WO2005056037A1 - Preparations pour inhalation dosees de proteines et de peptides - Google Patents

Preparations pour inhalation dosees de proteines et de peptides Download PDF

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WO2005056037A1
WO2005056037A1 PCT/GB2004/005206 GB2004005206W WO2005056037A1 WO 2005056037 A1 WO2005056037 A1 WO 2005056037A1 GB 2004005206 W GB2004005206 W GB 2004005206W WO 2005056037 A1 WO2005056037 A1 WO 2005056037A1
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formulation according
formulation
protein
dnase
pvp
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PCT/GB2004/005206
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WO2005056037B1 (fr
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Marc Barry Brown
Stuart Allen Jones
Gary Peter Martin
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Medpharm Limited
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    • 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
    • A61K9/008—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy comprising drug dissolved or suspended in liquid propellant for inhalation via a pressurized metered dose inhaler [MDI]

Definitions

  • the present invention relates to glycosidically stabilised preparations of therapeutic materials for use in metered dose inhalation devices, and methods for their preparation.
  • Pulmonary delivery has been employed for many years for drugs intended to have localised, rather than systemic, effects.
  • nebulisers metered dose inhalers (MDI) and dry powder inhalers (DPI).
  • MDI metered dose inhalers
  • DPI dry powder inhalers
  • Nebulisers are particularly effective for the administration of aqueous formulations of drug to non-ambulatory patients.
  • Drug solution is converted into microdroplets which are inhaled by the patient, these microdroplets providing the facility to deliver the drug in a variety of dose volumes, ranging from several milligrams to grams.
  • nebulisers are generally large and unsuitable for ambulatory use, and there is a problem with the potential instability of drugs in aqueous solution, as well as during the process of nebulisation. In addition, reproducible dosing can be difficult with these devices.
  • MDIs are the most widely used pharmaceutical inhalation devices.
  • the formulations used in these devices routinely comprise drug, propellants, and stabilising excipients.
  • the drug is formulated together with the excipients and then combined with the propellants, under pressure, to form either a suspension or solution formulation. Fine, respirable particles of drug are then produced as a consequence of the break up of droplets expelled from the device under pressure, followed by extremely rapid evaporation of the propellants.
  • the amount of drug is controlled by delivering a pre-metered volume of propellant/drug mixture.
  • DPIs lie in their ability to dispense large quantities of drug from a stable, powder formulation.
  • MDIs are able to dispense formulation in a more controlled, and more effective manner, but are more susceptible to physical instability changes. A loss of physical stability can lead to particle aggregation and a lowering in the respirable fraction, or both (Yamashita et al., 1998).
  • MDIs are propellant-based delivery systems which, until recently, relied on the use of chlorofluorocarbons, or CFCs [trichlorofluoromethane (CFC-11) dichlorofluoromethane (CFC-12) and 1,2-dichlorotetrafluoroethane (CFC-114)], in varying ratios, as the principal component of the formulation.
  • CFCs chlorofluorocarbons
  • CFC-11 dichlorofluoromethane
  • CFC-114 1,2-dichlorotetrafluoroethane
  • HFA-134a tetrafluoroethane
  • HFA-227 heptafluoropropane
  • Both of these hydrofluoroalkanes have boiling points substantially below 0°C, unlike CFC-11 (23.8°C).
  • the HFAs have poor solvency for those surfactants commonly employed as excipients in CFC-based MDIs, thereby further complicating the formulation design.
  • the two most commonly employed formulation strategies for new HFA based MDIs include either the addition of a co-solvent, such as ethanol, to generate a solution MDI, or the incorporation of novel stabilising excipients that are soluble in HFAs to form a suspension MDI.
  • Addition of a co-solvent to a drug-propellant mix can enhance the solubility of the drug to a point where it is completely dissolved in the HFA vehicle.
  • a solution MDI generates respirable particles in a different manner to more traditional suspension formulations.
  • particles of a defined size have already been manufactured and simply require safe storage and delivery by the device.
  • a solution uses the design of the device and the energy created by the evaporating solvent to form the particles upon actuation of the metering valve.
  • the size of the particles ejected from a solution MDI is, therefore, heavily dependent on the actuation orifice diameter and the device design (Lewis et al, 1998).
  • optimisation of these two parameters can potentially produce a dramatic increase in the delivery efficiency of the MDI compared to suspension based formulations (LeBelle et al., 1996; Stein, 1999).
  • Blondino and Byron investigated the effects of a solution formulation on the chemical stability of a model drug acetylsalicyclic acid. Results from this work indicated that inclusion of a co-solvent to enhance the drug-excipient-propellant compatibility also increased the chemical degradation of the drug. In this study, this was found to be dependent on the concentration of surfactant. Furthermore, within a solution formulation, the drug is exposed to the significant levels of dissolved water taken up in the HFA propellant (Vervaet and Byron, 1999), and this can also induce chemical degradation. Manufacturing an MDI formulation as a solution tends, therefore, to lose the prime advantage of the dosage form, which should be to provide a protective, apolar environment, which enhances both chemical and physical stability.
  • a suspension based MDI overcomes the fundamental flaws associated with solution formulations.
  • a physically stable suspension of a therapeutic agent within a propellant provides a protective environment from which particles can be combined with numerous excipients to potentially achieve a versatile range of drug delivery properties.
  • many therapeutic agents require additional stabilising excipients to overcome the problems associated with long-term physical stability within the formulation.
  • the traditional excipients cannot be used for this purpose due to the switch of MDI propellants from CFCs to HFAs.
  • the formulation and delivery of macromolecules is substantially more difficult than for the more commonly used low molecular weight organic compounds.
  • One of the major reasons for this is added complexity of the structural make up of macromolecules.
