EP1931374A2 - Formulations de pth et procedes d'utilisation - Google Patents

Formulations de pth et procedes d'utilisation

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Publication number
EP1931374A2
EP1931374A2 EP06749687A EP06749687A EP1931374A2 EP 1931374 A2 EP1931374 A2 EP 1931374A2 EP 06749687 A EP06749687 A EP 06749687A EP 06749687 A EP06749687 A EP 06749687A EP 1931374 A2 EP1931374 A2 EP 1931374A2
Authority
EP
European Patent Office
Prior art keywords
pth
formulation
dosage form
administration
less
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06749687A
Other languages
German (de)
English (en)
Inventor
Henry R. Costantino
Steven C. Quay
Michael V. Templin
Ching-Yuan Li
Anthony Sileno
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Marina Biotech Inc
Original Assignee
MDRNA Inc
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
Priority claimed from US11/246,450 external-priority patent/US20060052306A1/en
Priority claimed from US11/246,406 external-priority patent/US20060052305A1/en
Priority claimed from US11/347,551 external-priority patent/US20060127320A1/en
Priority claimed from US11/390,940 external-priority patent/US20060189533A1/en
Application filed by MDRNA Inc filed Critical MDRNA Inc
Publication of EP1931374A2 publication Critical patent/EP1931374A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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, i.e. 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • Osteoporosis can be defined as a systemic skeletal disease characterized by low bone mass, microarchitectural deterioration of bone tissue, and increased bone fragility and susceptibility to fracture. It most commonly affects older populations, primarily postmenopausal women.
  • osteoporosis poses a serious health problem.
  • the National Osteoporosis Foundation has estimated that 44 million people are experiencing the effects of osteoporosis or osteopenia.
  • osteoporosis will affect more than 52 million people and, by 2020, more than 61 million people.
  • the prevalence of osteoporosis is greater in Caucasians and Asians than in African-Americans, perhaps because African-Americans have a higher peak bone mass.
  • Women are affected in greater numbers than men because men have a higher peak bone density.
  • the rate of bone turnover increases, resulting in accelerated bone loss because of the lack of estrogen after menopause.
  • the goal of pharmacological treatment of osteoporosis is to maintain or increase bone strength, to prevent fractures throughout the patient's life, and to minimize osteoporosis-related morbidity and mortality by safely reducing the risk of fracture.
  • the medications that have been used most commonly to treat osteoporosis include calcium, and vitamin D, estrogen (with or without progestin), bisphonates, selective estrogen receptor modulators (SERMs), and calcitonin.
  • PTH Parathyroid hormone
  • BMD bone mineral density
  • PTH has multiple actions on bone, some direct and some indirect.
  • PTH increases the rate of calcium release from bone into blood.
  • the chronic effects of PTH are to increase the number of bone cells both osteoblasts and osteoclasts, and to increase the remodeling bone. These effects are apparent within hours after PTH is administered and persist for hours after PTH is withdrawn.
  • PTH administered to osteoporotic patients leads to a net stimulation of bone formation especially in trabecular bone in the spine and hip resulting in a highly significant reduction in fractures.
  • Parathyroid hormone is a secreted, 84 amino acid residue polypeptide having the amino acid sequence Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly- Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp- Val-His-Asn-Phe VaI Ala Leu GIy Ala Pro Leu Ala Pro Arg Asp Ala GIy Ser GIn Arg Pro Arg Lys Lys GIu Asp Asn VaI Leu VaI GIu Ser His GIu Lys Ser Leu GIy GIu Ala Asp Lys Ala Asn VaI Asp VaI Leu TIu Lys Ala Lys Ser GIn (
  • PTH (1-34), also called teriparatide, is currently on the market under the brand name FORTEO , Eli Lilly, Indianapolis, Indiana for the treatment of postmenopausal women with osteoporosis who are at high risk of fracture.
  • This drug is administered by a once daily subcutaneous injection of 20 ⁇ g in a solution containing acetate buffer, mannitol, and m-cresol in water, pH 4.
  • many people are adverse to injections, and thus become non-compliant with the prescribed dosing of the PTH.
  • FORTEO ® (Eli Lilly, U.S.), or FORSTEO (Eli Lilly, UK), is manufactured by recombinant DNA technology using an Escherichia coli strain.
  • PTH (1-34) has a molecular weight of 4117.87 daltons.
  • Reviews on PTH (1-34) and its clinical uses are published, including, e.g., Brixen et ah, 2004; Dobnig, 2004; Eriksen and Robins, 2004; Quattrocchi and Kourlas 2004, are hereby incorporated by reference.
  • FORTEO ® is currently licensed in the US and Europe (as FORSTEO).
  • teriparatide has been evaluated in over 2800 patients in doses ranging from 5 to 100 ⁇ g per day in short term trials. Doses of up to 40 ⁇ g per day have been given for up to two years in long term trials. Adverse events associated with FORSTEO were usually mild and generally did not require discontinuation of therapy. The most commonly reported adverse effects were dizziness, leg cramps, nausea, vomiting and headache. Mild transient hypercalcemia has been reported with FORSTEO which is usually self limiting within 6 hours.
  • FORTEO ® is administered as a daily subcutaneous injection.
  • the following Cmax and AUC values are described for various doses of FORTEO (20 ug is the commercially approved dose).
  • PTH formulations are reconstituted from fresh or lyophilized hormone, and incorporate various carriers, excipients and vehicles.
  • PTH formulations are often prepared in water-based vehicles such as saline, or water which is acidified typically with acetic acid to solubilize the hormone.
  • Many reported formulations also incorporate albumin as a stabilizer (see, for example, Reeve et al., Br. Med. J., 1980, 280:6228; Reeve et al., Lancet, 1976, 1:1035; Reeve at al., Calcif. Tissue Res., 1976, 21:469; Hodsman et al., Bone Miner 1990, 9(2):137; Tsai et al., J. Clin.
  • Tissue Res., 21, Suppl., 469-477 a human PTH (1-38) preparation reconstituted into a saline vehicle (see Hodsman et al., 1991, 14(1), 67-83); and a bovine PTH (1-34) preparation in aqueous vehicle pH adjusted with acetic acid and containing albumin.
  • the International Reference preparation for human PTH (1-84) consists of 100 ng of hormone ampouled with 250 ⁇ g human serum albumin and 1.25 mg lactose (1981), and for bovine PTH (1-84) consists of 10 ⁇ g lyophilized hormone in 0.01 M acetic acid and 0.1% w/v mannitol (see Martindale, The Extra Pharmacoepia, The Pharmaceutical Press, London, 29th Edition, 1989 at p. 1338).
  • a formulation aimed at improving the stability for a lyophilized preparation of h-PTH (1-34) is reported in EP 619 119 using a combination of sugar and sodium chloride.
  • U.S. Pat. No. 5,496,801 describes a freeze-dried composition for the natural hormone, PTH (1-84), containing mannitol as an excipient and a citrate source as a non- volatile buffering agent.
  • U.S. Patent No. 6,770,623 describes stabilized teriparatide formulations.
  • the '623 formulations require a buffer.
  • the buffering agent includes any acid or salt combination which is pharmaceutically acceptable and capable of maintaining the aqueous solution at a pH range of 3 to 7, preferably 3-6, e.g., acetate, tartrate, or citrate sources.
  • the concentration of buffer may be in the range of about 2 mM to about 500 mM.
  • U.S. Patent No. 5,407,911 describes the use of dipotassium glycyrrhizate as an emulsifying agent for nasal administration of PTH. Polysorbate 80 was determined to be inferior when used in the intranasal PTH formulations because it caused a precipitate and instability in the formulation.
  • parathyroid hormone requires the development of a formulation that is acceptable in terms of storage stability and ease of preparation.
  • a parathyroid hormone formulation presents challenges not commonly encountered by the pharmaceutical industry.
  • PTH is particularly sensitive to oxidation, deamidation, and hydrolysis, and requires that its N-terminal and C-terminal sequences remain intact in order to preserve bioactivity.
  • Formulating proteins is generally more difficult that formulating small molecules, because proteins are more susceptible to degradation (see Arakawa et al.
  • FORTEO is a liquid pharmaceutical formulation of teriparatide that requires a buffer for its stability.
  • PTH or PTH analogs that will be suitable for systemic delivery, but not cause significant local effects on the nasal tissue (i.e., not having an effect on nasal toxicity).
  • One aspect of the invention is an aqueous pharmaceutical formulation for intranasal delivery of PTH, comprising PTH(I -34) and a nonionic surface active agent.
  • the surface active agent is selected from the group consisting of nonionic polyoxyethylene ether, polysorbate 80, polysorbate 20, polyethylene glycol, cetyl alcohol, polyvinylpyr r olidone, polyvinyl alcohol, poloxamer F68, poloxamer F 127, and lanolin alcohol.
  • the surface active agent is polysorbate 80.
  • polysorbate 80 is present at less than about 50 mg/niL in the formulation.
  • polysorbate 80 is present at less than about 10 mg/mL in the formulation.
  • polysorbate 80 is present at less than about 1 mg/mL in the formulation.
  • the polyol is selected from the group consisting of sucrose, mannitol, sorbitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose, glycerin, and polyethylene glycol.
  • the polyol is sorbitol.
  • a preservative is selected from the group consisting of chlorobutanol, methyl paraben, propyl paraben, butyl paraben, benzalkonium chloride, benzethonium chloride, sodium benzoate, sorbic acid, phenol, and ortho-, meta- or paracresol.
  • the formulation has a pH of about 3 to about 6.
  • the formulation has a pH of about 5.0 or less.
  • the formulation has a pH of about 4.0 or less.
  • the aqueous solution is in the form of liquid droplets.
  • the liquid droplets have an average volume- mean particle size (Dv,50) between about 1 micron and 1000 microns.
  • the liquid droplets have an average volume-mean particle size (Dv,50) between about 5 microns and 500 microns. In another embodiment, the liquid droplets have an average volume-mean particle size (Dv,50) between about 10 microns and 100 microns. In another embodiment, administration in a human subject achieves a maximum serum concentration of PTH, post-dosing (Cmax), of at least 10 ⁇ g/mL.
  • Another aspect of the invention is a method for treating osteoporosis in a mammal, comprising administering intranasally a therapeutically effective amount of a PTH formulation to the mammal wherein the formulation comprises PTH(I -34) and a nonionic surface active agent.
  • the surface active agent is selected from the group consisting of nonionic polyoxyethylene ether, polysorbate 80, polysorbate 20, polyethylene glycol, cetyl alcohol, polyvinylpyrolidone, polyvinyl alcohol, poloxamer F68, poloxamer F127, and lanolin alcohol.
  • the formulation has a pH of about 3-6.
  • a dose containing about 1 ⁇ g to about 1000 ⁇ g of a PTH(l-34) is administered to the mammal. In another embodiment, a dose containing about 20 ⁇ g to about 400 ⁇ g of PTH(I -34) is administered to the mammal. In another embodiment, the mammal is a human. In another embodiment, administration of the PTH formulation results in an increase in plasma levels of calcium. In another embodiment, the increase in plasma levels of calcium are associated with the anabolic effects of PTH. In another embodiment, the increase in plasma levels of calcium are not the result of increased bone catabolism. In another embodiment, the increase in plasma levels of calcium are not the result of increased bone catabolism. In another embodiment, administration of the PTH formulation results in an increase in bone mass. In another embodiment, administration of the PTH formulation results in an increase in bone strength. In another embodiment, administration of the PTH formulation results in an increased resistance to bone fracture. In another embodiment, administration of the PTH formulation does not produce histological changes in nasal tissue.
