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
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
  • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M15/00—Inhalators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/53—Colony-stimulating factor [CSF]
  • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Definitions

• Aerosolization is an important method of drug delivery.
• An aerosolized drug is inhaled by the patient, bringing the drug into contact with the patients lungs. Because the airways in the lungs provide a large surface area for adsorption, aerosolization is useful not only for local delivery of drugs (e.g., bronchodilators and other anti-asthma drugs), but also for systemic administration. In fact, aerosolization is believed to be a preferred route for the non invasive systemic administration of certain drugs, including proteins. In general, the size of the aerosolized particle is adjusted to control whether the particle reaches the small airways and alveoli of the lungs (necessary for systemic administration) or is delivered throughout the respiratory tract (localized delivery) .
• Aerosolized drugs are generated by dispersing either a dry particulate or an aqueous or non aqueous solution containing the drug into a gaseous medium.
• a dry particulate the particles are suspended in propellants which evaporate after the suspension is released from a pressurized device into the air.
• the dry particulate can be aerosolized directly from a dry powder inhaler device. Where the drug is aerosolized from an aqueous solution, the drug solution is converted into a fine spray of minute water droplets.
• Aerosolized delivery of a drug can be by means of a single metered dose or through continuous delivery.
• One means for metered dose delivery of an aerosolized drug is a device referred to as a metered dose inhaler (MDI) .
• MDI metered dose inhaler
• an MDI When actuated, an MDI disperses a suspension of fine particles from a pressurized container. The patient inhales simultaneously upon actuating the inhaler, thus drawing the aerosolized drug into contact with the patient's lungs.
• the drug delivered through an MDI is aerosolized from a dry particulate, but it is also possible to deliver a single metered dose of a drug aerosolized from an aqueous solution.
• an MDI requires a certain amount of coordination and technique that may not be reasonably expected from certain patients, such as the very young, the elderly and the infirm.
• drugs that can be readily formulated for use in an MDI.
• continuous delivery of an aerosolized drug is often preferred.
• the most common method for continuous delivery of an aerosolized drug is by a nebulizer which administers the drug to a patient who inhales the drug through normal breathing over an extended period of time.
• an aqueous solution of the drug is continuously converted to a spray within the nebulizer, with only a small amount (approximately 1%) of the aqueous spray leaving the nebulizer directly for delivery to the patient at any given time.
• the aqueous spray that does not escape from the nebulizer impacts on the walls or baffles of the nebulizer and drains back to the fluid reservoir at the bottom of the nebulizer where it is again aerosolized into an aqueous spray until the reservoir is depleted or until drug administration is otherwise terminated.
• aerosolized drugs such as bronchodilators, corticosteroids, antibiotics, antihistamines and mucolytics
• bronchodilators corticosteroids
• antibiotics antibiotics
• antihistamines antihistamines
• mucolytics mucolytics
• protein-based drugs such as growth factors and cytokines
• aerosolization is considered to be a preferred alternative delivery route for proteins that are impeded by oral delivery due to barriers such as instability toward proteolytic enzymes.
• aerosolized delivery of proteins introduces new challenges due to the delicate nature of these proteins as compared with more traditional drugs.
• a potential problem with the aerosolized delivery of certain proteins is the delicate nature of the quaternary, and also secondary and tertiary, structure of the protein molecule which, if disrupted, leads to aggregation and degradation of the protein, resulting in loss of biological activity.
• the physical stresses inherent in aerosolization such as the formation of a large area of air-water interface, destabilize the structure of many proteins .
• This problem is exacerbated in the case of continuous flow delivery, where approximately 99% of the drug in aqueous solution is refluxed, i.e., aerosolized from aqueous solution more than once before it is delivered to the patient. This refluxing of the drug solution introduces additional physical stresses to the protein with each aerosolization cycle. It is an object of the present invention to provide a method for preventing activity loss and degradation and/or aggregation of proteins aerosolized from an aqueous solution.
• the present invention provides a method for stabilizing proteins aerosolized from an aqueous solution.
• the aerosolized proteins are stabilized by adding a water soluble polar organic compound, such as polyethylene glycol, or surfactant to the aqueous solution prior to aerosolization.
• the present invention further provides a stabilized aqueous protein solution containing a polar organic compound or surfactant and a system for aerosolizing the stabilized aqueous protein- containing solution.
• FIGURE 1 is a graph plotting the fraction of initial activity of LDH versus time of aerosolization.
