WO2005056037A1 - Metered dose inhalation preparations of proteins and peptides - Google Patents

Abstract

Glycosidically stabilised proteins and peptides have substantially greater stability in the presence of hydrofluoroalkane propellants for dispensing from metered dose inhalers, when formulated with PVP and a polyhydroxylated polyalkene, such as PVA.

Description

METERED DOSE INHALATION PREPARATIONS OF PROTEINS AND PEPTIDES

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. Essentially, there are three types of device available for pulmonary delivery, and these are nebulisers, metered dose inhalers (MDI) and dry powder inhalers (DPI). Each of these has its benefits and its drawbacks.

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. However, 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. In general, 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.

The suitability of MDIs to deliver peptide and protein pharmaceuticals has not been well established, and there are concerns for the physical and chemical stability of formulated proteins and peptide particles in propellant mixtures. For these reasons, and the ability to deliver more substantial quantities, DPIs have been generally preferred for the initial research into pulmonary delivery of proteins and peptides.

However, unlike MDIs, the ergonomics of DPIs are manufacture-dependent and, as a result, this can cause confusion amongst patients, which can lead to poor efficacy of therapy. In one study, 40% of patients who had been taught how to use a Turbuhaler^®, and who had used it for between 8 months to 8 years, used it so poorly that it was unlikely that the patients were obtaining any therapeutic benefit from the inhaled drugs (Prime et al., 1997).

In addition, where the amount of drug to be delivered is not an issue, then the benefits of using DPIs over MDIs is equivocal. In recent studies, there was no evidence that DPIs were any more effective in delivering corticosteroids and β-2 agonist bronchodilators in asthma than MDIs (Prime et al., 1997).

Furthermore, the aerodynamic performance of MDI and DPI devices containing the same glucocorticoid was compared in vitro, and it was established that the fine particle mass (FPM) delivered by the DPI was flow rate dependent and significantly lower than that achieved using the MDI (Terzano, 2001).

Thus, the primary advantage of DPIs lies in their ability to dispense large quantities of drug from a stable, powder formulation. By contrast, 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. With the universal, phased withdrawal of the use of CFCs, the only two propellants currently approved for inhalation are tetrafluoroethane (HFA-134a) and heptafluoropropane (HFA-227). Both of these hydrofluoroalkanes have boiling points substantially below 0°C, unlike CFC-11 (23.8°C). In addition, the HFAs have poor solvency for those surfactants commonly employed as excipients in CFC-based MDIs, thereby further complicating the formulation design.

To date, 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. As a consequence, a solution MDI generates respirable particles in a different manner to more traditional suspension formulations. Within a suspension MDI, particles of a defined size have already been manufactured and simply require safe storage and delivery by the device. However, 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). Several research groups have demonstrated that 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).

There are, however, several fundamental flaws with formulating an MDI as a solution, including: lack of specific drug targeting; reduced chemical and physical stability; and, a loss of control over the dissolution rate (Leach et al., 2002). The lack of control over the specific targeting within the deep lung has recently been studied by Hochhaus et al (1998). This group described mean pulmonary residence time as the major influence on pulmonary targeting of steroids. They showed that solution MDIs had a much lower pulmonary resonance time compared to suspension formulations and suggested that this could result in a lack of lung steroid receptor specificity, hence an increased chance of side effects (Hochhaus et al., 1998). However, by far the most difficult problem to overcome when manufacturing solution MDIs is the reduction in the chemical stability of the drug (Sonie et al., 1992).

Blondino and Byron (1998) 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. However, 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.

To date, 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). However, 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. Further, 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.

The advantages of delivering proteins using MDI or DPI devices would be significant, if the technological challenges can be overcome. It is of primary importance to maintain the stability of peptide and protein drugs during processing and storage, as well as ensuring the efficiency and reproducibility of the deposition of drug particles during use by the patient. Particular considerations for MDIs include; the production of particles with controlled particle size and stability, and compatibility between propellants and the proteins and peptides. Such factors ensure that the suspension and biological stability can be maintained over the required shelf life.