  • Another aspect of the invention is a method for treating osteoporosis in a mammal comprising administering intranasally a therapeutically effective amount of a PTH formulation to the mammal, wherein the PTH formulation comprises PTH(I -34) and one or more excipients selected from the group consisting of a solubilizing agent, a chelating agent, and one or more polyols.
  • the formulation further comprises a surface active agent.
  • the surface active agent is selected from the group consisting of nonionic polyoxyethylene ether, bile salts such, sodium glycocholate, deoxycholate, derivatives of fusidic acid, sodium taurodihydrofusidate, L- ⁇ -phosphatidylcholine didecanoyl (DDPC), polysorbate 80, polysorbate 20, a polyethylene glycol, cetyl alcohol, pplyvinylpyrolidone, a polyvinyl alcohol, lanolin alcohol, and sorbitan monooleate.
  • the surface-active agent is DDPC.
  • one or more polyols are selected from the group consisting of sucrose, mannitol, sorbitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose, glycerin and polyethylene glycol.
  • the polyol is sorbitol.
  • the chelating agent is ethylene diamine tetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA). In another embodiment, the chelating agent is EDTA.
  • the solubilizing agent is selected from the group consisting of a cyclodextran, hydroxypropyl- ⁇ - cyclodextran, sulfobutylether- ⁇ -cyclodextran, and methyl- ⁇ -cyclodextran. In another embodiment, the solubilizing agent is a cyclodextran.
  • Another aspect of the invention is method for treating osteoporosis in a mammal comprising administering intranasally a therapeutically effective amount of a PTH formulation to the mammal, wherein the PTH formulation comprises PTH(I -34) and a nonionic surface active agent, and wherein a time to maximum plasma concentration, T max , of PTH(I -34) following administration of said formulation to the mammal is less than 30 minutes.
  • a C max greater than about 300 pg/ml results from a single administration of said formulation.
  • a dosage form of PTH comprising an aqueous pharmaceutical formulation of PTH and a nonionic detergent for aerosolized intranasal delivery of PTH having a bioavailability of about 5% or greater, wherein the formulation comprises a therapeutically effective amount of PTH(l-34) and a polysorbate, and wherein least 90% of the PTH can be recovered after storage for 24 weeks at 5°C.
  • the PTH dosage form having greater than about 90% recovery of the PTH after at least six months at 5 0 C storage.
  • the PTH dosage form having greater than about 90% recovery of the PTH after one year at 5 0 C storage.
  • the PTH dosage form having greater than about 90% recovery of the PTH after being in use for greater than about five days. In another embodiment, the PTH dosage form having greater than about 90% recovery of PTH at 30°C/65% relative humidity between all sprays.
  • the pH is about 5.0 or less. In another embodiment, the pH is about 4.5 or less. In another embodiment, the pH is about 4.0 or less. In another embodiment, the pH is about 3.5 or less.
  • the concentration of PTH is at least about 1 mg/ml. In another embodiment, the concentration of PTH is at least about 2 mg/ml. In another embodiment, the concentration of PTH is at least about 6 mg/ml. In another embodiment, the concentration of PTH is at least about 10 mg/ml.
  • the dosage form is suitable for intra-nasal administration to achieve a dose of from about 2 ⁇ g to about 1000 ⁇ g of said PTH. In another embodiment, the dosage form is suitable for intra-nasal administration to achieve a dose of from about 100 ⁇ g to about 600 ⁇ g of said PTH.
  • the polysorbate is present at least about 1 mg/mL in the formulation. In another embodiment, the polysorbate is present at least about 10 mg/mL in the formulation. In another embodiment, the polysorbate is present at least about 50 mg/mL in the formulation.
  • a preservative is present. In another embodiment, the preservative is chlorobutanol.
  • a dosage form of PTH comprising an aqueous pharmaceutical formulation for aerosolized intranasal delivery of PTH having a bioavailability of about 10% or greater, wherein the formulation comprises a therapeutically effective amount of PTH(I -34), methyl- ⁇ -cyclodextrin, didecanoylphosphatidyl choline, and ethylenediaminetetraacetic acid, and wherein least 90% of the PTH can be recovered after storage for 24 weeks at 5°C.
  • the PTH dosage form having greater than about 90% recovery of PTH after at least six months at 5 0 C storage.
  • the PTH dosage form having greater than about 90% recovery of PTH after one year at 5 0 C storage.
  • the PTH dosage form having greater than about 90% recovery of the PTH after being in use for greater than about five days. In another embodiment, the PTH dosage form having greater than about 90% recovery of PTH at 30°C/65% relative humidity between all sprays.
  • the pH is about 5.0 or less. In another embodiment, the pH is about 4.5 or less. In another embodiment, the pH is about 4.0 or less. In another embodiment, the pH is about 3.5 or less.
  • the concentration of PTH is at least about 1 mg/ml. In another embodiment, the concentration of PTH is at least about 2 mg/ml. In another embodiment, the concentration of PTH is at least about 6 mg/ml. In another embodiment, the concentration of PTH is at least about 10 mg/ml.
  • the dosage form is suitable for intra-nasal administration to achieve a dose of from about 2 ⁇ g to about 1000 ⁇ g of said PTH. In another embodiment, the dosage form is suitable for intra-nasal administration to achieve a dose of from about 100 ⁇ g to about 600 ⁇ g of said PTH.
  • a preservative is present. In another embodiment, the preservative is chlorobutanol.
  • Another aspect of the invention is a method of delivering PTH to a human, comprising exposing a layer of mucosal cells to a PTH solution comprising PTH(I- 34) and a nonionic surface active agent. In one embodiment, the method utilizes a non-parenteral administration.
  • the method of administration is selected from the group consisting of intranasal, buccal, gastrointestinal, vaginal, and transdermal.
  • the method is an intranasal administration.
  • the intranasal administration comprises delivering an aerosol comprising droplets of between about 1 and about 700 microns in size.
  • intranasal administration comprises delivering an aerosol comprising about about 0.7 to about about 25 ⁇ g PTH per kg weight of the patient.
  • intranasal administration comprises delivering an aerosol comprising about 50 to about 800 ⁇ g PTH.
  • the method is an oral delivery.
  • oral delivery is a controlled release delivery wherein Tmax is less than about 40 minutes from the time of release.
  • Another aspect of the invention is a system for delivering PTH to a human by intranasal administration
  • an aqueous PTH solution comprising PTH(I -34) and a nonionic surface active agent in a bottle
  • a droplet-generating actuator attached to said bottle and fluidly connected to the PTH solution in the container, wherein said actuator produces a spray of the PTH solution through a tip of the actuator when said actuator is engaged, wherein said PTH spray has a spray pattern ellipticity ratio of from about 1.0 to about 1.4 when measured at a height of 3.0 cm from the actuator tip.
  • the PTH spray is comprised of droplets of the PTH solution wherein less than about 5% of the droplets are less than 10 ⁇ m in size.
  • the PTH spray is comprised of droplets of the PTH solution wherein less than about 1% of the droplets are less than 10 ⁇ m in size.
  • the PTH spray has a spray pattern major axis and minor axis of about 25 and about 40 mm, respectively.
  • the PTH spray is comprised of droplets of the PTH solution, wherein less than about 90% of the droplets are about 250 ⁇ m or less in size.
  • the PTH spray is comprised of droplets of the PTH solution, wherein less than about 90% of the droplets are about 120 ⁇ m or less in size.
  • the PTH spray is comprised of droplets of the PTH solution wherein less than about 50% of the droplets are about 75 ⁇ m or less in size. In another embodiment, the PTH spray is comprised of droplets of the PTH solution wherein less than about 50% of the droplets are about 50 ⁇ m or less in size. In another embodiment, the PTH spray is comprised of droplets of the PTH solution, wherein less than about 10% of the droplets are about 30 ⁇ m or less in size. In another embodiment, the PTH spray is comprised of droplets of the PTH solution, wherein less than about 10% of the droplets are about 20 ⁇ m or less in size. In another embodiment, the formulation is used in the treatment of osteoporosis or osteopenia.
  • PTH(I -34) is use of PTH(I -34) in the manufacture of a medicament for treating osteoporosis in a mammal, wherein the medicament comprises PTH(I -34) and a nonionic surface active agent.
  • the surface active agent is selected from the group consisting of nonionic polyoxyethylene ether, polysorbate 80, polysorbate 20, polyethylene glycol, cetyl alcohol, polyvinylpyrolidone, polyvinyl alcohol, poloxamer ⁇ 68, poloxamer F 127, and lanolin alcohol.
  • the formulation has a pH of about of about 3-6.
  • a dose containing 1 ⁇ g to 1000 ⁇ g of a PTH(I -34) is administered to the mammal. In another embodiment, a dose containing 20 ⁇ g to 400 ⁇ g of PTH(I -34) is administered to the mammal. In another embodiment, the mammal is a human. In another embodiment, administration of the PTH formulation results in an increase in plasma levels of calcium. In another embodiment, the increase in plasma levels of calcium are associated with the anabolic effects of PTH. In another embodiment, the increase in plasma levels of calcium are not the result of increased bone catabolism. In another embodiment, the increase in plasma levels of calcium are not the result of increased bone catabolism. In another embodiment, administration of the PTH formulation results in an increase in bone mass. In another embodiment, administration of the PTH formulation results in an increase in bone strength. In another embodiment, administration of the PTH formulation results in an increased resistance to bone fracture. In another embodiment, administration of the PTH formulation does not produce histological changes in nasal tissue.
  • Another aspect of the invention is use of PTH(I -34) in the manufacture of a medicament for treating osteoporosis in a mammal, wherein the medicament comprises a therapeutically effective amount of PTH(l-34) and one or more excipients selected from the group consisting of a solubilizing agent, a chelating agent, and one or more polyols.
  • the formulation further comprises a surface active agent.
  • the surface active agent is selected from the group consisting of nonionic polyoxyethylene ether, bile salts such, sodium glycocholate, deoxycholate, derivatives of fusidic acid, sodium taurodihydrofusidate, L- ⁇ -phosphatidylcholine didecanoyl (DDPC), polysorbate 80, polysorbate 20, a polyethylene glycol, cetyl alcohol, polyvinylpyrolidone, a polyvinyl alcohol, lanolin alcohol, and sorbitan monooleate.
  • the surface-active agent is DDPC.
  • one or more polyols are selected from the group consisting of sucrose, mannitol, sorbitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose, glycerin and glycol.
  • the polyol is sorbitol.
  • the chelating agent is ethylene diamine tetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA). In another embodiment, the chelating agent is EDTA.
  • the solubilizing agent is selected from the group consisting of a cyclodextran, hydroxypropyl- ⁇ -cyclodextran, sulfobutylether- ⁇ -cyclodextran, and methyl- ⁇ -cyclodextran. In another embodiment, the solubilizing agent is a cyclodextran.
  • Another aspect of the invention is use of PTH(I -34) in the manufacture of a medicament for treating osteoporosis in a mammal, wherein the medicament comprises PTH(l-34) and a nonionic surface active agent, and wherein a time to maximum plasma concentration, T max , of PTH(I -34) following administration of said formulation to the mammal is less than 30 minutes.
  • T max time to maximum plasma concentration
  • a C max greater than 300 pg/ml results from a single administration of said formulation.
  • Figure 1 Mean Plasma Concentration versus Time for Periods 1-5: (Linear Graph).