  • Proteins for example, have up to four levels of structural hierarchy including primary, secondary, tertiary and quaternary structures. If such compounds are to be used as therapeutic agents, they must be stored in a formulation and delivered to the site of action with minimal changes to these structural properties, as failure to do so could result in reduction or complete loss of therapeutic activity, and may also lead to immunogenicity, through changes in conformation leading to failure to recognise the protein, or peptide, as 'self.
  • rhDNase I recombinant human deoxyribonuclease I
  • rhDNase I is the only therapeutic protein specifically formulated for delivery to the lung.
  • rhDNase I is a hydrophilic glycosylated molecule with a molecular weight of ⁇ 33 kDa. It is commercially available as Pulmozyme ® , in the form of a nebuliser solution. It breaks down the viscosity of lung secretions of cystic fibrosis patients by digesting the endogenous DNA, which can be present at levels of up to 14 mg ml "1 in some cases. This digestion reduces the viscosity and facilitates the removal of the mucus from the lung (Gonda, 1996).
  • atomisation using a nebuliser can deliver less than 30% of the drug to the lungs (Clarke et al, 1993), while the machine is bulky and difficult to use.
  • Pulmozyme ® in solution is highly susceptible to heat degradation and has to be stored below 8°C and hence would not be considered an ideal formulation.
  • glycosidically stabilised macromolecules such as proteins and peptides
  • have substantially greater stability in the presence of HFAs when formulated with polyhydroxylated polyalkenes, such as PVA, and polyvinylpyrrolidone (PVP).
  • PVA polyhydroxylated polyalkenes
  • PVP polyvinylpyrrolidone
  • the present invention provides a formulation of a therapeutic substance suitable for delivery to a patient by a metered dose inhalation device, the formulation comprising a substantially dry powder preparation of the substance, in association with a stabilising amount of a glycoside, polyvinylpyrrolidone, and a polyhydroxylated polyalkene, in combination with one or more propellants therefor.
  • the present invention provides a formulation of a therapeutic substance suitable for delivery to a patient by a metered dose inhalation device, the substance being in association with a stabilising amount of a glycoside and being formulated in one or more propellants and/or cosolvent, characterised in that the therapeutic substance is first prepared as a substantially dry powder in the presence of polyvinylpyrrolidone and a polyhydroxylated polyalkene, prior to formulation with propellant.
  • Preferred such substances are proteins and peptides, especially those comprising one or more regions of ⁇ -helix. More preferred are enzymes, especially those whose activity is dependent on one or more regions of ⁇ -helix.
  • polymers such as PVP may usefully be reported in terms of the Fikentscher K-value, derived from solution viscosity measurements, generally at 25°C.
  • Fikentscher K-value derived from solution viscosity measurements, generally at 25°C.
  • the relationship between the viscosity in water at 25°C, the K-value, and the approximate molecular weight of PVP is shown in the Table, below.
  • PVP with K values of up to 120 and beyond are known, it is generally preferred to employ those with K values of up to 50, preferably no more than K30, with those having a K value of no more than 20 being most preferred.
  • polyvinylpyrrolidone K15 is employed in the present invention, although it will be appreciated that the K value is not a guarantee of the uniformity of the molecular weight of the individual PVP molecules, the K value providing a guide to the average molecular weight (MW).
  • Preferred therapeutic substances are peptides and proteins, and especially those capable of having a therapeutic effect via respiratory, nasal or generally naso- pharyngeal surface membrane administration from a pressurised propellant.
  • the protein or peptide may act in situ, or systemically.
  • a particularly preferred substance is DNase I, preferably human or humanised DNase I, especially DNase I substantially indistinguishable from naturally occurring human DNase I in amino acid sequence or tertiary structure. Human DNase I is most preferred. While human DNase I is the most preferred, the present invention further extends to formulations comprising other DNases, including human DNase II and bovine DNase.
  • DNase I for example, can be formulated with PVP, a polyhydroxylated polyalkene and a glycoside in an MDI to retain both biological activity and structural integrity during the production of respirable particles and formulating the particles with HFA propellant.
  • PVP polyhydroxylated polyalkene
  • HFA glycoside
  • the sugar and the polymers in combination, protect the protein from both heat-induced denaturation during spray- drying and solvent induced changes upon formulation.
  • formulations of the invention are less likely to be immunogenic, as the additives tend to stabilise the conformation of the active molecule.
  • the formulations of the invention can be used with portable MDI devices which are easy to use.
  • the stabilisation of the protein allows it to be stored at room temperature.
  • the delivery efficiency also tends to be higher than with nebulisers, while the delivered protein also generally has significantly greater activity than in a nebulisable formulation.
  • Therapeutic substances are generally any substances suitable for administration via an MDI device for therapeutic purposes, whether for prophylaxis or treatment.
  • therapeutic substances suitable for use in the formulations of the present invention are peptides and proteins.
  • the majority of peptides and proteins are not conformationally stable over long periods, and lose activity, or physical stability, often both. This loss of activity arises not only through degeneration of the peptide or protein, but also from aggregation of the suspended formulation particles, which serves to reduce the fine particle mass critical for the treatment of the patient.
  • the molecules may be stabilised by the presence of suitable glycosidic compounds, particularly the lower oligosaccharides, particularly the di-, tri-, and tetra- saccharides.
  • suitable glycosidic compounds particularly the lower oligosaccharides, particularly the di-, tri-, and tetra- saccharides.
  • glycosides and “glycosidic compounds” are used interchangeably herein.
  • the composition of the oligosaccharide is not critical to the present invention, and the molecule may comprise a furanosyl residues, pyranosyl residues, straight chain elements, or mixtures thereof.
  • sucrose comprises a furanosyl and a pyranosyl residue
  • mannitol comprises a pyranosyl residue and a straight chain element.
  • suitable disaccharides include lactose, isomaltose, cellobiose, maltose and trehalose, of which trehalose is preferred.