• FIGURE 2 is a non-denaturing electrophoretic gel showing G-CSF aerosolized from an aqueous solution containing PEG and an aqueous solution not containing PEG ("control") .
• FIGURE 3 is a graph plotting the percent degradation of G-CSF versus time of aerosolization both with 1% PEG and without PEG (“control”) .
• FIGURE 4 is an SDS electrophoretic gel showing
• FIGURE 5 is a graph plotting the fraction of initial activity of LDH versus time of aerosolization for LDH aerosolized from an aqueous solution containing PEG and an aqueous solution not containing PEG ("control") .
• FIGURE 6 is a graph plotting the fraction of initial activity of LDH versus time of aerosolization for LDH aerosolized from an aqueous solution containing various concentrations of PEG and an aqueous solution not containing PEG ("control") .
• FIGURE 7A is graph showing the effect of different molecular weights of PEG, at a concentration of 0.001%, on stabilization of G-CSF during aerosolization.
• FIGURE 7b is graph showing the effect of different molecular weights of PEG, at a concentration of 1%, on stabilization of G-CSF during aerosolization.
• the present invention provides a method for protecting proteins against aerosolization-induced degradation, aggregation and/or loss of protein activity.
• the present invention can be used in any situation where aerosolization of a protein is desired, including pulmonary delivery of an aerosolized protein- based drug to a patient.
• the present invention further provides a stabilized aqueous protein- containing solution that is resistant to degradation, aggregation and/or loss of protein activity upon aerosolization and a system for aerosolizing the stabilized aqueous protein-containing solution.
• the system for aerosolizing the stabilized protein-containing solution will be used in the pulmonary administration of an aerosolized drug to a patient.
• aerosolized proteins are protected from activity loss, degradation and/or aggregation upon aerosolization by the addition of a polar organic compound or surfactant to the aqueous solution containing the protein.
• Polar Organic Compound means an organic compound having a moderate non-polar moiety in an undefined region of the molecule (i.e., the non-polar moiety is not localized) .
• Polar organic compounds are capable of reducing the surface tension of water.
• Surfactant means a compound having both a polar moiety and a strong non-polar moiety, each of which is contained in a separate localized region of the molecule, such that the surfactant can reduce interfacial tension between two immiscible phases by aligning at the interface and can form micelles. Because of the localization of the polar and non-polar moieties in surfactants, these compounds decrease the surface tension of water to a much greater extent than polar organic compounds.
• Protein refers to any protein that is dispersed in the aqueous solution of the present invention. Ordinarily, the protein will be a protein of therapeutic utility that is intended for inhalation therapy.
• the protein may be chemically synthesized or purified from a natural source, but will typically be a recombinant form of a naturally occurring protein.
• the protein may also be chemically modified, usually by the covalent attachment of a chemical moiety to the protein molecule to enhance its therapeutic effect, such as extending the therapeutic effect of the protein.
• aerosolization of proteins can result in a time-dependent loss of protein activity.
• Aerosolization adversely affects protein stability due to several factors, including the large amount of air- water interface created in the aerosolization process.
• the degree of loss of protein activity from aerosolization will vary from protein to protein depending on the structure and stability of the particular protein and the means used to achieve aerosolization.
• shear forces from the air pressure of the jet disrupt the protein in addition to the interaction of the protein with the air-water interface created in the aerosolization process. Drying may also cause denaturation of the protein as it becomes concentrated in the evaporating droplets.
• the degree of activity loss from aerosolization using a jet nebulizer is related to both the air pressure of the jet and the starting volume of aqueous solution in the reservoir.
• shear forces are not present in the same form, but ultrasonic waves can denature the protein in other ways (e.g., heating) .
• Partial degradation does not prevent efficacious use of the protein as a therapeutic, but does require the administration of a greater amount of the protein to achieve an efficacious level of drug than would be required if the protein were delivered entirely in its active form.
• the immunogenicity of aggregated protein will be different from the biologically active form, thus causing an immune response from the patient to whom the aerosolized drug is administered.
• the exact mechanism by which proteins suffer activity loss and degradation and/or aggregation during aerosolization is unknown.
• Analysis of an aqueous solution containing the drug granulocyte colony stimulating factor (G-CSF) demonstrated that G-CSF itself decreases the surface tension of water even before aerosolization, indicating an orientation of this protein at the air-water interface.