  • Figure 2 Ratio of C max to Mean, Low Dose PTH Formulations versus FORTEO ® .
  • the hormone is parathyroid hormone and the mammal is a human.
  • the parathyroid hormone peptide is PTH (1-34), also known as teriparatide.
  • PTH 1-34
  • Tregear, U.S. Pat. No. 4,086,196 described human PTH analogues and claimed that the first 27 to 34 amino acids are the most effective in terms of the stimulation of adenylyl cyclase in an in vitro cell assay.
  • PTH operates through activation of two second messenger systems, G s -protein activated adenylyl cyclase (AC) and G q -protein activated phospholipase Cp. The latter system results in a stimulation of membrane-bound protein kinase C (PKC) activity.
  • GPC membrane-bound protein kinase C
  • the PKC activity has been shown to require PTH residues 29 to 32 (Jouis Subscribe et al. (1994) J. Bone Mineral Res. 9, (1179-1189). It has been established that the increase in bone growth, i.e. the effect which is useful in the treatment of osteoporosis, is coupled to the ability of the peptide sequence to increase AC activity.
  • the native PTH sequence, PTH (1-84) (SEQ ID NO: 1), has been shown to have all of these activities.
  • the above described forms of parathyroid hormone are embraced by the terms "parathyroid hormone” or "PTH” or "PTH peptide” as used generically herein.
  • the parathyroid hormones may be obtained by known recombinant or synthetic methods, such as described in U.S. Pat. No.
  • the present invention is a method for treating osteoporosis or osteopenia in a mammal comprising transmucosally administering a formulation comprised of a PTH peptide, such that when 50 ⁇ g of the PTH is administered transmucosally to the mammal the concentration of the PTH peptide in the plasma of the mammal increases by at least 5 pmol, preferably at least 10 pmol per liter of plasma.
  • Intranasal delivery-enhancing agents are employed which enhance delivery of
  • the intranasal delivery-enhancing agent of the present invention may be a pH control agent.
  • the pH of the pharmaceutical formulation of the present invention is a factor affecting absorption of PTH via paracellular and transcellular pathways to drug transport.
  • the pharmaceutical formulation of the present invention is pH adjusted to between about pH 3.0 to 7.0.
  • the pharmaceutical formulation of the present invention is pH adjusted to between about pH 3.0 to 6.0.
  • the pharmaceutical formulation of the present invention is pH adjusted to between about pH 4.0 to 5.0.
  • the pH is 4.0 ⁇ 0.3.
  • the present invention provides improved methods and compositions for mucosal delivery of PTH peptide to mammalian subjects for treatment or prevention of osteoporosis or osteopenia.
  • appropriate mammalian subjects for treatment and prophylaxis according to the methods of the invention include, but are not restricted to, humans and non-human primates, livestock species, such as horses, cattle, sheep, and goats, and research and domestic species, including dogs, cats, mice, rats, guinea pigs, and rabbits.
  • a PTH peptide also includes the free bases, acid addition salts or metal salts, such as potassium or sodium salts of the peptides, and PTH peptides that have been modified by such processes as amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, cyclization and other well known covalent modification methods.
  • the nasal spray product manufacturing process generally includes the preparation of a diluent for PTH (1-34) nasal spray, which includes -85% water plus the components of the nasal spray formulation without PTH.
  • the pH of the diluent is then measured and adjusted to pH 4.0 ⁇ 0.3 with sodium hydroxide or hydrochloric acid, if necessary.
  • the PTH (1-34) nasal spray is prepared by the non-aseptic transfer of ⁇ 85% of the final target volume of the diluent to a screw cap bottle. An appropriate amount of PTH (1-34) is added and mixed until completely dissolved.
  • the pH is measured and adjusted to pH 4.0 ⁇ 0.3 with sodium hydroxide or hydrochloric acid, if necessary.
  • a sufficient quantity of diluent is added to reach the final target volume.
  • Screw-cap bottles are filled and caps affixed.
  • the above description of the manufacturing process represents a method used to prepare the initial clinical batches of drug product. This method may be modified during the development process to optimize the manufacturing process.
  • Currently marketed PTH requires sterile manufacturing conditions for compliance with FDA regulations.
  • Parenteral administration, including PTH for injection or infusion, requires a sterile (aseptic) manufacturing process.
  • Current Good Manufacturing Practices (GMP) for sterile drug manufacturing include standards for design and construction features (21 CFR ⁇ 211.42 (April 1, 2005)); standards for testing and approval or rejection of components, drug product containers, and closures ( ⁇ 211.84); standards for control of microbiological contamination ( ⁇ 211.113); and other special testing requirements ( ⁇ 211.167).
  • Non-parenteral (non-aseptic) products such as the intranasal product of the invention, do not require these specialized sterile manufacturing conditions.
  • the requirements for a sterile manufacturing process are substantially higher and correspondingly more costly than those required for a non-sterile product manufacturing process.
  • These costs include much greater capitalization costs for facilities, as well as a more costly manufacturing cost: extra facilites for sterile manufacturing include additional rooms and ventilation; extra costs associated with sterile manufacturing include greater manpower, extensive quality control and quality assurance, and administrative support.
  • manufacturing costs of an intranasal PTH product such as that of the invention, are far less than those of a parenterally administered PTH product.
  • the present invention satisfies the need for a non-sterile manufacturing process for PTH.
  • Mucosal delivery-enhancing agents are defined as chemicals and other excipients that, when added to an aqueous PTH formulation results in a formulation that produces a significant increase in transport of PTH peptide across a mucosa as measured by the maximum blood, serum, or cerebral spinal fluid concentration (C max ) or by the area under the curve, AUC, in a plot of concentration versus time.
  • a mucosa includes the nasal, oral, intestinal, buccal, bronchopulmonary, vaginal, and rectal mucosal surfaces and in fact includes all mucus-secreting membranes lining all body cavities or passages that communicate with the exterior. Mucosal delivery enhancing agents are sometimes called carriers.
  • mucosal delivery-enhancing agents include agents which enhance the release or solubility (e.g., from a formulation delivery vehicle), diffusion rate, penetration capacity and timing, uptake, residence time, stability, effective half- life, peak or sustained concentration levels, clearance and other desired mucosal delivery characteristics (e.g., as measured at the site of delivery, or at a selected target site of activity such as the bloodstream or central nervous system) of PTH peptide or other biologically active compound(s).
  • Enhancement of mucosal delivery can thus occur by any of a variety of mechanisms, for example by increasing the diffusion, transport, persistence or stability of PTH peptide, increasing membrane fluidity, modulating the availability or action of calcium and other ions that regulate intracellular or paracellular permeation, solubilizing mucosal membrane components (e.g., lipids), changing non-protein and protein sulfhydryl levels in mucosal tissues, increasing water flux across the mucosal surface, modulating epithelial junctional physiology, reducing the viscosity of mucus overlying the mucosal epithelium, reducing mucociliary clearance rates, and other mechanisms.
  • mucosal membrane components e.g., lipids
  • mucosal membrane components e.g., lipids
  • changing non-protein and protein sulfhydryl levels in mucosal tissues increasing water flux across the mucosal surface
  • modulating epithelial junctional physiology reducing the viscosity
  • a "mucosally effective amount of PTH peptide" contemplates effective mucosal delivery of PTH peptide to a target site for drug activity in the subject that may involve a variety of delivery or transfer routes.
  • a given active agent may find its way through clearances between cells of the mucosa and reach an adjacent vascular wall, while by another route the agent may, either passively or actively, be taken up into mucosal cells to act within the cells or be discharged or transported out of the cells to reach a secondary target site, such as the systemic circulation.
  • compositions of the invention may promote the translocation of active agents along one or more such alternate routes, or may act directly on the mucosal tissue or proximal vascular tissue to promote absorption or penetration of the active agent(s).
  • the promotion of absorption or penetration in this context is not limited to these mechanisms.
  • peak concentration (C max ) of PTH peptide in a blood plasma As used herein "peak concentration (C max ) of PTH peptide in a blood plasma”, "area under concentration vs. time curve (AUC) of PTH peptide in a blood plasma”, “time to maximal plasma concentration (t max ) of PTH peptide in a blood plasma” are pharmacokinetic parameters known to one skilled in the art. Laursen et ah, Eur. J. Endocrinology, 135: 309-315 (1996). The “concentration vs. time curve” measures the concentration of PTH peptide in a blood serum of a subject vs. time after administration of a dosage of PTH peptide to the subject either by intranasal, intramuscular, or subcutaneous route of administration.
  • C raax is the maximum concentration of PTH peptide in the blood serum of a subject following a single dosage of PTH peptide to the subject.
  • t max is the time to reach maximum concentration of PTH peptide in a blood serum of a subject following administration of a single dosage of PTH peptide to the subject.
  • a “buffer” is generally used to maintain the pH of a solution at a nearly constant value. A buffer maintains the pH of a solution, even when small amounts of strong acid or strong base are added to the solution, by preventing or neutralizing large changes in concentrations of hydrogen and hydroxide ions.
  • a buffer generally consists of a weak acid and its appropriate salt (or a weak base and its appropriate salt).
  • the appropriate salt for a weak acid contains the same negative ion as present in the weak acid, (see Lagowski, Macmillan Encyclopedia of Chemistry, Vol. 1, Simon & Schuster, New York, 1997 at p. 273-4).
  • Examples of commonly used buffer sources include the following: acetate, tartrate, or citrate.
  • the "buffer capacity" means the amount of acid or base that can be added to a buffer solution before a significant pH change will occur.
  • Non-infused administration means any method of delivery that does not involve an injection directly into an artery or vein, a method which forces or drives (typically a fluid) into something and especially to introduce into a body part by means of a needle, syringe or other invasive method.
  • Non-infused administration includes subcutaneous injection, intramuscular injection, intraparitoneal injection and the non-injection methods of delivery to a mucosa.
  • Osteoporosis is a systemic skeletal disease characterized by low bone mass, microarchitectural deterioration of bone tissue, and increased bone fragility and susceptibility to fracture. Osteopenia is a decreased calcification or density of bone, a descriptive term applicable to all skeletal systems in which the condition is noted.
  • Osteoporosis or osteopenia therapies and medical diagnosis include the administration of a clinically effective dose of PTH for the prevention and/or treatment of osteoporosis or osteopenia.
  • the instant invention provides improved and useful methods and compositions for nasal mucosal delivery of a PTH peptide to prevent and treat osteoporosis or osteopenia in mammalian subjects.
  • prevention and treatment of osteoporosis or osteopenia means prevention of the onset or lowering the incidence or severity of clinical osteoporosis by reducing increasing bone mass, decreasing bone resporption, or reducing the incidence of fractured bones in a patient.
  • the PTH peptide can also be administered in conjunction with other therapeutic agents such as bisphonates, calcium, vitamin D, estrogen or estrogen- receptor binding compounds, selective estrogen receptor modulators (SERMs), bone morphogenic proteins, or calcitonin.
  • SERMs selective estrogen receptor modulators
  • the present invention provides mucosal delivery of PTH peptide, formulated with one or more mucosal delivery-enhancing agents such as a nonionic surface active agent, wherein PTH peptide dosage release is substantially normalized and/or sustained for an effective delivery period of PTH peptide release ranging from approximately 0.1 to 2.0 hours; 0.4 to 1.5 hours; 0.7 to 1.5 hours; or 0.8 to 1.0 hours; following mucosal administration.
  • the sustained release of PTH peptide may be facilitated by repeated administration of exogenous PTH peptide utilizing methods and compositions of the present invention.