  • suitable oligosaccharides include raffinose, melezitose and stachyose. It will be appreciated that the present invention envisages the use of any of these, or other, oligosaccharides either individually or as mixtures.
  • a particularly preferred glycosidic compound is trehalose.
  • glycosidic compounds that may be used include such compounds as mannitol, xylitol, sorbitol, maltitol, isomalt and lactitol. Suitable amounts of the glycosidic compounds are, very approximately, on parity with the therapeutic substance, by weight. More generally, the amount of glycosidic compounds may vary between about 30% and 400% by weight of the therapeutic substance.
  • glycosidic compounds are preferably simply carbohydrate compounds, but the present invention also includes derivatives thereof, including the glucuronides. It is an advantage of the present invention that, by combination with a glycoside, PVP, and a suitably substituted polyhydroxylated polyalkene, the therapeutic substances are now able to be provided in formulations which are stable, even in the presence of haloalkane propellants. It is a particular advantage that such stability is demonstrated in the presence of HFAs, but it will be appreciated that such stability is also demonstrated in the presence of other propellants, such as CFCs, and alkanes, such as butane and propane or combinations of said propellants.
  • propellants such as CFCs, and alkanes, such as butane and propane or combinations of said propellants.
  • the combination glycoside, PVP and polyhydroxylated polyalkene serves to lend substantial stability to therapeutic substances, and appears to be especially useful to stabilise proteins and peptides containing one or more regions of ⁇ -helix. It is a further advantage that formulations of the invention are particularly well suited to deliver MDI particles to the lungs, as shown by the delivery of large quantities of particles to the second stage of a twin-stage impinger.
  • peptide includes any molecule made of a plurality of amino acids, whether naturally occurring or synthetic.
  • the invention further extends to peptide mimetics, which may be considered to be substances resembling peptides and having the activity or other property of a peptide, such as the ability to interact with a given binding site, but which are modified or otherwise synthesised in such a manner as to provide a desirable feature, such as resistance to digestion.
  • Mimetics may simply comprise terminal blocking groups, for example, and/or peptide bonds replaced by bonds resistant to hydrolysis, and/or side groups substituted.
  • Preferred propellants are the haloalkanes, and it is preferably envisaged that HFAs are used as propellants for MDIs in formulations of the present invention.
  • HFAs are used as propellants for MDIs in formulations of the present invention.
  • the backbone of the propellant will generally be an alkane, whether substituted or unsubstituted, and may be straight or branched. Where branched, it is preferred that there only be one branch. Straight chains of the lower alkanes are preferred, especially C 2-4 .
  • the preferred HFAs for use in the present invention are HFA- 134a and HFA-227.
  • Suitable polyhydroxylated polyalkenes for use in the present invention preferably have the structure
  • R is the same or different from one monomeric unit to the next, and is hydrogen, lower alkyl, lower alkenyl, lower alkanoyl, lower alkenoyl or is a bridging group between adjacent monomers, such as a lower diacyl group.
  • lower is meant 1 to 6 carbon atoms, other than the carbonyl carbon, where present, with 1 to 4 being more preferred, and 1 or 2 being more preferred.
  • suitable polyhydroxylated polyalkenes include PVA, PVAc (polyvinylalcohol and polyvinylacetate, respectively), polyvinyl alcohol-co-vinyl acetate (PVAA), poly(vinyl butyral) and poly(vinyl alcohol-co-ethylene).
  • PVA is generally prepared by the hydrolysis of PVAc, and the level of hydrolysis may be as low as about 40% through to substantially complete hydrolysis, such as 98% or higher. Low levels of hydrolysis correspond to lower levels of hydrophilicity/higher levels of hydrophobicity, which can affect the formulations of the present invention. While levels of 98% hydrolysis are useful, it is generally preferred that the level of hydrolysis be in the region of 50 to 90%, with a level of about 80% being a preferred embodiment.
  • the size of the polyhydroxylated polyalkene compounds is not critical to the present invention, and PVA may range from a molecular weight of 9kDa through to about 500kDa, with 9kDa to 50kDa being more preferred. Where PVA is used as the sole polyhydroxylated polyalkene, then a preferred molecular weight is in the region of lOkDa. It will be appreciated that molecular weights for the polyhydroxylated polyalkenes are necessarily highly approximate, as the methods for their preparation necessarily result in a spread of molecular sizes.
  • PVP/PVA copolymers are also available, and may be employed in the present invention, as a substitute for either or both of PVA and PVP.
  • Plasdone ® copolyvidonum is a synthetic water-soluble copolymer consisting of N-vinyl-2- pyrrolidone and vinyl acetate in a random 60:40 ratio, and is also known as Copolyvidonum Ph Eur, Copolyvidon DAB, and Copolyvidone JSPI, BP.
  • the K-value for Plasdone S-630 copolyvidonum is specified as being between 25.4 and 34.2, and is similar to Plasdone K-29/32 povidone.
  • Suitable amounts of each of the PVP and the polyhydroxylated polyalkene excipients range from about 5% to about 200% by weight of the therapeutic substance, although there is little advantage to be seen in the provision of large amounts of either.
  • a suitable amount ofeach excipient, or excipient typewhere more than one polyhydroxylated polyalkene is used is between about 10% and about 50% by weight of the therapeutic substance, with a range of about 20% to about 40% being preferred.
  • aqueous vehicle Prior to formulation with the haloalkane propellant, it is preferred to blend the therapeutic agent with the glycosidic compound and polyhydroxylated polyalkene in an aqueous vehicle, prior to drying.
  • the aqueous vehicle may be any suitable, and will typically be selected from saline or a suitable buffer such as phosphate buffered saline (PBS), although deionised water may also be used, if desired.