The stabilisation of proteins using compounds, such as sugars, has enabled these complex macromolecules to be processed using a wide variety of manufacturing techniques with a minimal loss in therapeutic activity (Allison et al., 1999; Aoudia and Zana, 1998a; Aoudia and Zana, 1998b; Byron et al., 1996; Guiavarc'h et al., ; rmamura et al., 2003). Of the numerous processing methods used, spray-drying is one of the most suitable to produce inhalable particles, as the surface morphology of the particle can be manipulated (Berggren, 2003; Chan et al., 1997; Harlow, 1993; Prinn et al., 2002; Stahl et al, 2002). However, proteins cannot commonly be spray-dried alone, as the heat used to dry the particles denatures them, so that additional stabilising excipients are required to protect the molecule during the particulate manufacture. Although there have been many previous studies investigating the ability of compounds to protect against the stresses induced during protein manipulation, little has been done to investigate the effects of such excipients on the performance of the final formulation. Although sugars can protect against temperature-induced changes during processing, they do little to protect against solvent-induced protein unfolding, hydrolysis, or aggregation-induced denaturation, within a formulation. There is, therefore, a requirement to not only incorporate excipients to protect the protein during manufacture into a suitable particle, but also to maximise stability in the final delivery device.

The compatibility of HFA propellants with protein powders has been investigated in a number of previous studies. For example, Quinn et al.(l999) found that protein MDI formulations retained the biological activity of tested peptides and proteins, such as calcitonin and DNase I, and found that the conformation of lysozyme underwent no change in the presence of HFA- 134a, as analysed by Fourier transform Raman spectroscopy (Quinn et al., 1999). Other work also suggests that MDI protein formulations might be efficient in terms of aerodynamic performance and reproducibility, in terms of dosimetry (Gonda, 1992).

Nevertheless, it remains desirable to maximise the stability and activity of therapeutic peptides and proteins, and it is an object to provide MDI formulations of protein having both suitable chemical and physical stability during manufacture and storage. Such MDIs would have substantial advantages over DPIs for the delivery of appropriate therapeutic substances.

In co-pending PCT/GB2003/004836 there is disclosed peptides or proteins combined with a glycoside and a polyhydroxylated polyalkene, such as polyvinyl alcohol (PVA), which are protected during preparation by spray-drying.

Surprisingly, we have now found that 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).

Accordingly, in a first aspect, 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. In an alternative aspect, 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 α-helix. More preferred are enzymes, especially those whose activity is dependent on one or more regions of α-helix.

The properties of polymers such as PVP may usefully be reported in terms of the 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.

Relationship of viscosity, K-Value, and approximate molecular weight for PVP

Although 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. In a preferred embodiment, 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.

In particular, we have now found that 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. Without any additives, there is a dramatic loss in activity together with structural changes when DNase I is spray-dried. In the presence of all three additives, substantially all of the naturally occurring activity can be retained, and this appears to increase beyond the basal rate, in the presence of HFA134a, for example. Thus, it appears that the sugar and the polymers, in combination, protect the protein from both heat-induced denaturation during spray- drying and solvent induced changes upon formulation.

It is also an advantage that the formulations of the invention are less likely to be immunogenic, as the additives tend to stabilise the conformation of the active molecule.

Where reference is had to DNase I, herein, it will be appreciated that such reference includes reference to all suitable therapeutic substances, unless otherwise apparent, or indicated.

It is a further advantage that the formulations of the invention can be used with portable MDI devices which are easy to use. In addition, 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. In general, 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. The terms "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. For example, sucrose comprises a furanosyl and a pyranosyl residue, whilst mannitol comprises a pyranosyl residue and a straight chain element. Other suitable disaccharides include lactose, isomaltose, cellobiose, maltose and trehalose, of which trehalose is preferred. Other 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.

Other 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.

It will be appreciated that the 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.

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 α-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.

It will be appreciated that the term "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. However, it will be appreciated that the invention also extends to the use of CFCs and other alkanes, for example. 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

-(CH₂-CHOR)_n-

where 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. By "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.

Examples of 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. For example, 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. In general, a suitable amount ofeach excipient, or excipient typewhere more than one polyhydroxylated polyalkene is used, for example, is between about 10% and about 50% by weight of the therapeutic substance, with a range of about 20% to about 40% being preferred.

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.

It will be appreciated that some formulations may comprise two or more populations of particles for administration. In such instances, 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 lμm and 50 μm, more particularly between about 1 μm and 12 μm, and even more particularly between about 1 μm and 10 μm. The dried powder is then brought into contact with the propellants under conditions suitable for storing in a reservoir useful in an MDI.

It is a particular advantage of the present invention that the stability of the particles prepared as described above is considerably greater than anything provided in the art, and preferred formulations of the present invention comprise only the active ingredient(s), glycoside(s), PVP, polyhydroxylated polyalkene(s), and propellant(s). Thus, formulations of the present invention provide long-term stability of activity of the therapeutic substance, as well as ensuring consistency of dosing with time.

It will be appreciated that 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 α-interferon, β-interferon and γ-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 αi- antitrypsin.

As shown in the accompanying Examples, 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.