  • the present invention provides improved mucosal (e.g., nasal) delivery of a formulation comprising PTH peptide in combination with one or more mucosal delivery- enhancing agents and an optional sustained release-enhancing agent or agents.
  • Mucosal delivery-enhancing agents of the present invention yield an effective increase in delivery, e.g., an increase in the maximal plasma concentration (C max ) to enhance the therapeutic activity of mucosally-administered PTH peptide.
  • C max maximal plasma concentration
  • a second factor affecting therapeutic activity of PTH peptide in the blood plasma and CNS is residence time (RT).
  • Sustained release-enhancing agents in combination with intranasal delivery-enhancing agents, increase C max and increase residence time (RT) of PTH peptide.
  • PEG polyethylene glycol
  • the present invention provides an improved PTH peptide delivery method and dosage form for treatment or prevention of osteoporosis or osteopenia in mammalian subjects.
  • various delivery-enhancing agents are employed which enhance delivery of PTH peptide into or across a mucosal surface.
  • delivery of PTH peptide across the mucosal epithelium can occur "transcellularly” or “paracellularly.”
  • the extent to which these pathways contribute to the overall flux and bioavailability of the PTH peptide depends upon the environment of the mucosa, the physico-chemical properties the active agent, and the properties of the mucosal epithelium.
  • Paracellular transport involves only passive diffusion, whereas transcellular transport can occur by passive, facilitated, or active processes.
  • hydrophilic, passively transported, polar solutes diffuse through the paracellular route, while more lipophilic solutes use the transcellular route.
  • Absorption and bioavailability (e.g., as reflected by a permeability coefficient or physiological assay), for diverse, passively and actively absorbed solutes, can be readily evaluated, in terms of both paracellular and transcellular delivery components, for any selected PTH peptide within the invention.
  • the relative contribution of paracellular and transcellular pathways to drug transport depends upon the pKa, partition coefficient, molecular radius and charge of the drug, the pH of the luminal environment in which the drug is delivered, and the area of the absorbing surface.
  • the paracellular route represents a relatively small fraction of accessible surface area of the nasal mucosal epithelium.
  • the methods and compositions of the invention provide for significantly enhanced transport of biotherapeutics into and across mucosal epithelia via the paracellular route. Therefore, the methods and compositions of the invention successfully target both paracellular and transcellular routes, alternatively, or within a single method or composition.
  • While the mechanism of absorption promotion may vary with different mucosal delivery-enhancing agents of the invention, useful reagents in this context will not substantially adversely affect the mucosal tissue and is selected according to the physicochemical characteristics of the particular PTH peptide or other active or delivery-enhancing agent.
  • delivery-enhancing agents that increase penetration or permeability of mucosal tissues will often result in some alteration of the protective permeability barrier of the mucosa.
  • delivery-enhancing agents for coordinate administration or combinatorial formulation with PTH peptide of the invention are selected from absorption promoting small hydrophilic molecules, including but not limited to, dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones.
  • absorption promoting small hydrophilic molecules including but not limited to, dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones.
  • long-chain amphipathic molecules for example, deacylmethyl sulfoxide, azone, sodium laurylsulfate, oleic acid, and the bile salts, may be employed to enhance mucosal penetration of the PTH peptide.
  • surfactants e.g., nonionic surface active agents such as polysorbates
  • surfactants may be employed as adjunct compounds, processing agents, or formulation additives to enhance intranasal delivery of the PTH peptide.
  • Agents such as DMSO, polyethylene glycol, and ethanol can, if present in sufficiently high concentrations in delivery environment (e.g., by pre-administration or incorporation in a therapeutic formulation), enter the aqueous phase of the mucosa and alter its solubilizing properties, thereby enhancing the partitioning of the PTH peptide from the vehicle into the mucosa.
  • Additional mucosal delivery-enhancing agents that are useful within the coordinate administration and processing methods and combinatorial formulations of the invention include, but are not limited to, mixed micelles; enamines; nitric oxide donors (e.g., S-nitroso-N-acetyl-DL-penicillamine, NORl, NOR4 ⁇ which are preferably co-administered with an NO scavenger such as carboxy-PITO or doclofenac sodium); sodium salicylate; glycerol esters of acetoacetic acid (e.g., glyceryl- 1, 3 -diacetoacetate or l,2-isopropylideneglycerine-3-acetoacetate); and other release-diffusion or intra- or trans-epithelial penetration-promoting agents that are physiologically compatible for mucosal delivery.
  • nitric oxide donors e.g., S-nitroso-N-acetyl-DL-penicillamine, NORl,
  • Other delivery-enhancing agents are selected from a variety of carriers, bases and excipients that enhance mucosal delivery, stability, activity, or trans-epithelial penetration of the PTH peptide.
  • carriers, bases and excipients that enhance mucosal delivery, stability, activity, or trans-epithelial penetration of the PTH peptide.
  • cyclodextrins and ⁇ -cyclodextrin derivatives e.g., 2- hydroxypropyl- ⁇ -cyclodextrin and heptakis(2,6-di-O-methyl- ⁇ -cyclodextrin).
  • cyclodextrins and ⁇ -cyclodextrin derivatives e.g., 2- hydroxypropyl- ⁇ -cyclodextrin and heptakis(2,6-di-O-methyl- ⁇ -cyclodextrin).
  • These compounds optionally conjugated with one or more of the active ingredients and further optionally formulated in an oleaginous base
  • Additional delivery-enhancing agents adapted for mucosal delivery include medium-chain fatty acids, including mono- and diglycerides (e.g., sodium caprate—extracts of coconut oil, Capmul), and triglycerides (e.g., amylodextrin, Estaram 299, Miglyol 810).
  • medium-chain fatty acids including mono- and diglycerides (e.g., sodium caprate—extracts of coconut oil, Capmul), and triglycerides (e.g., amylodextrin, Estaram 299, Miglyol 810).
  • compositions of the present invention may be supplemented with any suitable delivery-enhancing agent that facilitates absorption, diffusion, or penetration of PTH peptide across mucosal barriers.
  • the penetration promoter may be any promoter that is pharmaceutically acceptable.
  • compositions are provided that incorporate one or more delivery-enhancing agents that promote penetration selected from sodium salicylate and salicylic acid derivatives (acetyl salicylate, choline salicylate, salicylamide.); amino acids and salts thereof (e.g.
  • monoaminocarboxlic acids such as glycine, alanine, phenylalanine, proline, hydroxyproline; hydroxyamino acids such as serine; acidic amino acids such as aspartic acid, glutamic acid; and basic amino acids such as lysine — inclusive of their alkali metal or alkaline earth metal salts); and N-acetylamino acids (N-acetylalanine, N-acetylphenylalanine, N-acetylserine, N-acetylglycine, N-acetyllysine, N- acetylglutamic acid, N-acetylproline, N-acetylhydroxyproline.) and their salts (alkali metal salts and alkaline earth metal salts).
  • emulsifiers e.g. sodium oleyl phosphate, sodium lauryl phosphate, sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters.
  • caproic acid lactic acid, malic acid and citric acid and alkali metal salts thereof, pyrrolidonecarboxylic acids, alkylpyrrolidonecarboxylic acid esters, N-alkylpyrrolidones, proline acyl esters, and the like.
  • improved nasal mucosal delivery formulations and methods allow delivery of PTH peptide and other therapeutic agents within the invention across mucosal barriers between administration and selected target sites.
  • Certain formulations are specifically adapted for a selected target cell, tissue or organ, or even a particular disease state.
  • formulations and methods provide for efficient, selective endo- or transcytosis of PTH peptide specifically routed along a defined intracellular or intercellular pathway.
  • the PTH peptide is efficiently loaded at effective concentration levels in a carrier or other delivery vehicle, and is delivered and maintained in a stabilized form, e.g., at the nasal mucosa and/or during passage through intracellular compartments and membranes to a remote target site for drug action (e.g., the blood stream or a defined tissue, organ, or extracellular compartment).
  • the PTH peptide may be provided in a delivery vehicle or otherwise modified (e.g., in the form of a prodrug), wherein release or activation of the PTH peptide is triggered by a physiological stimulus (e.g. pH change, lysosomal enzymes).
  • a physiological stimulus e.g. pH change, lysosomal enzymes.
  • the PTH peptide is pharmacologically inactive until it reaches its target site for activity.
  • the PTH peptide and other formulation components are nontoxic and non-immunogenic.
  • carriers and other formulation components are generally selected for their ability to be rapidly degraded and excreted under physiological conditions.
  • formulations are chemically and physically stable in dosage form for effective storage.
  • Various additional preparative components and methods, as well as specific formulation additives, are provided herein which yield formulations for mucosal delivery of aggregation-prone peptides and proteins, wherein the peptide or protein is stabilized in a substantially pure, unaggregated form using a solubilization agent.
  • a range of components and additives are contemplated for use within these methods and formulations.
  • Exemplary of these solubilization agents are cyclodextrins (CDs), which selectively bind hydrophobic side chains of polypeptides. These CDs have been found to bind to hydrophobic patches of proteins in a manner that significantly inhibits aggregation. This inhibition is selective with respect to both the CD and the protein involved.
  • Such selective inhibition of protein aggregation provides additional advantages within the intranasal delivery methods and compositions of the invention.
  • Additional agents for use in this context include CD dimers, trimers and tetramers with varying geometries controlled by the linkers that specifically block aggregation of peptides and protein.
  • solubilization agents and methods for incorporation within the invention involve the use of peptides and peptide mimetics to selectively block protein-protein interactions.
  • the specific binding of hydrophobic side chains reported for CD multimers is extended to proteins via the use of peptides and peptide mimetics that similarly block protein aggregation.
  • a wide range of suitable methods and anti-aggregation agents are available for incorporation within the compositions and procedures of the invention.
  • mucus is a viscoelastic, gel-like substance consisting of water, electrolytes, mucins, macromolecules, and sloughed epithelial cells. It serves primarily as a cytoprotective and lubricative covering for the underlying mucosal tissues. Mucus is secreted by randomly distributed secretory cells located in the nasal epithelium and in other mucosal epithelia. The structural unit of mucus is mucin.
  • This glycoprotein is mainly responsible for the viscoelastic nature of mucus, although other macromolecules may also contribute to this property.
  • macromolecules include locally produced secretory IgA, IgM, IgE, lysozyme, and bronchotransferrin, which also play an important role in host defense mechanisms.
  • the coordinate administration methods of the instant invention optionally incorporate effective mucolytic or mucus-clearing agents, which serve to degrade, thin, or clear mucus from intranasal mucosal surfaces to facilitate absorption of intranasally administered biotherapeutic agents.
  • a mucolytic or mucus-clearing agent is coordinately administered as an adjunct compound to enhance intranasal delivery of PTH.
  • an effective amount of a mucolytic or mucus-clearing agent is incorporated as a processing agent within a multi-processing method of the invention, or as an additive within a combinatorial formulation of the invention, to provide an improved formulation that enhances intranasal delivery of biotherapeutic compounds by reducing the barrier effects of intranasal mucus.
  • a variety of mucolytic or mucus-clearing agents are available for incorporation within the methods and compositions of the invention.
  • mucolytic and mucus clearing agents can often be classified into the following groups: proteases (e.g., pronase, papain) that cleave the protein core of mucin glycoproteins; sulfhydryl compounds that split mucoprotein disulfide linkages; and detergents (e.g., Triton X-IOO, Tween 20) that break non-covalent bonds within the mucus.