  • PBS phosphate buffered saline
  • formulations may comprise two or more populations of particles for administration.
  • the glycosides and polyhydroxylated polyalkenes may be selected as appropriate to each substance, and combined with propellant once prepared. It is also possible that, where there are two or more active substances, any two or more may be formulated together.
  • the powdered products resulting from the drying of the aqueous preparation may be achieved by any suitable drying process, including freeze-drying, spray-drying, spray-freeze-drying, supercritical drying, co-precipitation and air-drying. Of these, spray-drying and spray-freeze-drying are preferred, as these result in fine powders which generally require no further processing. However, if required, the dried products may be further processed to reduce the size of the resulting particles to an appropriate level. In particular, it is preferred that the aerodynamic diameter of the particles of the powder used in the formulations of the present invention is between about l ⁇ m and 50 ⁇ m, more particularly between about 1 ⁇ m and 12 ⁇ m, and even more particularly between about 1 ⁇ m and 10 ⁇ m.
  • the dried powder is then brought into contact with the propellants under conditions suitable for storing in a reservoir useful in an MDI.
  • formulations of the present invention provide long-term stability of activity of the therapeutic substance, as well as ensuring consistency of dosing with time.
  • the present invention further provides a powdered formulation of a therapeutic agent, a glycoside, PVP, and a polyhydroxylated polyalkene suitable for incorporation with a haloalkane propellant for dispensing from a metered dose inhaler.
  • the present invention further provides a metered dose inhalation device provided with a reservoir comprising a haloalkane propellant prepared with a therapeutic substance, a glycoside, PVP, and a polyhydroxylated polyalkene.
  • Doses delivered by the MDIs of the present invention will be readily determined by those skilled in the art and as appropriate to the condition to be treated. In general, doses will vary with the size and age of the patient and can be readily determined by calculating the concentration of the active ingredient in the propellant preparation.
  • Suitable macromolecular compounds for use as therapeutic agents include antibodies, interferon, such as ⁇ -interferon, ⁇ -interferon and ⁇ -interferon, enzymes such as proteases and ribonucleases, especially DNase I, hormones, such as insulin, LHRH, granulocyte-colony stimulating factor, calcitonin, heparin, human growth hormone, euprolide acetate and parathyroid hormone and gene products such as CFTR, and ⁇ i- antitrypsin.
  • interferon such as ⁇ -interferon, ⁇ -interferon and ⁇ -interferon
  • enzymes such as proteases and ribonucleases, especially DNase I
  • hormones such as insulin, LHRH, granulocyte-colony stimulating factor, calcitonin, heparin, human growth hormone, euprolide acetate and parathyroid hormone and gene products such as CFTR, and ⁇ i- antitrypsin.
  • PVA, PVP and trehalose together, retained the biological integrity of the protein whilst maintaining consistently high dosing in the second stage of the twin-stage impinger apparatus. Combinations lacking one or more excipients provided significantly inferior results. Whilst raw DNase I spray-dried alone out-performed the PVA, PVP and trehalose formulation in terms of delivery efficiency, it lost 40% of its biological activity, so cannot be considered to be viable as a pulmonary dosage form. DNase I stabilised with PVA and trehalose had a consistently low second stage deposition in the twin-stage impinger and was, therefore, not considered as effective, in delivering the protein, as the DNase I, trehalose, PVA and PVP formulation.
  • Figure 2 shows a combination of enzyme activity data and twin-stage impinger data to predict the quantity of active enzyme delivered to the lung
  • Figure 3 shows the biological activity of DNase I SD over a 24 week period, when stored in an HFA 134a, metered dose inhaler. Three samples were taken at each time point;
  • Figure 4 shows the biological activity of DNase I formulated with trehalose (DT) over a 24 week period, when stored in an HFA 134a metered dose inhaler. Three samples were taken at each time point;
  • Figure 5 shows the biological activity of DNase I formulated with trehalose and PVA. The formulation was suspended in an HFA 134a MDI over a 24 week period. Three samples were taken at each time point;
  • Figure 6 shows the biological activity of DNase I formulated with trehalose, PVP and PVA.
  • the formulation was suspended in an HFA 134 MDI over a 24 week period. Three samples were taken at each time point;
  • Figure 7 shows the twin-stage impinger assessment of DNase I spray-dried alone.
  • the formulation was suspended in an HFA 134 MDI over a 24 week period;
  • Figure 8 shows the twin-stage impinger assessment of DNase I spray-dried with trehalose.
  • the formulation was suspended in an HFA 134 MDI over a 24 week period;
  • Figure 9 shows the twin-stage impinger assessment of DNase I spray-dried with trehalose and PVA.
  • the formulation was suspended in an HFA 134 MDI over a 24 week period;
  • Figure 10 shows the twin-stage impinger assessment of DNase I spray-dried with trehalose, PVA and PVP.
  • the formulation was suspended in an HFA 134a MDI over a 24 week period;
  • Figure 11 shows the combination of the twin-stage impinger and biological activity data of the DNase I MDI formulations. All the data was measured after the formulations were suspended in a HFA 134a MDI for a 24 week period;
  • Figure 12 shows the comparison of the intensity of the alpha helix band in the second derivative FTIR spectra and the % relative biological activity of DNase I.
  • the combination of trehalose, PVA and PVP was found to be superior in conserving the biological activity of high purity DNase I during microparticulate manufacture, compared to either trehalose when used alone or PVA and trehalose used in combination. It was the only formulation to stay within the specification for retention of biological activity. Whilst DNase I spray-dried alone exhibited excellent stage 2 deposition within the twin-stage impinger, 40% of the protein was denatured. Further, while the combination of PVA and trehalose served to enhance the biological stability of high purity DNase I, compared to the spray-dried protein alone, its stage 2 deposition in the twin-stage impinger was lower than that of DNase I simply stabilised with trehalose.
