterials having incorporated therein bioactive factors through enzymatically degradable linkages
US20080207505A1 (en) * 2005-01-12 2008-08-28 James Kenneth D Bna Conjugates and Methods of Use
US20070212414A1 (en) * 2006-03-08 2007-09-13 Penwest Pharmaceuticals Co. Ethanol-resistant sustained release formulations
PL2136850T3 (en) * 2007-04-13 2012-07-31 Kuros Biosurgery Ag Polymeric tissue sealant
WO2008151512A1 (en) 2007-06-12 2008-12-18 Institute Of Pharmacology And Toxicology Academy Of Military Medical Sciences P.L.A. Site-specific pegylated linear salmon calcitonin derivatives
US20090124650A1 (en) * 2007-06-21 2009-05-14 Endo Pharmaceuticals, Inc. Method of Treating Pain Utilizing Controlled Release Oxymorphone Pharmaceutical Compositions and Instructions on Effects of Alcohol
JP5657011B2 (en) * 2009-10-29 2015-01-21 コリア アドバンスド インスティチュート オブ サイエンス アンド テクノロジィ Conjugate of catechol polyethylene glycol derivative and protein or peptide, and production method thereof
WO2015005459A1 (en) * 2013-07-10 2015-01-15 生化学工業株式会社 Pharmaceutical composition for respiratory administration
KR101964376B1 (en) 2017-09-29 2019-04-01 주식회사 나이벡 Pharmaceutical composition and Biomaterial Containing Fusion Peptide Comprising Parathyroid hormone(PTH) bonded Bone-selective Peptide

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5359030A (en) * 1993-05-10 1994-10-25 Protein Delivery, Inc. Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same
WO1998052614A2 (en) * 1997-05-21 1998-11-26 The Board Of Trustees Of The Leland Stanford Junior University Composition and method for enhancing transport across biological membranes

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61267528A (en) * 1984-11-26 1986-11-27 Yamanouchi Pharmaceut Co Ltd Transnasal calcitonin agent containing absorbefacient
US5707644A (en) * 1989-11-04 1998-01-13 Danbiosyst Uk Limited Small particle compositions for intranasal drug delivery
FR2675807B1 (en) * 1991-04-23 1994-07-01 Medgenix Group Sa CONJUGATE OF CALCITONIN AND POLYETHYLENE GLYCOL.
EP0588255A1 (en) * 1992-09-12 1994-03-23 Dott Limited Company Physiologically active peptide compositions
US5681811A (en) * 1993-05-10 1997-10-28 Protein Delivery, Inc. Conjugation-stabilized therapeutic agent compositions, delivery and diagnostic formulations comprising same, and method of making and using the same
IL118003A0 (en) * 1996-04-23 1996-08-04 Yeda Res & Dev Novel vip fragments and pharmaceutical compositions comprising them
US6165509A (en) * 1998-09-01 2000-12-26 University Of Washington Pegylated drug complexed with bioadhesive polymer suitable for drug delivery and methods relating thereto

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5359030A (en) * 1993-05-10 1994-10-25 Protein Delivery, Inc. Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same
WO1998052614A2 (en) * 1997-05-21 1998-11-26 The Board Of Trustees Of The Leland Stanford Junior University Composition and method for enhancing transport across biological membranes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1204427A4 *