  • proteases e.g., pronase, papain
  • sulfhydryl compounds that split mucoprotein disulfide linkages
  • detergents e.g., Triton X-IOO, Tween 20
  • Additional compounds in this context include, but are not limited to, bile salts and surfactants, for example, sodium deoxycholate, sodium taurodeoxycholate, sodium glycocholate, and lysophosphatidylcholine.
  • bile salts in causing structural breakdown of mucus is in the order: deoxycholate > taurocholate > glycocholate.
  • Other effective agents that reduce mucus viscosity or adhesion to enhance intranasal delivery according to the methods of the invention include, e.g., short-chain fatty acids, and mucolytic agents that work by chelation, such as N-acylcollagen peptides, bile acids, and saponins (the latter function in part by chelating Ca 2+ and/or Mg 2+ which play an important role in maintaining mucus layer structure).
  • Additional mucolytic agents for use within the methods and compositions of the invention include N-acetyl-L-cysteine (ACS), a potent mucolytic agent that reduces both the viscosity and adherence of bronchopulmonary mucus and is reported to modestly increase nasal bioavailability of human growth hormone in anesthetized rats (from 7.5 to 12.2%).
  • ACS N-acetyl-L-cysteine
  • These and other mucolytic or mucus-clearing agents are contacted with the nasal mucosa, typically in a concentration range of about 0.2 to 20 rnM, coordinately with administration of the biologically active agent, to reduce the polar viscosity and/or elasticity of intranasal mucus.
  • mucolytic or mucus-clearing agents may be selected from a range of glycosidase enzymes, which are able to cleave glycosidic bonds within the mucus glycoprotein, ⁇ -amylase and ⁇ -amylase are representative of this class of enzymes, although their mucolytic effect may be limited.
  • bacterial glycosidases which allow these microorganisms to permeate mucus layers of their hosts may have a stronger effect.
  • non-ionogenic detergents are generally also useful as mucolytic or mucus-clearing agents. These agents typically will not modify or substantially impair the activity of therapeutic polypeptides.
  • mucosal tissues e.g., nasal mucosal tissues
  • mucociliary transport in the respiratory tract is a particularly important defense mechanism against infections.
  • ciliary beating in the nasal and airway passages moves a layer of mucus along the mucosa to removing inhaled particles and microorganisms.
  • Ciliostatic agents within the methods and compositions of the invention, increase the residence time of mucosally (e.g., intranasally) administered PTH.
  • delivery is significantly enhanced in certain aspects by the coordinate administration or combinatorial formulation of one or more ciliostatic agents that function to reversibly inhibit ciliary activity of mucosal cells, to provide for a temporary, reversible increase in the residence time of the mucosally administered active agent(s).
  • the foregoing ciliostatic factors are all candidates for successful employment as ciliostatic agents in appropriate amounts (depending on concentration, duration and mode of delivery) such that they yield a transient (i.e., reversible) reduction or cessation of mucociliary clearance at a mucosal site of administration to enhance delivery of PTH peptide, analogs and mimetics, and other biologically active agents disclosed herein, without unacceptable adverse side effects.
  • Certain surface active agents are readily incorporated within the mucosal delivery formulations and methods of the invention as delivery-enhancing agents. These agents, which may be coordinately administered or combinatorially formulated with PTHand other delivery-enhancing agents disclosed herein, may be selected from a broad assemblage of known surface active agents.
  • Examples of surface-active agent are nonionic polyoxyethylene ether, bile salts, sodium glycocholate, deoxycholate, derivatives of fusidic acid, sodium taurodihydrofusidate, L- ⁇ -phosphatidylcholine didecanoyl (DDPC), polysorbate 80, polysorbate 20, a polyethylene glycol, cetyl alcohol, polyvinylpyrolidone, a polyvinyl alcohol, lanolin alcohol, and sorbitan monooleate.
  • the mechanisms of action of these various classes of surface active agents typically include solubilization of a biologically active agent.
  • nonionic surface active agent has no charge group in its head.
  • nonionic surface active agents are nonionic polyoxyethylene ether, polysorbate 80, polysorbate 20, polyethylene glycol, cetyl alcohol, polyvinylpyrolidone, polyvinyl alcohol, poloxamer F68, poloxamer F 127, and lanolin alcohol.
  • bile salts and some fusidic acid derivatives reportedly inhibit proteolytic degradation of proteins by nasal homogenates at concentrations less than or equivalent to those required to enhance protein absorption. This protease inhibition may be especially important for peptides with short biological half-lives.
  • the present invention provides a pharmaceutical composition that contains PTH in combination with delivery-enhancing agents disclosed herein formulated in a pharmaceutical preparation for mucosal delivery.
  • the combinatorial formulations and/or coordinate administration methods herein incorporate an effective amount of PTH which may adhere to charged glass thereby reducing the effective concentration in the container.
  • Silanized containers for example, silanized glass containers, are used to store the finished product to reduce adsorption of the PTH to a glass container.
  • a kit for treatment of a mammalian subject comprises a stable pharmaceutical composition of PTH formulated for mucosal delivery to the mammalian subject wherein the composition is effective for treating or preventing osteoporosis or osteopenia.
  • the kit further comprises a pharmaceutical reagent bottle to contain the PTH.
  • the pharmaceutical reagent bottle is composed of pharmaceutical grade polymer, glass or other suitable material.
  • the pharmaceutical reagent bottle is, for example, a silanized glass bottle.
  • the kit further comprises an aperture for delivery of the composition to a nasal mucosal surface of the subject.
  • the delivery aperture is composed of a pharmaceutical grade polymer, glass or other suitable material.
  • the delivery aperture is, for example, a silanized glass.
  • a silanization technique combines a special cleaning technique for the surfaces to be silanized with a silanization process at low pressure.
  • the silane is in the gas phase and at an enhanced temperature of the surfaces to be silanized.
  • the method provides reproducible surfaces with stable, homogeneous and functional silane layers having characteristics of a monolayer.
  • the silanized surfaces prevent binding to the glass of polypeptides or mucosal delivery enhancing agents of the present invention.
  • the procedure is useful to prepare silanized pharmaceutical reagent bottles to hold PTH peptide compositions of the present invention.
  • Glass trays are cleaned by rinsing with double distilled water (ddH 2 O) before using.
  • the silane tray is then be rinsed with 95% EtOH, and the acetone tray is rinsed with acetone.
  • Pharmaceutical reagent bottles are sonicated in acetone for 10 minutes. After the acetone sonication, reagent bottles are washed in ddH 2 O tray at least twice. Reagent bottles are sonicated in 0.1 M NaOH for 10 minutes. While the reagent bottles are sonicating in NaOH, the silane solution is made under a hood.
  • PTH may be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to the eyes, ears, skin or other mucosal surfaces.
  • compositions according to the present invention are often administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by a variety of methods known to those skilled in the art.
  • Preferred systems for dispensing liquids as a nasal spray are disclosed in U.S. Patent No. 4,511,069, hereby incorporated by reference.
  • the formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in U.S. Patent No. 4,511,069.
  • Additional aerosol delivery forms may include, e.g., compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the biologically active agent dissolved or suspended in a pharmaceutical solvent, e.g., water, ethanol, or a mixture thereof.
  • a pharmaceutical solvent e.g., water, ethanol, or a mixture thereof.
  • Nasal and pulmonary spray solutions of the present invention typically comprise PTH, formulated with a surface active agent, such as a nonionic surfactant (e.g., polysorbate-80), and water.
  • a surface active agent such as a nonionic surfactant (e.g., polysorbate-80)
  • Another embodiment of the present invention comprises PTH, formulated with methyl- ⁇ -cyclodextrin, EDTA, didecanoylphosphatidyl choline (DDPC), and water.
  • the nasal spray solution further comprises a propellant.
  • the pH of the nasal spray solution is optionally between about pH 3.0 and 6.0, preferably 4.0 ⁇ 0.3.
  • Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases.
  • Suitable preservatives include, but are not limited to, phenol, methyl paraben, paraben, m-cresol, thiomersal, chlorobutanol, benzylalkonimum chloride, and the like.
  • Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphatidyl cholines, and various long chain diglycerides and phospholipids.
  • Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid, and the like.
  • gases include, but are not limited to, nitrogen, helium, chlorofiuorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and the like.
  • the biologically active agent can be combined with various pharmaceutically acceptable additives, as well as a base or carrier for dispersion of the active agent(s).
  • local anesthetics e.g., benzyl alcohol
  • isotonizing agents e.g., sodium chloride, mannitol, sorbitol
  • adsorption inhibitors e.g., Tween 80
  • solubility enhancing agents e.g., cyclodextrins and derivatives thereof
  • stabilizers e.g., serum albumin
  • reducing agents e.g., glutathione
  • the tonicity of the formulation is typically adjusted to a value at which no substantial, irreversible tissue damage is induced in the nasal mucosa at the site of administration.
  • the tonicity of the solution is adjusted to a value of about 1/3 to 3, more typically 1/2 to 2, and most often 3/4 to 1.7.
  • PTH formulations may also contain a hydrophilic low molecular weight compound as a base or excipient.
  • a hydrophilic low molecular weight compound provide a passage medium through which a water-soluble active agent, such as PTH, may diffuse through the base to the body surface where PTH is absorbed.
  • the hydrophilic low molecular weight compound optionally absorbs moisture from the mucosa or the administration atmosphere and dissolves the water-soluble active
  • the molecular weight of the hydrophilic low molecular weight compound is generally not more than 10000 and preferably not more than 3000.
  • Exemplary hydrophilic low molecular weight compound include polyol compounds, such as oligo-, di- and monosaccarides such as sucrose, mannitol, sorbitol, lactose, L- arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose, glycerin, and polyethylene glycol.
  • hydrophilic low molecular weight compounds useful as carriers within the invention include N-methylpyrrolidone, and alcohols (e.g. oligovinyl alcohol, ethanol, ethylene glycol, and propylene glycol) These hydrophilic low molecular weight compounds can be used alone or in combination with one another or with other components of the intranasal formulation.
  • compositions of the invention may alternatively contain as pharmaceutically acceptable carriers substances as required to approximate physiological conditions, such as tonicity adjusting agents, wetting agents and the like, for example, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.
  • pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • Therapeutic compositions for administering PTH can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • polyol for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the biologically active agent can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts, and gelatin.
  • Mucosal administration according to the invention allows effective self- administration of treatment by patients, provided that sufficient safeguards are in place to control and monitor dosing and side effects. Mucosal administration also overcomes certain drawbacks of other administration forms, such as injections, that are painful and expose the patient to possible infections and may present drug bioavailability problems.
  • systems for controlled aerosol dispensing of therapeutic liquids as a spray are well known.
  • metered doses of active agent are delivered by means of a specially constructed mechanical pump valve, U.S. Patent No. 4,511,069.
  • PTH may be administered to the subject intranasally once daily.
  • a therapeutically effective dosage of the PTH may include repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate or prevent osteoporosis or osteopenia.
  • Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, dog, non-human primate, and other accepted animal model subjects known in the art.
  • effective dosages can be determined using in vitro models (e.g., immunologic and histopathologic assays). Using such models, only ordinary calculations and adjustments are typically required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the biologically active agent(s) (e.g., amounts that are intranasally effective, transdermally effective, intravenously effective, or intramuscularly effective to elicit a desired response).
  • in vitro models e.g., immunologic and histopathologic assays.