  • bovine form of the protein provides an excellent model.
  • the sequences of the human and bovine forms are 77% homologous and the crystal structures can be superimposed upon each other (Quan et al, 1999).
  • highly purified bovine DNase I was reformulated in a metered dose inhaler preparation, and the ability of trehalose, PVP, and polyvinyl alcohol to stabilise bovine DNase I during manufacture using spray- drying and formulation in a metered dose inhaler was assessed, by comparison with spray-drying the raw enzyme alone.
  • DNase I isolated from the bovine pancreas, high purity, RNAse free, 14200 U/mg (defined by Sigma Aldrich as Genotech® units) Sigma Aldrich, Gillingham, UK] formulations were manufactured using the Bucchi 191 mini spray-dryer (Bucchi, Darmstadt, Germany).
  • the aspiration rate was set as 70%, the material feed rate was 3 ml min "1 and the inlet temperature was set to 95 °C.
  • the feed suspension was pumped through a spray atomisation nozzle that combined the liquid with a 700 ml hr "1 airflow.
  • the outlet temperature was found to be in the range of 65-70°C.
  • the DNase I spray-drying feed solutions were made up in 100 ml of 0.15 M NaCl buffer.
  • the PVA was 80% hydrolysed with a molecular weight (M w ) of 8,000- 10,000 (Sigma Aldrich, Gillingham, UK).
  • M w molecular weight
  • the trehalose was in the dihydrate form (Sigma Aldrich, Gillingham, UK).
  • the metered dose inhalers were manufactured by adding the direct equivalent of 15.0 mg of the raw drug (DNase I) into a PET canister (BesPack, Kings Lynn, UK).
  • a 25 ⁇ L canister valve (BesPack, Kings Lynn, UK) was crimped in place using the Pamasol MDI filler (Pamasol, Pfaffikon, Switzerland) and 15.0 g of HFA 134a (Dupont, Willington, Germany) or 17.0 g HFA 227 (Solvay, Frankfurt, Germany) was pressure-filled into the can via the valve.
  • the formulation was then sonicated in an ultrasonication bath (Decon, Hove, UK) for 15 s to ensure particle separation and stored, valve up, at room temperature.
  • the denatured DNase I used as a positive control was simply manufactured by placing 5.0mg of the protein in a 180°C oven for 10 minutes.
  • the spray-dried powders were assessed using the Mastersizer X laser diffraction particle size analyser (Malvern Instruments Ltd, Malvern, UK). The Malvern was set up using the liquid dispersion system. Mixtures of 1% lecithin (Sigma Aldrich, Gillingham, UK) and cyclohexane (Merck, Poole, UK) were used as the dispersion media. Samples were prepared by sonicating 2 mg of powder in 2 ml of the dispersion media for 30 seconds. The particle size was measured using the 63 mm (0.5 - 110 ⁇ m) lens set at a focal length of 145 mm, whilst stirring the cell on 75% of full power. The samples were added dropwise in to the stirred cell until the desired obscuration was achieved. Each sample was measured in triplicate and 3 batches from each sample were analysed.
  • Mastersizer X laser diffraction particle size analyser Malvern Instruments Ltd, Malvern, UK. The Malvern was set up using the liquid dispersion
  • the biological activity of DNase I was monitored by assessing the enzyme's ability to digest the substrate, DNA.
  • the substrate was made up in an acetate buffer (0.1 M, pH 5.0), containing 5 mM Mg 2+ . This was prepared by dissolving 1.165 g of anhydrous sodium acetate (BDH, Merck labs, Darmstadt, Germany), 0.355 g of acetic acid (Sigma Aldrich, Gillingham, UK), and 0.203 g of MgCl 2 .6H 2 O (Sigma Aldrich, Gillingham, UK), in 150 ml of purified water.
  • BDH anhydrous sodium acetate
  • acetic acid Sigma Aldrich, Gillingham, UK
  • MgCl 2 .6H 2 O Sigma Aldrich, Gillingham, UK
  • a DNase I standard 2,000 Kunitz units mg "1 (Sigma Aldrich, Gillingham, UK), was used as a calibrant for the activity assay. This standard was reconstituted by dissolving it in 1.0 ml of 0.15 M NaCl solution. The solution was further diluted to obtain five separate standard solutions within the concentration range of 20 - 80 units ml "1 . All dilutions were performed using 0.15 M NaCl solution.
  • a lambda 5 UV spectrophotometer (Perkin-Elmer, Beaconsfield, UK) was adjusted to a wavelength of 260 nm and 2.5 ml of substrate was placed into a cuvette (10 mm light path) and incubated in a thermostatic cell (25°C) for 3-4 minutes to allow • temperature equilibration. Then, 0.5 ml of diluted standard, or sample, was added and the solutions were immediately mixed by inversion. The increase in A 260 ( ⁇ A 260 ) minutes was recorded as a function of time for 10-12 minutes. An activity calibration curve was constructed by plotting the maximum ⁇ A 260 vs. Kunitz units mg "1 of the standard DNase I vials.
  • the DNase I samples were diluted to attain a ⁇ A 260 within the calibration range and, hence, measure the equivalent Kunitz units.
  • the Pierce Protein Assay® was then used to quantify the protein, thereby to obtain the activity per mg. This was compared to the lyophilised raw DNase I to produce the % relative activity.
  • the twin-stage impinger (Radleys, Saffron, UK) was set up as per the United States Pharmacopoeia specification. Stage 2 of the twin-stage impinger device mimics the desired site of deposition within the human lung.
  • the DNase I formulations used distilled water as washing agent and the solvent in the apparatus.
  • the airflow was set to 60 ml min "1 and the inhalers were actuated 20 times. Between each actuation there was a five second pause with the pump running. The pump was then stopped, the canister removed and shaken for five seconds before the sequence repeated. Each of the stages were washed individually upon completion of the 20 canister actuations.