Cited By (70)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001024831A3 (en) * 1999-10-04 2002-03-07 Shearwater Corp Polymer stabilized neuropeptides
WO2001024831A2 (en) * 1999-10-04 2001-04-12 Shearwater Corporation Polymer stabilized neuropeptides
US8440623B2 (en) 1999-10-04 2013-05-14 Nektar Therapeutics Polymer stabilized neuropeptides
US8008435B2 (en) 1999-10-04 2011-08-30 Nektar Therapeutics Polymer stabilized neuropeptides
US6815530B2 (en) 2001-06-04 2004-11-09 Nobex Corporation Substantiallymonodispersed mixtures of polymers having polyethylene glycol moieties
US8329216B2 (en) 2001-07-06 2012-12-11 Endo Pharmaceuticals Inc. Oxymorphone controlled release formulations
US8309122B2 (en) 2001-07-06 2012-11-13 Endo Pharmaceuticals Inc. Oxymorphone controlled release formulations
US8034618B2 (en) 2001-12-18 2011-10-11 Eldgenossische Technische Hochschule Zurich PTH containing cell growth matrix
EP1477496A1 (en) * 2002-02-01 2004-11-17 Chugai Seiyaku Kabushiki Kaisha Peg-conjugated pth or peg-conjugated pth derivate
EP1477496A4 (en) * 2002-02-01 2006-08-02 Chugai Pharmaceutical Co Ltd Peg-conjugated pth or peg-conjugated pth derivate
US8633153B2 (en) 2002-04-04 2014-01-21 Amgen Inc. Transthyretin variants
US9675610B2 (en) 2002-06-17 2017-06-13 Grünenthal GmbH Abuse-proofed dosage form
US10369109B2 (en) 2002-06-17 2019-08-06 Grünenthal GmbH Abuse-proofed dosage form
EP1569676A4 (en) * 2002-11-08 2008-10-08 Biocon Ltd Calcitonin drug-oligomer conjugates, and uses thereof
EP1569676A2 (en) * 2002-11-08 2005-09-07 Nobex Corporation Calcitonin drug-oligomer conjugates, and uses thereof
WO2005007714A1 (en) * 2003-07-18 2005-01-27 Japan Exlan Company Limited Polymer sustainedly releasing amino acid derivative, cosmetic and fiber construct containing the polymer, method of producing the same and method of regenerating the same
US10058548B2 (en) 2003-08-06 2018-08-28 Grünenthal GmbH Abuse-proofed dosage form
US10130591B2 (en) 2003-08-06 2018-11-20 Grünenthal GmbH Abuse-proofed dosage form
US8114383B2 (en) 2003-08-06 2012-02-14 Gruenenthal Gmbh Abuse-proofed dosage form
US9629807B2 (en) 2003-08-06 2017-04-25 Grünenthal GmbH Abuse-proofed dosage form
US7786133B2 (en) 2003-12-16 2010-08-31 Nektar Therapeutics Chemically modified small molecules
US9388104B2 (en) 2003-12-16 2016-07-12 Nektar Therapeutics Chemically modified small molecules
US8034825B2 (en) 2003-12-16 2011-10-11 Nektar Therapeutics Chemically modified small molecules
US8067431B2 (en) 2003-12-16 2011-11-29 Nektar Therapeutics Chemically modified small molecules
US11129794B2 (en) 2003-12-16 2021-09-28 Nektar Therapeutics Chemically modified small molecules
US11844865B2 (en) 2004-07-01 2023-12-19 Grünenthal GmbH Abuse-proofed oral dosage form
US8323889B2 (en) 2004-07-01 2012-12-04 Gruenenthal Gmbh Process for the production of an abuse-proofed solid dosage form
EP1779864A4 (en) * 2004-07-14 2010-10-20 Chugai Pharmaceutical Co Ltd Pth-containing preparation for transmucosal administration
EP1779864A1 (en) * 2004-07-14 2007-05-02 Chugai Seiyaku Kabushiki Kaisha Pth-containing preparation for transmucosal administration
US9101596B2 (en) 2004-07-19 2015-08-11 Biocon Limited Cation complexes of insulin compound conjugates, formulations and uses thereof
US7872095B2 (en) 2004-07-19 2011-01-18 Biocon Limited Insulin-oligomer conjugates, formulations and uses thereof
US9102758B2 (en) 2004-07-19 2015-08-11 Biocon Limited Insulin-oligomer conjugates, formulations and uses thereof
US7875700B2 (en) 2004-07-19 2011-01-25 Biocon Limited Cation complexes of insulin compound conjugates, formulation and uses thereof
US10675278B2 (en) 2005-02-04 2020-06-09 Grünenthal GmbH Crush resistant delayed-release dosage forms
US10729658B2 (en) 2005-02-04 2020-08-04 Grünenthal GmbH Process for the production of an abuse-proofed dosage form
WO2006135930A3 (en) * 2005-06-13 2007-05-31 Nastech Pharm Co Transmucosal delivery of peptide derivatives
WO2006135930A2 (en) * 2005-06-13 2006-12-21 Nastech Pharmaceutical Company Inc. Transmucosal delivery of peptide derivatives
US8580919B2 (en) 2005-12-16 2013-11-12 Nektar Therapeutics Polymer conjugates of GLP-1