  • a therapeutically effective amount of the biologically active agent(s) e.g., amounts that are intranasally effective, transdermally effective, intravenously effective, or intramuscularly effective to elicit a desired response.
  • the actual dosage of biologically active agents will of course vary according to factors such as the disease indication and particular status of the subject (e.g., the subject's age, size, fitness, extent of symptoms, and susceptibility factors), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the biologically active agent(s) for eliciting the desired activity or biological response in the subject. Dosage regimens may be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the biologically active agent are outweighed in clinical terms by therapeutically beneficial effects.
  • a non-limiting range for a therapeutically effective amount of a PTH peptide within the methods and formulations of the invention is 0.7 ⁇ g/kg to about 25 ⁇ g/kg.
  • an intranasal dose of PTH peptide is administered at dose high enough to promote the increase in bone mass but low enough so as not to induce any unwanted side-effects such as nausea.
  • a preferred intranasal dose of PTH (1-34) is about 1 to about 10 ⁇ g/kg weight of the patient, most preferably about 6 ⁇ g/kg weight of the patient.
  • a patient will receive about 1 to about 1000 ⁇ g, more preferably about between 20 to 800 ⁇ g, most preferably 100 ⁇ g to about 600 ⁇ g with 300 ⁇ g being a dose that is considered to be highly effective.
  • a non-limiting range for a therapeutically effective amount of a biologically active agent within the methods and formulations of the invention is between about 0.001 pmol to about 100 pmol per kg body weight, between about 0.01 pmol to about 10 pmol per kg body weight, between about 0.1 pmol to about 5 pmol per kg body weight, or between about 0.5 pmol to about 1.0 pmol per kg body weight.
  • Per administration it is desirable to administer at least one microgram of PTH, more typically between about 10 ⁇ g and 5.0 mg, and in certain embodiments between about 100 ⁇ g and 1.0 or 2.0 mg to an average human subject. For certain oral applications, doses as high as 0.5 mg per kg body weight may be necessary to achieve adequate plasma levels.
  • An intranasal dose of a parathyroid hormone will range from 1 ⁇ g to 1000 ⁇ g of parathyroid hormone, preferably 20 ⁇ g to 800 ⁇ g, more preferably 100 ⁇ g to 600 ⁇ g with 300 ⁇ g being a dose that is considered to be highly effective.
  • the invention provides compositions and methods for intranasal delivery of PTH peptide, wherein the PTH peptide is repeatedly administered through an intranasal effective dosage regimen that involves multiple administrations of the PTH peptide to the subject during a daily or weekly schedule to maintain a therapeutically effective elevated and lowered pulsatile level of PTH peptide during an extended dosing period.
  • the compositions and method provide PTH peptide that is self-administered by the subject in a nasal formulation between one and six times daily to maintain a therapeutically effective elevated and lowered pulsatile level of PTH peptide during an 8 hour to 24 hour extended dosing period.
  • kits, packages and multicontainer units containing the above described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects.
  • these kits include a container or formulation that contains PTH in combination with mucosal delivery enhancing agents disclosed herein formulated in a pharmaceutical preparation for mucosal delivery.
  • the intranasal formulations of the present invention can be administered using any spray bottle (i.e., a bottle with an actuator, spray pump).
  • a nasal spray bottle is the, "Nasal Spray Pump w/ Safety Clip", which delivers a dose of 0.1 mL per squirt and has a diptube length of 36.05 mm (Pfeiffer of America, Princeton, NJ).
  • Intranasal doses of a PTH peptide such as parathyroid hormone can range from 0.1 ⁇ g/kg to about 1500 ⁇ g/kg.
  • the particle size of the spray is between 10 - 100 ⁇ m (microns) in size, preferably 20 - 100 ⁇ m in size.
  • parathyroid hormone peptides can be administered intranasally using a nasal spray or aerosol. This is surprising because many proteins and peptides have been shown to be sheared or denatured due to the mechanical forces generated by the actuator in producing the spray or aerosol. In this area the following definitions are useful.
  • Aerosol - A product that is packaged under pressure and contains therapeutically active ingredients that are released upon activation of an appropriate valve system.
  • Metered aerosol - A pressurized dosage form comprised of metered dose valves, which allow for the delivery of a uniform quantity of spray upon each activation.
  • Powder aerosol - A product that is packaged under pressure and contains therapeutically active ingredients in the form of a powder, which are released upon activation of an appropriate valve system.
  • Spray aerosol - An aerosol product that utilizes a compressed gas as the propellant to provide the force necessary to expel the product as a wet spray; it generally applicable to solutions of medicinal agents in aqueous solvents.
  • Spray - A liquid minutely divided as by a jet of air or steam.
  • Nasal spray drug products contain therapeutically active ingredients dissolved or suspended in solutions or mixtures of excipients in nonpressurized dispensers.
  • Metered spray - A non-pressurized dosage form consisting of valves that allow the dispensing of a specified quantity of spray upon each activation.
  • Suspension spray A liquid preparation containing solid particles dispersed in a liquid vehicle and in the form of course droplets or as finely divided solids.
  • plume geometry the spray's divergence angle
  • spray pattern the spray's cross-sectional ellipticity, uniformity and particle/droplet distribution (spray pattern)
  • time evolution of the developing spray have been found to be the most representative performance quantities in the characterization of a nasal spray pump.
  • plume geometry and spray pattern measurements are key identifiers for verifying consistency and conformity with the approved data criteria for the nasal spray pumps. Definitions Plume Height - the measurement from the actuator tip to the point at which the plume angle becomes non-linear because of the breakdown of linear flow.
  • Ellipticity Ratio the ratio of the major axis to the minor axis D 10 - the diameter of droplet for which 10% of the total liquid volume of sample consists of droplets of a smaller diameter ( ⁇ m)
  • D 50 the diameter of droplet for which 50% of the total liquid volume of sample consists of droplets of a smaller diameter ( ⁇ m), also known as the mass median diameter Dgo - the diameter of droplet for which 90% of the total liquid volume of sample consists of droplets of a smaller diameter ( ⁇ m)
  • Span measurement of the width of the distribution, the smaller the value, the narrower the distribution. Span is calculated as ⁇ ⁇ 90 " 10 ' .
  • % RSD - percent relative standard deviation the standard deviation divided by the mean of the series and multiplied by 100, also known as % CV.
  • a nasal spray device can be selected according to what is customary in the industry or acceptable by the regulatory health authorities.
  • One example of a suitable device is described in described in U.S. Application No. 10/869,649 (S. Quay and G. Brandt: Compositions and methods for enhanced mucosal delivery of Y2 receptor- binding peptides and methods for treating and preventing obesity).
  • an intranasal dose of a PTH peptide parathyroid hormone is administered at dose high enough to promote an increase in bone mass, but low enough so as not to induce any unwanted side-effects such as nausea.
  • a preferred intranasal dose of a PTH is about 1 ⁇ g - 10 ⁇ g/kg weight of the patient, most preferably about 6 ⁇ g/kg weight of the patient.
  • a patient will receive 1 ⁇ g to 1000 ⁇ g, more preferably about between 20 ⁇ g to 800 ⁇ g, most preferably 100 ⁇ g to about 600 ⁇ g with 300 ⁇ g being the dose that is considered to be highly effective.
  • a PTH peptide such as parathyroid hormone (1-34) is preferably administered once a day. The following examples are provided by way of illustration, not limitation. EXAMPLES
  • the MatTek cell line is normal, human-derived tracheal/bronchial epithelial cells (Epi AirwayTM Tissue Model). Cells were cultured for 24-48 hours before using to produce a tissue insert.
  • Each tissue insert was placed in an individual well containing 1 ml media. On the apical surface of the inserts, 100 ⁇ l of test formulation was applied, and the samples were shaken for 1 h at 37 °C. The underlying culture media samples were taken at 20, 40, and 60 minutes and stored at 4 ° C for up to 48 hours for lactate dehydrogenase (LDH, cytotoxicity) and sample penetration (PTH HPLC evaluations). The 60-min samples were used for lactate dehydrogenase (LDH, cytotoxicity). Transepithelial electrical resistance (TER) was measured before and after the 1-h incubation. Following the incubation, the cell inserts were analyzed for cell viability via the mitochondrial dehydrogenase (MDH) assay. A reverse phase high pressure liquid chromatography method was used to determine the Teriparatide concentration in the tissue permeation assay.
  • TER measurements were accomplished using the Endohm-12 Tissue Resistance Measurement Chamber connected to the EVOM Epithelial Voltohmmeter (World Precision Instruments, Sarasota, FL) with the electrode leads.
  • the electrodes and a tissue culture blank insert were equilibrated for at least 20 minutes in MatTek medium with the power off prior to checking calibration.
  • the background resistance was measured with 1.5 ml Media in the Endohm tissue chamber and 300 ⁇ l Media in the blank insert.
  • the top electrode was adjusted so that it was close to, but not in contact with, the top surface of the insert membrane. Background resistance of the blank insert was about 5-20 ohms.
  • 300 ⁇ l of MatTek medium was added to the insert followed by placement in the Endohm chamber. Resistance was expressed as (resistance measured - blank) X 0.6 cm 2 .
  • MTT assay MTT-100, MatTek kit.
  • Thawed and diluted MTT concentrate was pipetted (300 ⁇ l) into a 24-well plate. Tissue inserts were gently dried, placed into the plate wells, and incubated at 37°C for 3 hours. After incubation, each insert was removed from the plate, blotted gently, and placed into a 24-well extraction plate.
  • the cell culture inserts were immersed in 2.0 ml of the extractant solution per well (to completely cover the sample). The extraction plate was covered and sealed to reduce evaporation of extractant. After an overnight incubation at room temperature in the dark, the liquid within each insert was decanted back into the well from which it was taken, and the inserts discarded.
  • the extractant solution 200 ⁇ l in at least duplicate
  • the amount of cell death was assayed by measuring the loss of lactate dehydrogenase (LDH) from the cells using a CytoTox 96 Cytoxicity Assay Kit
  • LDH analysis of the apical media was evaluated. The appropriate amount of media was added to the apical surface in order to total 250 ⁇ L, taking into consideration the initial sample loading volume. The inserts was shaken for 5 minutes. 150 ⁇ L of the apical media was removed to eppendorf tubes and centrifuged at 10000 rpm for 3 minutes. 2 ⁇ L of the supernatant was removed and added to a 96 well plate. 48 uL of media was used to dilute the supernatant to make a 25x dilution. For LDH analysis of the basolateral media, 50 ⁇ L of sample was loaded into a 96-well assay plates.
  • Fresh, cell-free culture medium was used as a blank. Fifty microliters of substrate solution was added to each well and the plates incubated for 30 minutes at room temperature in the dark. Following incubation, 50 ⁇ l of stop solution was added to each well and the plates read on an optical density plate reader at 490 ran.
  • results of the MTT assays showed no significant reduction of cell viability when cells were treated with all formulations.
  • Media applied to the apical side did not show an effect on cell viability whereas the Triton X treated group showed significant reduction of cell viability, as expected.
  • the results of the LDH assays showed no significant cytotoxicity was observed when cells were treated with all formulations.
  • Media control applied to the apical side did not show cytotoxicity whereas Triton X treated group showed significant cytotoxicity, as expected.
  • a preferred formulation containing non-GRAS enhancers is exemplified by the combination of 45 mg/ml M- ⁇ -CD, 1 mg/ml DDPC, and 1 mg/ml EDTA. It is also preferred that the formulation contain a suitable solvent such as water, a preservative, such as sodium benzoate, chlorobutanol or benzalkonium chloride, and a tonicifiers such as a sugar or polyol such as trehalose or a salt such as sodium chloride. Alternatively, the formulation could contain other enhancers including alternative solubilizers, surface-active agents and chelators.