  • the device was washed into a 50 ml volumetric with stages
  • the working reagent was prepared by mixing 25 parts of Micro BCA reagent A and 24 parts of reagent B with 1 part of reagent C. An aliquot of 150 ⁇ L of each standard or test sample was transferred into a 96-well microplate in duplicate. 150 ⁇ L of the working reagent was subsequently added to each well and the plate mixed on the shaker for 30 seconds. The plate was covered and incubated at 50°C for 90 minutes, after which it was cooled to room temperature and the UV absorbance in each well determined at 562 nm using a UV plate reader. The response of each enzyme was determined by comparing the nominal concentration and the BSA protein standard.
  • Fluorescence emission and Rayleigh light scattering were both assessed using a LS-50 fluorescence spectrophotometer with a thermostatic cell set at 5°C (Perkin- Elmer, Beaconsf ⁇ eld, UK).
  • the excitation wavelength was set to 270 nm and the emission was monitored over a range of 250 nm to 450 nm.
  • the excitation slit width was set as 4 nm and the emission slit width 8 nm.
  • the spectra were attained at a rate of 150 nm. All the samples were made up in a.0.15 M NaCl solution (Sigma Aldrich, Gillingham, UK). The samples were each scanned five times and averaged. The spectra from the solvent were subtracted from each result.
  • the area under the light scattering peak (maximum cc. 270nm) and the fluorescence peak (maximum cc. 335nm) were integrated from each sample and compared.
  • the light source variance was assessed and, if appropriate, corrected for, using Nile Red (Sigma Aldrich, Gillingham, UK) as a standard.
  • the product from the spray-drying process was collected and weighed into a glass vial.
  • the samples were stored under phosphorous pentoxide desiccation at room temperature for 24 hours prior to MDI manufacture.
  • the PVP was K15, M w 13,000 (Sigma Aldrich, Gillingham, UK).
  • the particle size measurements of the spray-dried material indicated that all three of the batches were of a suitable respirable size, i.e. less than 10 ⁇ m, as shown in Table 7.
  • the smallest mean particle size (1.94 ⁇ 0.14 ⁇ m) was produced the DTPP formulation.
  • the DTPVA formulation of the Comparative Example had a slightly lower yield than DTI : 1 and a higher particle size.
  • Formulation of the unsupplemented DNase I particles, DO1 :1, with HFA 134a had no significant effect on the activity of the enzyme, as shown in Table 7, above. However, addition of DT 1:1 to HFA 134a improved activity, although not reverting the enzyme back to full activity, unlike DTPVA in the Comparative Example. Formulation of DTPP 1:1 :1:1 with HFA 134a, however, enhanced the enzyme's activity to surpass the potency of the original material.
  • In vitro prediction of particle deposition using the twin-stage impinger apparatus defines the fine particle fraction (FPF) as the particles collected on stage 2 of the device.
  • Stage 2 has a size cut off MMAD of ⁇ 6.4 ⁇ m.
  • All three sets of the spray-dried DNase I particles produced a high FPF in the twin-stage impinger apparatus when suspended in HFA propellants, as shown in Figure 1.
  • DO1 :0 delivered a significantly higher (p ⁇ 0.05, ANOVA) FPF compared to either the DT 1 : 1 or the DTPP 1:1:1:1 formulation (which were not significantly different (p > 0.05, ANOVA) from each other).
  • DPVA was not significantly different from DT or DTPP (p > 0.05, ANOVA).
  • DO1 :0 delivers a high proportion of particles to stage two, only 60% of these retained biological activity.
  • the DT 1:1 134a formulation retained almost full biological activity and the DTPP 1:1:1:1 134 showed greater activity than the raw material. Therefore, although DO1 :0 had the best aerodynamic characteristics, the enhanced enzyme activity attained with DTPP 1:1:1:1 134 made this the most efficient formulation. Combining activity and deposition resulted in a prediction of almost 60% of the enzyme activity reaching the lung i.e. on stage 2 of the TSI device, compared with DTPVA which delivered ca. 50%.
  • Rayleigh light scattering is measured at 90° to the incident light.
  • the Rayleigh emission from particulates within solutions occurs at the same wavelength at which it was applied to a sample.
  • the intensity of the Rayleigh light scattering increases. Therefore, measurement of Rayleigh light emission has been previously be used to monitor the aggregation of protein solutions. Aggregation follows secondary structure break down in a protein and therefore may be indicative of protein denaturation.
  • the tryptophan residue in a protein is known to be fluorescent. Although this is not a unique property of amino acids (both tyrosine and phenylalanine also fluoresce) the fluorescence of the tryptophan residue is uniquely sensitive to its micro-environment. Structural changes in a protein such as unfolding or aggregation can lead to change in the micro-environment of the tryptophan residue, which results in ' a change in fluorescent intensity, due to quenching or intensity maximum, due to a variation in hydrophobicity of the micro-environment. Hence, monitoring of Rayleigh light scattering (which can be performed in a single scan on a fluorescence spectrophotometer) and fluorescence can both indicate structural changes on both a macro and micro-environmental level.
  • DOl :0 134 showed no significant change in Rayleigh light scattering. However, it did show a significant drop (p ⁇ 0.05, ANOVA) in fluorescence emission from a peak area of 1546667 to a peak area of 1165807. DT 1 :1 in 134a also showed a drop in fluorescence intensity compared to the spray-dried material, coupled with a significant drop (p ⁇ 0.05, ANOVA) in Rayleigh light scattering.
  • DTPP 1 : 1 : 1 : 1 in 134a did not ' observe a significant change (p >0.05) in Rayleigh light scattering compared to the spray-dried material (DTPP 1 :1 :1:1).