• G-CSF drug granulocyte colony stimulating factor
• adsorption of proteins at the air- water interface in an aqueous solution involves at least partial unfolding of the protein to expose the hydrophobic regions of the protein to the non-polar air phase.
• Analysis of an aqueous solution of G-CSF demonstrated homogeneity of the protein at this point (i.e., prior to aerosolization), with the protein appearing as a single band on a non-denaturing electrophoretic gel and no evidence of aggregation being evident. This indicates that the surface adsorption of G-CSF prior to aerosolization is probably a reversible process.
• G-CSF was observed to be irreversibly denatured, consequently suffering aggregation, chemical degradation, or both.
• G-CSF is a potent growth factor of granulocyte lineage that enhances the number of white blood cells. Because of this activity, it has a beneficial clinical application in treating a number of diseases and conditions, including infectious disease.
• the current commercial route for administering G-CSF has been intravenous injection exclusively, but efficacy has been demonstrated in animals by means of pulmonary administration. (International Patent Publication No. WO 92/16192.)
• Much of the aerosolized G-CSF can be expected to reach the patient in inactive form as covalent and non-covalent dimers and higher molecular weight aggregates and other degraded forms.
• a marked stabilization of aerosolized proteins against degradation was surprisingly observed when certain polar organic compounds or surfactants, as defined above, were included in the aqueous solution from which the proteins were aerosolized in accordance with the teachings of the present invention.
• This marked stability was demonstrated in connection with both the enzyme lactate dehydrogenase (LDH) and the aforementioned G-CSF.
• Preferred polar organic compounds found to have a protective effect on aerosolized proteins include polyethylene glycol (PEG) and methyl pentanediol (MPD) . It is more preferred that the polar organic compound be PEG.
• the polar organic compound be PEG 1000 (i.e., PEG with an average molecular weight of 1000) , although other molecular weight PEGs are also effective, although the lower molecular weight PEGs must be used at a higher concentration to achieve optimum protection of the aerosolized protein.
• PEG 1000 i.e., PEG with an average molecular weight of 1000
• a preferred surfactant is Tween 80.
• Polyethylene glycol is not known for its ability to stabilize proteins.
• PEG is better known for its ability to destabilize proteins due to its weak hydrophobicity.
• This compound is also known to salt-out proteins due to its large excluded volume. Ibid.
• PEG is strongly excluded from the surface of proteins, due to steric exclusion principles. This thermodynamically unfavorable interaction between PEG and proteins from whose surface PEG is excluded, results in decreased solubility of the proteins.
• PEG would be expected to destabilize proteins and decrease the solubility of proteins such as G-CSF, thus encouraging protein aggregation.
• PEG has surprisingly been found to stabilize both LDH and G-CSF during nebulization from an aqueous solution. This may be due to the repeating nature of the amphiphilic units created by the non-localized polar and non-polar regions of the PEG molecule. In fact, PEG decreases the surface tension of water, although to a much lesser extent than common surfactants such as SDS and Tween. Thus, PEG is adsorbed at air-water interface, which may exclude proteins, such as G-CSF, from coming to the surface.
• the polar organic compound be employed at a concentration sufficient to reduce the surface tension of water to no greater than about 65 dynes/centimeter.
• the polar organic compound it is more preferred to use the polar organic compound at a concentration capable of reducing the surface tension of water to no greater than about 48 dynes/centimeter.
• the polar organic compounds contemplated for use in connection with the present invention are to be contrasted with certain surfactants, also known to decrease surface tension, that have been suggested for reducing or preventing surface induced aggregation of the protein caused by aerosolization of the protein from an aqueous solution.
• surfactants include "conventional" surfactants such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty acid esters, with an especially preferred surfactant being polyoxyethylene sorbitan monooleate.
• International Patent Publication No. WO 92/16192 are examples of surfactants.
• these surfactants have been concluded as not being necessary for the aerosolization of G-CSF from an aqueous solution. Ibid.
• Surfactants such as Tween may also be capable of lowering surface tension, in fact to a significantly greater extent than PEG or other polar organic compounds contemplated for use in the practice of the present invention.
• surfactants particularly cationic surfactants
• cationic surfactants lower surface tension, they also bind strongly to the protein, particularly in micelle form, thus altering its stability.
• the tendency of surfactants to cause foaming makes it somewhat difficult to utilize the surface tension reducing properties of these compounds in air-jet nebulization in many instances. Nevertheless, it may sometimes be preferred to use a surfactant to stabilize a protein against degradation.