  • a preferred formulation containing GRAS enhancers is exemplified by the combination of 1 mg/mL Tween-80, 100 mg/mL ethanol and 1 mg/ml EDTA. It is also preferred that the formulation contain a suitable co-solvent such as water, a preservative, such as sodium benzoate, chlorobutanol or benzalkonium chloride, and a tonicifiers such as a sugar or polyol such as trehalose or a salt such as sodium chloride. Alternatively, the formulation could contain other GRAS enhancers including alternative surface-active agents, co-solvents, and chelators.
  • compositions containing GRAS enhancers are exemplified by inclusion of 1 mg/mL Tween-80 (polysorbate 80). It is also preferred that the formulation contain a suitable co-solvent such as water, a preservative, such as sodium benzoate, chlorobutanol or benzalkonium chloride, and a tonicifiers such as a sugar or polyol such as trehalose or a salt such as sodium chloride. Alternatively, the formulation could contain other GRAS enhancers such as alternative surface-active agents.
  • a suitable co-solvent such as water
  • a preservative such as sodium benzoate, chlorobutanol or benzalkonium chloride
  • a tonicifiers such as a sugar or polyol such as trehalose or a salt such as sodium chloride.
  • the formulation could contain other GRAS enhancers such as alternative surface-active agents.
  • a PTH formulation will be supplied as a liquid in a bottle for intranasal administration via an actuator.
  • Formulations containing 1-10 mg/mL PTH at pH 4.0- 4.5 were tested for "as-sold" stability.
  • "As-sold" stability studies are defined as those studies involving formulation stored within a closed (i.e., capped) bottle, placed at specific storage or accelerated temperature conditions for specified amounts of time.
  • Formulation excipients were selected from the group consisting of PTH; methyl- ⁇ - cyclodextrin (M- ⁇ -CD); ethylenediaminetetraacetic acid (EDTA); didecanoylphosphatidyl choline (DDPC); chlorobutanol (CB); sodium benzoate (NaBZ), polysorbate 80, and sorbitol.
  • the initial pH of the formulations was adjusted to pH 4.0 or 4.5 with sodium hydroxide or hydrochloric acid, as necessary.
  • the formulations that were tested are shown in Table 2.
  • the reported storage conditions for injectable FORTEO ® is 2-8° C for up to 28 days (four weeks).
  • the storage stability of PTH formulations # 1, # 3, # 4, and # 7 was monitored at regular intervals by determining the remaining percentage of PTH relative to initial using HPLC. All four formulations used in the stability studies included CB as preservative and were at a pH of 4.0.
  • the results in Tables 3 and 4 show PTH intranasal formulations # 1, # 3, # 4, and # 7 may be safely stored at 5° C and 25° C for at least four weeks without a significant decrease in stability.
  • Formulations # 1, # 3, # 4, and # 7 remained stable for at least 24 weeks when stored at 5° C.
  • Formulation #7 was the most stable of the tested formulations at 5° C and 25° C. Storage conditions of PTH intranasal formulations at 5° C for at least 24 weeks is longer than the current recommended storage conditions for injectable FORTEO.
  • the percent PTH remaining from initial was determined at I 5 2, 3, and 4 week timepoints.
  • the 30° C data without buffer is compared to the injectable formulation data containing buffer from U.S. Pat. No. 6,770,623 (the '623 formulation).
  • the '623 formulation contained 0.1 mg/mL rhPTH (1-34), 50 mg/mL mannitol, 2.5 mg/mL m-cresol, 0.52 mg/mL acetic acid and 0.12 mg/mL sodium acetate.
  • Formulations # 1 and # 4 without a buffer at 30° C had stability similar to the '623 formulation with buffer at 30° C. At 50° C, Formulations # 1 , # 3, # 4 and # 7 have a greater stability than the '623 formulation. Formulation # 7 was the most stable compared to other formulations tested at 40° C and 50° C. Table 5. Percent Stability with and without buffer at 30° C
  • PTH formulations # 1 and # 4 were also tested for in-use and spray stability at both 5 0 C and 30 0 C storage temperatures over a 29-day period. Results include % Peptide Recover and % Total Peptide Impurity. "In-use" studies are those in which an actuator is present and the bottles were primed five times initially, and then actuated once daily by hand after subjecting to the storage temperatures. All bottles were returned to the 5°C and 30°C stability chamber after 30 minute exposure to room temperature. All bottles were actuated daily, and the actuated samples were collected and stored at -20°C until scheduled for HPLC measurements.
  • HPLC measurements are scheduled for in-use (i.e., in the bottle with an actuator present) and spray (i.e., measured from the spray produced by the actuator in the bottle) samples at Day 0, Day 8, Day 15, Day 22 and Day 29.
  • spray i.e., measured from the spray produced by the actuator in the bottle samples at Day 0, Day 8, Day 15, Day 22 and Day 29.
  • the HPLC measurements for stability are shown in Table 8 (% Peptide Recovery) and Table 9 (% Total Impurity).
  • the PTH solution is provided in a multi-unit dose container to deliver a metered dose of 0.1 mL of drug product per actuation.
  • Hydrochloric acid is added for pH adjustment to meet target pH of 4.0 ⁇ 0.2 or 4.5 ⁇ 0.2, as appropriate.
  • the stability of the formulations was monitored at regular intervals.
  • This study was a single-site, open-label, active controlled, 5 period crossover, dose ranging study involving 6 healthy male and 6 healthy female volunteers.
  • the commercially available formulation of teriparatide, FORSTEO was the active control.
  • the five study periods were as follows:
  • Period 1 All subjects received FORSTEO (injection) 20 ⁇ g subcutaneously.
  • Period 2 All subjects received 500 ⁇ g intranasal dose of teriparatide, 100 microliter spray of intranasal formulation as described in Example 5, Formulation #6, Table 2.
  • Period 3 All subjects received 200 ⁇ g intranasal dose of teriparatide, 100 microliter spray of intranasal formulation as described in Example 5, Formulation #3 Table 2.
  • Period 4 All subjects received a 1000 ⁇ g intranasal dose of teriparatide, 100 microliter spray of intranasal formulation as described in Example 5, Formulation #7 Table 2.
  • Period 5 All subjects received a 400 ⁇ g intranasal dose of teriparatide, 2 X
  • Plasma samples for PK were collected at 0 (i.e., pre-dose), 5, 10, 15, 30, 45, 60, 90 minutes and 2, 3, and 4 hours post-dose and analyzed using a validated method. Because the bioassay is fully cross reactive with endogenous PTH(I -84), all data was corrected for pre-dose values. When this correction resulted in a negative post-dose value, all such negative values were set to 'missing'. Values reported as ⁇ LLOQ were set to half LLOQ in order to evaluate PK and change from baseline. Standard pharmacokinetic parameters, including AUCi ast , AUQnf, C max , tyi, t max , and K e were calculated using WinNonlin.
  • Intra-subject variability of the pharmacokinetic profiles was evaluated for the test versus the reference using analysis of variance methods.
  • An analysis of variance (ANOVA) was performed based on a 2-period design and incorporating a main effect term for each of the two products under consideration (Snedecor GW and Cochran WG, One- Way Classifications ⁇ Analysis of Variance. In: Statistical Methods, 6 th ed.: Iowa State University Press, Ames, IA, (1967) pp. 258-98). (Subject (Sequence) was a random effect in the model with all others fixed.) A separate model was created for each dose of teriparatide nasal spray versus the reference.
  • the 90% confidence intervals were generated for the ratio of test dose/reference with respect to C max , AUQast, and AUCj n f. These values were natural log (ln)-transformed prior to analysis. The corresponding 90% confidence intervals for the geometric mean ratio were obtained by taking the antilog of the 90% confidence intervals for the difference between the means on the log scale. These analyses were not performed to demonstrate bioequivalence but were for informational purposes only. As a result, no adjustment to the confidence level for each of the paired comparisons was made to account for multiplicity of analysis. This study is hypothesis-generating only. For t max , the analyses were run using Wilcoxon's signed-rank test (Steinijans VW and Diletti E (1983) Eur J Clin Pharmacol.
  • AUCi nf AUCi ast + C t /K e where C t is the last measurable concentration and K e is the apparent terminal phase rate constant.
  • K e Apparent terminal phase rate constant, where K e is the magnitude of the slope of the linear regression of the log concentration versus time profile during the terminal phase. ti /2 Apparent terminal phase half-life (whenever possible), where X ⁇ a - (In2)/K e .
  • Figures 1 and 2 show the mean plasma concentration versus time for periods 1-5, and the ratio of C max to mean, low dose formulations versus Forsteo, respectively.
  • a summary of arithmetic mean pharmacokinetic parameters for each formulation and dose of teriparatide are presented in Table 12.
  • the mean t max was 0.68 versus 0.57 and 0.17 hours for the FORSTEO and low dose nasal formulations of Formulation # 6 and # 3, respectively.
  • the C max was 1.6 and 2.4 fold higher than FORSTEO for each low dose formulation.
  • the AUCi ast was 1.23 and 1.45 fold higher than FORSTEO for each low dose formulation.
  • both nasal formulations had a greater C max and AUC as compared to FORSTEO.
  • the t max occurred sooner after dosing for the nasal formulations, particularly for Formulation # 3.
  • a tm of approximately 1 hour was very similar for the nasal formulations compared to FORSTEO, except for the low dose Formulation # 3, where there may be an apparent outlier for subject numbers 1 and 5. If the two subjects are removed the tm is 1.5 hours, the same as FORSTEO.
  • the apparent difference in elimination rates may reflect slower wash-in for the subcutaneous product and Formulations # 6 and # 7 when compared with Formulation # 3.
  • Both nasal formulations have very similar ty 2 compared to FORSTEO.
  • Formulation # 3 also showed good dose linearity from 200 to 400 ⁇ g dose based on the clearance rate and regression analysis.
  • Formulation # 3 was less variable than Formulations #6 and # 7 and FORSTEO based on % coefficient of variation. Accordingly, the intranasal formulations of the invention exceed the C max and AUC values for the currently marketed subcutaneous product. This demonstrates that the levels of the marketed product can be exceeded by a nasally administered product, and also that the concentrations of PTH in nasal formulations can be decreased if it is desired to more closely approximate the plasma concentrations of the currently approved product.
  • the droplet size and spray characterization of two teriparatide intranasal formulations were evaluated using the Pfeiffer 0.1 ml Nasal Spray Pump 65550 with 36 mm dip tube.
  • the droplet size distribution is characterized by laser diffraction using a Malvern MasterSizer S modular particle size analyzer and a MightyRunt automated actuation station. Single spray droplet distribution is volume weighted measurement.
  • the Spray Pattern is characterized using a Spray VIEW NSP High Speed Optical Spray Characterization System and Spray VIEW NSx Automated Actuation System. The data are shown in Table 18.
  • the ellipticity ratio is 1.3 and 1.4 for formulation # 5 and # 2, respectively.
  • EXAMPLE 9 The anabolic effects of synthetic human PTH 1-34 and recombinant PTH 1-34 (Forteo ,® , EIi LiIIy U.S.) were studied in male rats. A common vehicle (composed of glacial acetic acid, m-cresol, sterile water, sodium acetate and mannitol) was used for each treatment Group and Vehicle control.