  • the fluorescence intensity showed a small but significant (p ⁇ 0.05, ANOVA) change of the fluorescence signal after incorporation of the DNase I particles in HFA 134a propellant.
  • the changes in both Rayleigh light scattering and fluorescence intensity infer that all the spray-dried particles changed, to differing degrees, upon suspension in HFA 134a propellant. Only minimal changes occurred with the DTPP 1:1:1:1 134a formulation, indicative of enhanced stability for the formulation.
  • DNase I isolated from the bovine pancreas, high purity, RNAse free, 14200 U/mg Sigma Aldrich, UK
  • formulations were manufactured using the Bucchi 191 mini spray-dryer (Bucchi, Germany).
  • the spray-drying feed solutions were made up in 100 ml of 0.15M NaCl buffer.
  • the DNase I was combined with additional excipients, as shown in Table 9, below, including: PVA 80% hydrolysed (Mw of 8,000-10,000, Sigma Aldrich, Gillingham, UK); trehalose dihydrate (Sigma Aldrich, Gillingham, UK); and PVP K15 (10,000 Mw Sigma Aldrich, Gillingham, UK).
  • the resulting, buffered solution was pumped through a spray atomisation nozzle that combined the liquid with a 700 ml hr-1 airflow delivered to the drying chamber.
  • the aspiration rate was set as 70%
  • the material feed rate was 3 ml min-1
  • the inlet temperature was set to 95 °C.
  • the outlet temperature was found to be in the range of 65-70°C.
  • the metered dose inhalers were manufactured by adding the direct equivalent of 15.0 mg of the raw drug (DNase I) into a PET canister (BesPack, Kings Lynn, UK). A 25 ⁇ L canister valve (BesPack, Kings Lynn, UK) was crimped in place using the Pamasol MDI filler (Pamasol, Pfaffikon, Switzerland) and 15.0 g of HFA 134a (Dupont, Willington, Germany) was pressure-filled into the can via the valve. The formulation was then sonicated in an ultrasonication bath (Decon, Hove, UK) for 15 seconds to ensure particle separation and stored, valve up, at room temperature.
  • the MDI DNase I formulations were stored valve up, at room temperature, for 24 weeks. Samples were released from the HFA reservoir, immediately after manufacture, at 2 weeks, 4 weeks 12 weeks and at 24 weeks, and the protein's secondary structure, activity and aggregation were determined, using FTIR and the ⁇ enzymatic assay described below. Twin-stage impinger assessment
  • the aerosol characteristics were determined using the twin-stage impinger as described above.
  • the solvent in the twin-stage apparatus was water, the airflow was set to 60 Lmin _1 , and the protein was quantified using the Pierce Protein Assay® (Perbio science, UK) as described below.
  • the Pierce Protein Assay® was performed as per the manufacturer's instructions.
  • BSA bovine serum albumin
  • the working reagent was prepared by mixing 25 parts of Micro BCA reagent A and 24 parts of reagent B with 1 part of reagent C. An aliquot of 150 ⁇ L of each standard or test sample was transferred into a 96-well microplate in duplicate. 150 ⁇ L of the working reagent was subsequently added to each well and the plate mixed on the shaker for 30 seconds.
  • the plate was covered and incubated at 50°C for 90 minutes, after which it was cooled to room temperature and the UV absorbance in each well determined at 562 nm using a UV plate reader. The response of each enzyme was determined by comparing the nominal concentration and the BSA protein standard.
  • the biological activity of DNase I was monitored by assessing the enzyme's ability to digest the substrate, DNA.
  • the substrate was made up in an acetate buffer (0.1 M, pH 5.0), containing 5 mM Mg 2+ . This was prepared by dissolving 1.165 g of anhydrous sodium acetate (BDH, Merck labs, Darmstadt, Germany), 0.355 g of acetic acid (Sigma Aldrich, Gillingham, UK), and 0.203 g of MgC12.6H 2 O (Sigma Aldrich, Gillingham, UK), in 150 ml of purified water.
  • BDH anhydrous sodium acetate
  • acetic acid Sigma Aldrich, Gillingham, UK
  • MgC12.6H 2 O Sigma Aldrich, Gillingham, UK
  • a lambda 5 UV spectrophotometer (Perkin-Elmer, Beaconsfield, UK) was adjusted to a wavelength of 260 nm and 2.5 ml of substrate was placed into a cuvette (10 mm light path) and incubated in a thermostatic cell (25°C) for 3-4 minutes to allow temperature equilibration. Then, 0.5 ml of diluted standard, or sample, was added and the solutions were immediately mixed by inversion. The increase in A260 ( ⁇ A260) minutes was recorded over 10-12 minutes. An activity calibration curve was constructed by plotting the maximum ⁇ A260 vs. Kunitz units mg "1 of the standard DNase I vials.
  • the DNase I samples were diluted to attain a ⁇ A260 within the calibration range and, hence, measure the equivalent Kunitz units.
  • the Pierce Protein Assay® was then used to quantify the protein, thereby to obtain the activity per mg. This was compared to the lyophilised raw DNase I to produce the % relative activity.
  • FTIR spectra were recorded on a Perkin-Elmer 1600 series spectrometer and analysed using PE-GRAMS/32 1600 software. The 2 nd derivative FTIR spectra was obtained using previously reported methods (Dong et al., 1992). Dry protein samples (approximately 0.5 mg protein) were mixed with about 300 mg of ground potassium bromide (Sigma Aldrich, UK) and compressed into a pellet. For each spectrum, 64 scans were collected in absorbance mode with a 4 cm "1 resolution, and subsequently a 64-scan background was immediately recorded.
  • the intensity of the amide I band of the resultant spectra was between 0.9 and 1.2, otherwise the spectra was discarded.
  • the spectra were smoothed with a nine-point Savitsky-Golay function to remove any possible white-noise.