• the surfactant be employed at a concentration sufficient to reduce the surface tension of water to no greater than about 40 dynes/centimeter.
• the polar organic compounds contemplated by the present invention have the advantage of being able to lower surface tension without binding to and destabilizing the protein. Also, in the case of PEG, the random coil formation of PEG in aqueous solution and the large hydrodynamic volume that even small molecular weight PEGs occupy may add to their ability to prevent proteins from reaching the air interface generated during nebulization.
• cytokines including various hematopoietic factors such as the aforementioned G-CSF, SCF, EPO, GM-CSF, CSF-1, the interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, IGFs, M-CSF, thymosin, TNF, or LIF.
• cytokines including various hematopoietic factors such as the aforementioned G-CSF, SCF, EPO, GM-CSF, CSF-1, the interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, IGFs, M-CSF, thymosin, TNF, or LIF.
• Interferons alpha-, beta-, gamma- or consensus interferons
• growth factors or hormones such as human or other animal growth hormones (for example, bovine, porcine, or chicken growth hormone), ET-1, FGF, KGF, EGF, IGF, and PDGF.
• Protease inhibitors such as metalloproteinase inhibitors are contemplated (such as TIMP-1, TIMP-2, or other proteinase inhibitors) .
• Nerve growth factors are also contemplated, such as BDNF and NT3.
• Plasminogen activators such as tPA, urokinase and streptokinase are also contemplated.
• peptide portions of proteins having all or part of the primary structure of the parent protein and at least one of the biological properties of the parent protein.
• Analogs such as substitution or deletion analogs, or those containing altered amino acids, such as peptidomimetics are also contemplated.
• Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products aerosolized from aqueous solution, including but not limited to nebulizers and metered dose inhalers, which are familiar to those skilled in the art. It will be appreciated, however, that the greatest advantage of the present invention lies in its use in connection with continuous aerosolization of proteins, such as by a nebulizer, because of the increased degree of degradation ordinarily encountered with continuous aerosolization of proteins.
• Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri, the A corn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado and the Collison 3-jet nebulizer, manufactured by BGI, Inc., Waltham, Massachusetts.
• Formulations suitable for use with a nebulizer will typically comprise the protein to be aerosolized (or chemically modified protein) dissolved in water.
• the concentration should be about 0.1 to 25 mg of G-CSF per ml of solution.
• the formulation may also include a buffer or may simply be aqueous HCl. Examples of buffers which may be used are sodium acetate, citrate and glycine.
• the buffer will have a composition and molarity suitable to adjust the solution to a pH in the range of 2.5 to 5.5. Generally, buffer molarities of from 1 mM to 50 mM are suitable for this purpose.
• a means for aerosolizing the stabilized aqueous protein-containing solution of the present invention may be provided along with the aqueous solution to provide a system for generating an aerosolized protein.
• the system of the present invention may be useful in any situation where the aerosolization of proteins is desired, it is anticipated that this system will be most useful in the pulmonary administration of aerosolized protein-based drugs.
• the present invention contemplates the administration of therapeutic amounts of aerosolized protein sufficient to achieve the desired therapeutic effect. What constitutes a therapeutically effective amount of the protein or in a particular case will depend on a variety of factors which the knowledgeable practitioner will take into account. including the desired therapeutic result, the severity of the condition or illness being treated, the physical condition of the subject, and so forth.
• a dosage regimen will be followed such that the normal blood neutrophil level for the individual undergoing treatment is restored, at least in cases of abnormally low or depressed blood neutrophil counts.
• the normal blood neutrophil level is about 5000 to 6000 neutrophils per microliter of blood.
• Neutrophil counts below 1000 in humans are generally regarded as indicative of severe neutropenia and as placing the subject at great risk to infection.
• Clinical studies with cancer patients suffering from chemotherapy-induced neutropenia have shown that subcutaneous injected doses of 3-5 ⁇ g G-CSF/kg every twenty-four hours are effective in elevating acutely deficient blood neutrophil levels above 1000.
• the operating conditions for delivery of a suitable inhalation dose will vary according to the type of mechanical device employed.
• the frequency of administration and operating period will be dictated chiefly by the amount of protein or other active composition per unit volume in the aerosol.
• the higher the concentration of protein in the nebulizer solution the shorter the operating period.
• Some devices such as MDIs may produce higher aerosol concentrations than others and thus will be operated for shorter periods to give the desired result.