US8293869B2 (en) 2005-12-16 2012-10-23 Nektar Therapeutics Polymer conjugates of GLP-1
EP1973952A4 (en) * 2006-01-23 2010-09-01 Kwangju Inst Sci & Tech Conjugate comprising pharmaceutical active compound covalently bound to mucoadhesive polymer and transmucosal delivery method of pharmaceutical active compound using the same
EP1973952A1 (en) * 2006-01-23 2008-10-01 Gwangju Institute of Science and Technology Conjugate comprising pharmaceutical active compound covalently bound to mucoadhesive polymer and transmucosal delivery method of pharmaceutical active compound using the same
US9289432B2 (en) 2007-01-19 2016-03-22 HANANJA EHF and UNIVERSITY OF ICELAND Methods and compositions for the delivery of a therapeutic agent
US10052333B2 (en) 2007-01-19 2018-08-21 University Of Iceland Methods and systems for the delivery of a therapeutic agent
US9687495B2 (en) 2007-01-19 2017-06-27 Hananja Ehf Methods and systems for the delivery of a therapeutic agent
WO2008089426A3 (en) * 2007-01-19 2009-02-12 Hananja Ehf Methods and compositions for the delivery of a therapeutic agent
WO2008089426A2 (en) * 2007-01-19 2008-07-24 Hananja Ehf Methods and compositions for the delivery of a therapeutic agent
US9241908B2 (en) 2007-10-16 2016-01-26 Biocon Limited Orally administrable solid pharmaceutical composition and a process thereof
US8226942B2 (en) 2007-12-28 2012-07-24 Kuros Biosurgery Ag PDGF fusion proteins incorporated into fibrin foams
US9750701B2 (en) 2008-01-25 2017-09-05 Grünenthal GmbH Pharmaceutical dosage form
US9161917B2 (en) 2008-05-09 2015-10-20 Grünenthal GmbH Process for the preparation of a solid dosage form, in particular a tablet, for pharmaceutical use and process for the preparation of a precursor for a solid dosage form, in particular a tablet
CN101531756B (en) * 2009-04-16 2011-06-15 山东大学 Method for preparing water soluble polyethylene glycol/polylactic segmented polycarbonate resin
US10493033B2 (en) 2009-07-22 2019-12-03 Grünenthal GmbH Oxidation-stabilized tamper-resistant dosage form
US9925146B2 (en) 2009-07-22 2018-03-27 Grünenthal GmbH Oxidation-stabilized tamper-resistant dosage form
US10080721B2 (en) 2009-07-22 2018-09-25 Gruenenthal Gmbh Hot-melt extruded pharmaceutical dosage form
US9636303B2 (en) 2010-09-02 2017-05-02 Gruenenthal Gmbh Tamper resistant dosage form comprising an anionic polymer
US10300141B2 (en) 2010-09-02 2019-05-28 Grünenthal GmbH Tamper resistant dosage form comprising inorganic salt
US10695297B2 (en) 2011-07-29 2020-06-30 Grünenthal GmbH Tamper-resistant tablet providing immediate drug release
US10201502B2 (en) 2011-07-29 2019-02-12 Gruenenthal Gmbh Tamper-resistant tablet providing immediate drug release
US10864164B2 (en) 2011-07-29 2020-12-15 Grünenthal GmbH Tamper-resistant tablet providing immediate drug release
US9655853B2 (en) 2012-02-28 2017-05-23 Grünenthal GmbH Tamper-resistant dosage form comprising pharmacologically active compound and anionic polymer
US10335373B2 (en) 2012-04-18 2019-07-02 Grunenthal Gmbh Tamper resistant and dose-dumping resistant pharmaceutical dosage form
US10064945B2 (en) 2012-05-11 2018-09-04 Gruenenthal Gmbh Thermoformed, tamper-resistant pharmaceutical dosage form containing zinc
US9737490B2 (en) 2013-05-29 2017-08-22 Grünenthal GmbH Tamper resistant dosage form with bimodal release profile
US10154966B2 (en) 2013-05-29 2018-12-18 Grünenthal GmbH Tamper-resistant dosage form containing one or more particles
US10624862B2 (en) 2013-07-12 2020-04-21 Grünenthal GmbH Tamper-resistant dosage form containing ethylene-vinyl acetate polymer
US10449547B2 (en) 2013-11-26 2019-10-22 Grünenthal GmbH Preparation of a powdery pharmaceutical composition by means of cryo-milling
US9913814B2 (en) 2014-05-12 2018-03-13 Grünenthal GmbH Tamper resistant immediate release capsule formulation comprising tapentadol
US9872835B2 (en) 2014-05-26 2018-01-23 Grünenthal GmbH Multiparticles safeguarded against ethanolic dose-dumping
US9855263B2 (en) 2015-04-24 2018-01-02 Grünenthal GmbH Tamper-resistant dosage form with immediate release and resistance against solvent extraction
US10842750B2 (en) 2015-09-10 2020-11-24 Grünenthal GmbH Protecting oral overdose with abuse deterrent immediate release formulations