Combination of the twin-stage impinger data together with the biological activity highlight that a formulations of the invention have a clear advantage over any of the formulations tested in this study in stabilising a hydrophilic protein in a hydrofluoroalkane MDI.

Without being limited by theory, it may be that the mechanism of stabilisation for the novel combination of excipients is related to the conservation of the higher order structure of protein, with FTIR studies suggesting that low quantities of alpha helix detected in the formulation correlated to low biological activity.

In the accompanying Figures:

Figure 1 shows impaction data for the three DNase I MDI formulations (error bars are equal to 1 standard deviation n=3);

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; and

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.

In the accompanying Examples, 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.

The present invention will now be further illustrated by the following, non- limiting Examples.

EXAMPLES

While the human form of deoxyribonuclease I (rhDNase I) is used for clinical applications, its manufacture and purification is costly. However, the 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). In the following Examples, 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.

EXAMPLE 1

Stabilisation of a DNase I during manufacture and formulation within an MDI

MATERIALS AND METHODS

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). 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 μL 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.

Particle size analysis

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 μm) 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.

Biological Activity

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 Mg²⁺. 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 MgCl₂.6H₂O (Sigma Aldrich, Gillingham, UK), in 150 ml of purified water. Two mg of fibrous DNA isolated from a calf thymus (Sigma Aldrich, Gillingham, UK) was dissolved in 52 ml of the acetate buffer by gently shaking overnight. The absorbance of this substrate solution at 260 nm was determined to be between 0.630 and 0.690.

Prior to assessing the test samples, 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 A₂₆₀ (ΔA₂₆₀) minutes was recorded as a function of time for 10-12 minutes. An activity calibration curve was constructed by plotting the maximum ΔA₂₆₀ vs. Kunitz units mg^"1 of the standard DNase I vials. The DNase I samples were diluted to attain a ΔA₂₆₀ 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.

Twin-Stage Impinger

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

1 and 2 being washed into 100 ml volumetric flasks. The resulting solutions were analysed using the Pierce Protein Assay® (Pierce Chemical Company, UK). All twin- stage runs were completed in triplicate.

. The Pierce Protein Assay® was performed as per the manufacturer's instructions. BSA was used as the protein standard and a set of BSA solutions between

2 and 20μg were prepared by diluting the 2.0 mg ml^"1 standard. 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 μL of each standard or test sample was transferred into a 96-well microplate in duplicate. 150 μL 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

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, Beaconsfϊeld, 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.

RESULTS

Stabilisation of DNase I using a glycoside and PVA

Table 1 Composition of the DNase I spray-dried formulations

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.

Table 2 Composition of the DNase I MDI formulations

Spray-dried material characterisation

The particle size measurements of the two spray-dried formulations indicated that both of the batches were of a suitable respirable size, i.e. less than 10 μm. The results are shown in Table 3. The smallest mean particle size (2.25 ± 0.05 μm) was produced by simply spray-drying the protein alone.

Table 3

DNase I spray-dryer manufacture yield and particle size distribution (Mean + SD, n=3).

Incorporation of trehalose and PVA increased the yield of the manufacture method from 15.40 % to 34.75 %.

The Effect of HFA on the DNase I Formulations

The biological activity of the enzyme, DNase I, was reduced by almost 40% when spray-dried alone from a 0.15 M NaCl solution. However, about 85% of the biological activity was retained when PVA and trehalose were incorporated into the feed solution. Results are shown in column 2 of Table 4, below. Table 4. Relative biological activity of the spray-dried DNase I particles before and after formulation in HFA propellants (Mean ± SD, n=3)

Stabilisation of a DNase I MDI with a glycoside and PVA/PVP

Three formulations were manufactured, as detailed in Table 5. The PVP was K15, M_w 13,000 (Sigma Aldrich, Gillingham, UK).

Table 5. Composition of the DNase I spray-dried formulations

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. Spray-dried material characterisation

The yields for the operation were improved with the addition of the excipients. Incorporation of trehalose and/or the polymer increased the yield of the manufacture method over two fold from 15.40 % to cc. 40 %, see Table 6.

Table 6.

DNase I spray dryer manufacture yield and particle size distribution (Mean ± SD, n=3).

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 μm, as shown in Table 7. The smallest mean particle size (1.94 ± 0.14μm) 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.