• LDH lactate dehydrogenase
• Aerosolization of LDH under each of the conditions set forth above resulted in an irreversible, time dependent loss of enzymatic activity in the reservoir solution, as demonstrated by electrophoretic/densitometric analysis.
• approximately 60% of the starting enzyme activity was lost after 60 minutes of nebulization at 40 psig, as shown in Figure 1.
• the loss of activity was reduced by progressively lowering the applied air pressure, but not substantially (see Figure 1) .
• a homogeneous 6% non-denaturing (i.e., in the absence of SDS) Novex gel was used to evaluate the aerosolized G-CSF.
• An electrode buffer contained 25 mM Tris and 20 mM glycine.
• the sample for the gel contained the same concentrations of 25 mM Tris and 20 mM glycine, along with 2.5% glycerol, 2.5% sucrose, and 0.025% ⁇ -bromophenol blue.
• a constant 50 V was applied to the gel, with a total running time of approximately 7 hours.
• Approximately 30 mg of G-CSF was loaded onto the gel. Each gel was then stained with Coomassie blue. After destaining, if necessary, the gels were subjected to densitometric analysis using an LKB Ultrascan® XL laser densitometer.
• PEG 1000 and 2-methyl-2,4- pentanediol were added to different aqueous solutions containing either LDH or G-CSF.
• Concentrations of 0.1%, 1% and 10% (w/v) PEG 1000 and 1% and 5% and 10% (w/v) MPD were used to determine the optimum concentrations of the polar organic compounds for the stabilization of the two proteins being studied.
• the resulting solutions were aerosolized, with the aerosolized proteins being analyzed by gel electorophoresis and densitometry, as set forth in the previous examples.
• the surface tension of aqueous solutions was measured using the ring method of Denuoy (Leukenheimer and Wantke, Colloid and Polymer Sci . , 259, 354 (1981) using a Kr ⁇ ss K10T surface tensiometer (Kruss Company, Hamburg, Germany) . All measurements were made at 20°C. For all samples, at least 30 minutes were allowed prior to measurement for equilibration of the surface concentrations of the various components. All values were adjusted for density as described by Harkins and Jordan, J. Amer. Chem. Soc , 52, 1772 (1930). The surface tension of pure water (Super Q®, Millipore) was measured prior to and after all sets of measurements to ensure accuracy. In addition, experiments were performed to ensure that potential protein deposition onto the tensiometer ring did not affect the measured values. The results are set forth in Table I.
• MPD exhibit a protective effect on the protein G-CSF during aerosolization, although the amount of the polar organic compound required to achieve a high level (i.e., greater than 90% retention of activity) is higher for MPD (5%) than for PEG (1%) .
• PEG 8000 average molecular weight of 8000
• PEG 400 average molecular weight of 400
• the polar organic compounds and/or surfactants were added prior to nebulization, which was then performed according to the protocol described in Example 1, above.
• MPD was tested for its ability to protect LDH and G-CSF during aerosolization. Like PEG, MPD is a protein stabilizer with weak surfactant activity. When LDH was formulated in 1% (w/v) MPD, the protein retained nearly 100% of the initial activity over the nebulization period relative to approximately only 33% retention in the absence of any additives. The stabilization effect of MPD against G-CSF degradation upon 10 minutes of nebulization is shown in Table II.
• sucrose did not provide any significant protection against degradation, and, in fact, had a tendency to further destabilize LDH activity.
• the effects of 1% (w/v) and 0.5 M (17%) sucrose on G-CSF stabilization were also determined after 10 minutes of nebulization, with the results being nearly identical to those obtained for LDH.
• sucrose did not have a protective effect against degradation during nebulization of G-CSF.
• sucrose demonstrated a potentially destabilizing effect.
• Tween 80 like the polar organic compounds PEG and MPD, stabilizes G-CSF against degradation in a strongly concentration-dependent manner. Essentially no stabilizing effect was observed below a surfactant concentration of 0.001% (w/v), although substantial stabilization was observed at around 0.01% (w/v). Virtually complete stabilization was observed at surfactant concentrations above 0.05% (w/v). A similar stabilization pattern was observed with respect to LDH activity in the presence of Tween 80.
• Table IV summarizes the results of surface tension measurements of aqueous solutions of G-CSF and of the various polar organic compounds and/or surfactants examined in this and preceding example.

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• 1994
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