  • the second derivative spectrum was obtained with Savitsky-Golay derivative function software for a five data point window and was smoothed with a seven-point Savitsky-Golay function.
  • the spectra of experimental samples in the amide I region (1600-1710 cm "1 ) were analysed. The baseline of the spectrum between 1710 and 1500 cm "1 was levelled and zeroed, then the spectrum of the sample was normalised for the area in the amide I region using Grams 3.0 software.
  • the biological activity of the spray-dried DNase I microparticles was compared to the raw DNase I, immediately after manufacture and at four time points after suspension within an HFA propellant, to isolate the effects of the manufacture process and of the HFA propellants.
  • Limits of ⁇ 10% were assigned to the DNase I formulations, as these limits are typically employed when assessing the stability of formulations containing protein therapeutics.
  • the DNase I spray-dried alone lost almost 40% of its original activity as a consequence of the spray-drying process, although suspension in HFA did little to degrade the protein any further. Indeed, the trend line shown in Figure 3 actually shows a gradual increase, rather than any further decline.
  • the biological activity of DNase I SD remained outside the determined specification throughout the stability study.
  • the formulation was suspended in a HFA 134a MDI over a 24 week period. Three samples were taken at each time point, while Figure 6 shows the biological activity of DNase I formulated with trehalose, PVP and PVA.
  • the formulation was suspended in a HFA 134 MDI over a 24 week period. Three samples were taken at each time point
  • the addition of PVA to the DNase I trehalose microparticle formulation did not eliminate the initial loss in biological activity of the enzyme caused by spray-drying.
  • the DTPVA134a formulation exhibited an enzymatic activity that represented ca. 80% of that seen with the original material, which was similar to the DT particles.
  • the PVA containing particles recovered the majority of its lost activity (c.f. Figure 5) upon suspension within HFA 134a.
  • the high relative biological activity of the DNase I was not maintained upon storage and it was again reduced to ca. 80% over the 26 weeks of the study.
  • the high stage 2 deposition for the DNase I SD implies that the particles show some physical stability within the HFA propellant alone.
  • the DNase I formulation delivered a significantly larger FPF (p ⁇ 0.05, ANOVA) for the first four of the five time points tested in the stability study.
  • Figure 7 illustrates that the linear trend fitted to the points in the stability work implies that the FPF is reducing over time.
  • Figure 11 combines the data from the activity experiments and the twin-stage impinger deposition work after 24 weeks on stability. This Figure clearly shows that a combination of PVA, PVP and trehalose is superior to any other combination of these excipients, to stabilise DNase I.
  • Inter-Beta sheet 161 0.106 - 0.353 4.84 Ihtra-B eta sheet 1635 0.137 0.370 16.48 Alpha helix 1656 0.131 0.362 11.61 Turn 1 1668 0.161 0.401 31.47 Turn 2 1683 0.029 0.173 - 9.99

Abstract

Selon l'invention, les protéines et les peptides glycosidiquement stabilisés présentent une stabilité sensiblement supérieure en présence de propulseurs d'hydrofluoroalcane en vue d'une distribution à partir d'aérosols-doseurs lorsqu'ils sont préparés avec de la PVP et un polyalcène polyhydroxylé, tel que le PVA.
PCT/GB2004/005206 2003-12-10 2004-12-10 Preparations pour inhalation dosees de proteines et de peptides WO2005056037A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008152398A2 (fr) * 2007-06-14 2008-12-18 Cipla Limited Formulations pour inhalation
WO2013114371A1 (fr) * 2012-02-01 2013-08-08 Protalix Ltd. Formulation en poudre sèche de dnase i

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020010318A1 (en) * 1997-10-03 2002-01-24 Amgen, Inc. Secretory leukocyte protease inhibitor dry powder pharmaceutical compositions
US20020106368A1 (en) * 2000-07-28 2002-08-08 Adrian Bot Novel methods and compositions to upregulate, redirect or limit immune responses to peptides, proteins and other bioactive compounds and vectors expressing the same
WO2002094200A2 (fr) * 2001-05-21 2002-11-28 Nektar Therapeutics Administration par voie pulmonaire d'insuline chimiquement modifiee
WO2003015750A1 (fr) * 2001-08-16 2003-02-27 Baxter International, Inc. Preparations de microparticules a base d'un agent propulseur

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020010318A1 (en) * 1997-10-03 2002-01-24 Amgen, Inc. Secretory leukocyte protease inhibitor dry powder pharmaceutical compositions
US20020106368A1 (en) * 2000-07-28 2002-08-08 Adrian Bot Novel methods and compositions to upregulate, redirect or limit immune responses to peptides, proteins and other bioactive compounds and vectors expressing the same
WO2002094200A2 (fr) * 2001-05-21 2002-11-28 Nektar Therapeutics Administration par voie pulmonaire d'insuline chimiquement modifiee
WO2003015750A1 (fr) * 2001-08-16 2003-02-27 Baxter International, Inc. Preparations de microparticules a base d'un agent propulseur

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008152398A2 (fr) * 2007-06-14 2008-12-18 Cipla Limited Formulations pour inhalation
WO2008152398A3 (fr) * 2007-06-14 2009-11-12 Cipla Limited Formulations pour inhalation
WO2013114371A1 (fr) * 2012-02-01 2013-08-08 Protalix Ltd. Formulation en poudre sèche de dnase i
WO2013114373A1 (fr) * 2012-02-01 2013-08-08 Protalix Ltd. Formulations liquides inhalables d'adnase i
US9603907B2 (en) 2012-02-01 2017-03-28 Protalix Ltd. Dry powder formulations of dNase I
US9603906B2 (en) 2012-02-01 2017-03-28 Protalix Ltd. Inhalable liquid formulations of DNase I

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