Also Published As

Publication number Publication date
EP1204427A1 (en) 2002-05-15
KR20010018158A (en) 2001-03-05
JP2003507344A (en) 2003-02-25
KR100345214B1 (en) 2002-07-25
AU6478400A (en) 2001-03-13
US6506730B1 (en) 2003-01-14
EP1204427A4 (en) 2007-12-19

Similar Documents

Publication Publication Date Title
US6506730B1 (en) Nasal transmucosal delivery of peptide conjugated with biocompatible polymers
US9682153B2 (en) Polymer conjugates of therapeutic peptides
US10420844B2 (en) Peptide pharmaceuticals
JP2851627B2 (en) Nasal administration of powdered polypeptides
US6703381B1 (en) Methods for delivery therapeutic compounds across the blood-brain barrier
US5482706A (en) Transmucosal therapeutic composition
US20110171312A1 (en) Modified therapeutic peptides, methods of their preparation and use
EP2710031B9 (en) Improved peptide pharmaceuticals for insulin resistance
Youn et al. Improved intrapulmonary delivery of site-specific PEGylated salmon calcitonin: optimization by PEG size selection
LT5153B (en) Pulmonary adminstration of chemically modified insulin
WO1990009167A1 (en) Composition and method for administration of pharmaceutically active substances
Na et al. Stability of PEGylated salmon calcitonin in nasal mucosa
Mezo et al. New derivatives of GnRH as potential anticancer therapeutic agents
JPH06228199A (en) Peptide binding body capable of passing through blood brain barrier
Youn et al. Improved peroral delivery of glucagon-like peptide-1 by site-specific biotin modification: design, preparation, and biological evaluation
Balasubramaniam et al. Structure− activity studies including a ψ (CH2-NH) scan of peptide YY (PYY) active site, PYY (22− 36), for interaction with rat intestinal PYY receptors: development of analogues with potent in vivo activity in the intestine
JP2009536670A (en) Compositions and methods for increasing cell permeability of compounds
AU2021399904A1 (en) Pharmaceutical composition of glp-1/glp-2 dual agonists
JP3263598B2 (en) Bioactive peptide composition for nasal absorption
JP2004002331A (en) Treatment of liver cancer
Shin et al. Nasal absorption and pharmacokinetic disposition of salmon calcitonin modified with low molecular weight polyethylene glycol
Mező New Derivatives of GnRH as Potential Anticancer Therapeutic Agents Gábor Mező", Marilena Manea “, Ildikó Szabó", Borbála Vincze" and Magdolna Kovács
NZ617824B2 (en) Improved peptide pharmaceuticals for insulin resistance

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2000952020

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000952020

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642