  • mice received either vehicle or one of two dose levels (16 ⁇ g/kg/d or 80 ⁇ g/kg/d) of synthetic or recombinant PTH 1-34 via subcutaneous (SQ) administration.
  • the animals were randomized into treatment groups (10 rats/group) based on body weight. Each animal was given once daily subcutaneous injections of vehicle or test PTH 1-34 treatment, starting on Day 1 and continuing for 21 consecutive days. Cage side observations were performed twice daily, and weekly body weight measurements were taken throughout the study. Animals were given a total of two doses of calcein, one dose six (6) and one dose two (2) days prior to scheduled necropsy. On Day 21, blood samples for pharmacokinetic analysis were collected from animals in select treatment groups. At the conclusion of the treatment period and after blood collection on Day 21, the animals were euthanized and bone specimens collected. The treatment groups are shown in Table 21.
  • Bone in the distal and midshaft regions of the right femur were analyzed using peripheral quantitative computed tomography (pQCT) and bone strength was determined via three-point bending at the femoral mid-shaft and in the marrow cavity of the distal femur.
  • the entire right tibia was subject to dual X-ray absorptiometry scan (DXA).
  • Bone 5 mineral content, area, and density of four areas of the tibia were analyzed separately (whole tibia and distal, midshaft and proximal tibia) by DXA.
  • PTH 1-34 (synthetic form) was administered once daily via intranasal instillation to rats for at least 13 weeks. For comparison, one group received commercially available recombinant PTH 1-34 via subcutaneous injection. Assessment of toxicity was based on mortality, clinical observations, ophthalmic 5 examinations, body weights, food consumption, clinical and anatomic pathology, and toxicokinetic evaluations. Two synthetic PTH 1-34 formulations were used in the study, PTH-072-1 and PTH-074 at low and high doses (formulations are shown in Table 22).
  • Placebo was 0.9% Sodium Chloride, USP (sterile saline). 5 *Four animals/sex from Groups 7 and 8 and four females in Group 9 received Calcein (10mg/kg via intraperitoneal injection) on Days 86 and 90.
  • the ti /2 for PTH 1-34 when administered in the PTH-072-1 formulation ranged from 14 to 21 minutes in male and female rats; T ma ⁇ ranged from 5 to 15 minutes for both males and females.
  • C max ranged from 5,041 pg/mL to 12,911 pg/mL in male rats and from 3,044 pg/mL to 5106 pg/mL in female rats.
  • AUQas t ranged from 100,038 pg-min/mL to 457,644 pg-min/mL in males and 58,890 pg-min/mL to 73,444 pg-min/mL in females.
  • the AUQ 3St values for male and female rats exceeded that in humans by 80-fold and 13-fold, respectively.
  • the ti /2 for PTH 1-34 when administered in the PTH-074-1 formulation ranged from 12 to 24 minutes; T max ranged from 5 to 30 minutes for both male and female rats.
  • C max ranged from 12,251 pg/mL to 35,964 pg/mL in male rats and from 3,679 pg/mL to 17,175 pg/mL in female rats.
  • AUCi ast ranged from 252,790 pg ⁇ min/mL to 1,010,348 pg-mm/mL in males and 78,059 pg-min/mL to 377,278 pg-min/rnL in females. In comparison to a clinical study with PTH-074-1 formulation, the AUCi ast values for male and female rats exceeded that in humans by 71 -fold and 27-fold, respectively.
  • the ti /2 for PTH 1-34 when administered by injection ranged from 15 to 23 minutes; T max was 5 minutes for female rats.
  • C max and AUCi as t ranged from 7,721 pg/mL to 12,200 pg/mL and from 140,945 pg-min/mL to 296,908 pg-min/mL, respectively.
  • PTH 1-34 related clinical signs, ophthalmic observations, body weight changes, or food consumption changes were observed, regardless of route of administration, dose level, or formulation.
  • No changes considered to be attributable to the intranasal administration of PTH 1-34 were observed in the nasal turbinate tissues from any animal in the study.
  • the nasal cavity was sectioned such that meaningful regions of the cavity were represented, and the soft (epithelial lining) or hard (bone and cartilage based structures) tissues of the nasal cavity were examined.
  • Toxicity and toxiokinetics OfPTH 1-34 was studied after administration of PTH 1-34 once daily by intranasal instillation to dogs for at least 13 weeks.
  • One additional group received recombinant PTH 1-34 by subcutaneous injection for comparison.
  • the dog study groups are shown in Table 24.
  • T max for PTH 1-34 ranged from 8 to 26 minutes. Cmax and AUCiast showed dose-dependence.
  • T max for PTH 1-34 ranged from 8 to 24 minutes.
  • T ma x for PTH 1-34 ranged from 13 to 26 minutes. Systemic exposure for subcutaneous injection, as determined by C max , AUQastj and AUQ nf , were intermediate between the low and high doses of PTHi -34 following intranasal administration.
  • the relative bioavailability for PTHi -34 was greater at the higher concentration dose for both intranasal formulations.
  • the relative bioavailability for PTHi -34 was greater in the PTH-072-1 formulation.
  • the T max , C max , and AUQ 8St for PTH 1-34 in each formulation were consistent with achieving peak levels soon after dosing and returning to baseline within a few hours post-dose; this general profile is desired for induction of anabolic actions of PTHi -34 .
  • nasal surface doses were approximately 0.9-fold for Day 1 and at least 1.5-fold by the end of the study.
  • nasal surface area doses were at least 1.0-fold on Day 1 and 3.8-fold or greater by the end of the study.
  • C max and AUCi ast for PTHi -34 were at least 7-fold and 10-fold, respectively, greater in the dog than that found in humans at representative doses.
  • Results were collected for mortality, clinical signs, gross nasal passage observations, ophthalmic findings, electrocardiogram measurements, blood pressure and heart rate differences, body weights, food consumption, clinical and anatomic pathology, and toxicokinetic evaluations. All animals in the study survived to scheduled necropsy. No PTHi -34 related clinical signs, ophthalmic findings, electrocardiogram differences, blood pressure and heart rate differences, body weights, or food consumption changes were noted.
  • the nasal cavity was sectioned such that meaningful regions of the cavity were represented, and the soft (e.g., epithelial lining) or hard tissues (e.g., bone and cartilage based structures) of the nasal cavity were examined.
  • PTH 1-34 injection but not the intranasal formulations, produced elevated serum calcium levels at the pre-dose time point.
  • the absolute level for group mean serum calcium and the frequency of statistically significant elevation was similar for the injection group and the two high does intranasal formulations, but slightly higher for the injection group.
  • the magnitude of change for the intranasal formulations was dose-dependent. Serum ionized calcium followed the same general pattern as total calcium.
  • the time and magnitude of the observed effect precludes the likelihood of catabolic effects. Instead, the biodynamic effect is one of an anabolic drug.
  • Such anabolic effects in animals are predictive of resistance to fracture in humans and used as predictors by the FDA.
  • Transiently elevated serum calcium is an expected action of PTH 1-34 , and there were no adverse clinical observations noted in association with the transient elevation in serum calcium.
  • the (gross) nasal passage examination showed an increased incidence of erythema in PTH 1-34 treated animals (both subcutaneous and intranasal administration) compared to placebo control.
  • PTH 1-34 is known to have actions on vascular tone, and erythema is likely a reflection of the pharmacology of PTH 1-34 .
  • An attenuation of the normal age-related decrease in serum alkaline phosphatase activity is another effect OfPTH 1-34 .
  • Mean serum alkaline phosphatase activity dropped approximately 47% and 48% on Day 93 for placebo control males and females, respectively. None of the PTH 1-34 treated groups (both subcutaneous and intranasal administration) showed a drop of greater than 30% in alkaline phosphatase activity. Attenuation of serum alkaline phosphatase activity was statistically significant in male dogs in both high dose intranasal groups as well as the males in the injection group.
  • Elevated serum calcium was observed with intranasal doses OfPTH 1-34 .
  • the elevated serum calcium is an anabolic effect of PTH.
  • Higher alkaline phosphatase activity in intranasal and subcutaneous PTH 1-34 treated animals was suggestive of osetoblast activity.
  • a higher incidence of minimally thickened trabecular bone was noted in femur and sternum of PTH 1-34 treated animals.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Rheumatology (AREA)
  • Endocrinology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Otolaryngology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Biochemistry (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Formulation pharmaceutique aqueuse de PTH, qui renferme la substance PTH(I -34) et un tensioactif non ionique, forme pharmaceutique contenant la formulation, système de délivrance de PTH à l'homme par exposition de couche de cellules mucosales à la formulation, et procédé d'utilisation de la formulation pour le traitement de l'ostéoporose chez un mammifère, de préférence avec un temps de concentration de plasma maximum, Tmax, de PTH(I-34) après l'administration inférieur à 30 minutes. On décrit aussi un procédé de traitement de l'ostéoporose chez un mammifère par administration intranasale de formulation PTH comprenant une quantité thérapeutiquement efficace de PTH(I-34) et un ou plusieurs excipients pouvant être agent de solubilisation, agent de chélation et un ou plusieurs polyols, PTH(I-34) et un tensioactif non ionique. On décrit encore une utilisation de PTH(l-34) pour l'élaboration de médicament utilisable dans le traitement de l'ostéoporose chez un mammifère, comprenant une quantité thérapeutiquement efficace de PTH(I-34) et un ou plusieurs excipients pouvant être tensioactif non ionique, agent de solubilisation, agent de chélation et un ou plusieurs polyols.
EP06749687A 2005-10-06 2006-04-10 Formulations de pth et procedes d'utilisation Withdrawn EP1931374A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US11/246,450 US20060052306A1 (en) 2004-05-10 2005-10-06 GRAS composition for enhanced mucosal delivery of parathyroid hormone
US11/246,406 US20060052305A1 (en) 2004-05-10 2005-10-06 Method of treating osteoporosis using intranasal parathyroid hormone
US11/347,551 US20060127320A1 (en) 2004-05-10 2006-02-03 Method of delivering parathyroid hormone to a human
US11/390,940 US20060189533A1 (en) 2004-05-10 2006-03-27 Stable pharmaceutical dosage forms of teriparatide
PCT/US2006/013377 WO2007044069A2 (fr) 2005-10-06 2006-04-10 Formulations de pth et procedes d'utilisation

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EP2197464A2 (fr) * 2007-09-11 2010-06-23 Mondobiotech Laboratories AG Utilisation du peptide sfllr-oh et du muramyl-dipeptide comme agents thérapeutiques
CA2698980A1 (fr) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent therapeutique
EP2052736A1 (fr) * 2007-10-26 2009-04-29 Nycomed Danmark ApS Formulations d'hormones parathyroïdiennes et leurs utilisations
KR101796604B1 (ko) * 2016-08-30 2017-11-10 목포대학교 산학협력단 위장관 흡수증진제를 함유하는 부갑상선 호르몬의 경구 투여 제형
CN110755380B (zh) * 2019-11-04 2021-09-24 中国人民解放军军事科学院军事医学研究院 一种甲状旁腺激素1-34鼻喷剂、其制备方法及用途

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GB9020544D0 (en) * 1990-09-20 1990-10-31 Sandoz Ltd Improvements in or relating to organic compounds
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ATE219374T1 (de) * 1996-02-01 2002-07-15 Chugai Pharmaceutical Co Ltd Medikamente für die behandlung oder vorbeugung von thrombocytopenie
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AU2006299887A1 (en) 2007-04-19
WO2007044069A2 (fr) 2007-04-19

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