The effect of HFA on the DNase I formulations

The biological activity of DNase I was reduced by almost 40% when spray-dried alone from a 0.15M NaCl solution. However, almost 80% of the biological activity was retained when trehalose was incorporated into the feed solution. However, the combination of the two polymers with trehalose and the DNase I lead to complete retention of the enzyme's activity, as shown in Table 7. In the Comparative Example, DTPVA retained ca. 82% activity after spray-drying. Table 7. Relative biological activity of the spray-dried DNase I particles before and after formulation in HFA 134a propellant (Mean ± SD, n=3).

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.

The biological activity data suggest that, when the protein is spray-dried alone, denaturation is occurring. The addition of trehalose minimises this damage and retains the majority of the protein's activity. However, formulation of DNase I with the sugar and two polymers is superior to trehalose alone, as it not only maintained the biological activity of the enzyme but also enhanced it. It is possible that the polymer/sugar matrix is either enhancing the actual enzyme reaction, or that the combination of excipients is holding or restoring the protein in or to its truly native state. Manufacture of the raw protein involves freeze drying to facilitate shipping and storage, which may have a detrimental effect on the enzyme, which the polymer and sugar combination reverses.

Aerodynamic assessment of the stabilised formulations Figure 1 shows impaction data for the three DNase I MDI formulations (error bars are equal to 1 standard deviation n=3).

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 μm. 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). However, the data cannot be analysed in isolation. Both the percentage of DNase I delivered to the second stage of the device and the activity of the particles delivered must be taken into account in order to obtain a true prediction of formulation efficiency. This is shown in Figure 2, which shows a combination of enzyme activity data and twin-stage impinger data to predict the quantity of active enzyme delivered to the lung.

Although the DO1 :0 formulation delivers a high proportion of particles to stage two, only 60% of these retained biological activity. The DT 1:1 134a formulation, on the other hand, 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%.

Fluorescence and Rayleigh light scattering assessment of the formulations

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. As more aggregates are formed within a solution 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.

Furthermore, 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.

Assessing the particles prior to suspension in HFA propellant highlighted that the DTPP 1:1:1:1 spray-dried formulation produced a significantly lower (p < 0.05, ANOVA) Rayleigh light scattering peak area compared to the DO1 :0 particles, implying less protein aggregates are present (Table 8). However, the spray-dried DNase I, DT 1:1, produced a significantly (p < 0.05, ANOVA) larger Rayleigh peak compared to the other two sets of particles. The largest peak area for the fluorescence emission of the tryptophan compared was provided by the DO1 :0 formulation followed by the DT 1 : 1. spray-dried material and then the DTPP 1:1:1:1. There was a significant difference (p< 0.05) between each of the spray-dried batches. Although the fluorescence maxima were significantly different (p < 0.05, ANOVA) for the two DNase I batches, the differences were so small that they were not considered indicative of changes in the protein structure. Table 8.

Integrated light scattering and fluorescence peaks for the DNase I spray-dried material. (Mean ± standard deviation, n=3).

Upon suspension of the DNase I particles in HFA, changes were observed with both the Rayleigh light scattering and the fluorescence emission spectra (again, the small shifts in peak maxima, though statistically significant, were not considered, as the shifts were so small that they were thought not to be indicative of structural changes). 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). However, 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.

EXAMPLE 2

Stability testing of the stabilised MDI formulations

A further study was performed to determine if physical changes occurred in the HFA MDI formulations over a 26 week time period, in order to ensure that the formulations of the invention produced a stable pharmaceutical product.

MATERIALS AND METHODS

Spray-drying the DNase I formulations

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 and the inlet temperature was set to 95 °C. The outlet temperature was found to be in the range of 65-70°C. Table 9 Particle size, manufacture yield, and DNase I content of the spray-dried formulations

Formulation DNase I Trehalose PVA PVP

DNase I SD 05 g - - -

DT 0.5 g 0.5 g

DTPVA134 0.5 g 0.5 g 0.5 g

DTPVAPVP 0.5 g 0.5 g 0.5 g 0.5 g

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 μL 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.

Stability Study

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.

DNase I quantification

The Pierce Protein Assay® was performed as per the manufacturer's instructions. BSA (bovine serum albumin) was used as the protein standard, and a set of BSA solutions of between 2 and 20μg were prepared by diluting the 2.0 mg ml^"1 standard. 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 μL of each standard or test sample was transferred into a 96-well microplate in duplicate. 150 μL 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.

Assay of DNase I biological activity

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 Mg²⁺. 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 MgC12.6H₂O (Sigma Aldrich, Gillingham, UK), in 150 ml of purified water. Two mg of fibrous DNA isolated from a calf thymus (Sigma Aldrich, Gillingham, UK) was dissolved in 52 ml of the acetate buffer by gently shaking overnight. The absorbance of this substrate solution, at 260 nm, was determined to be between 0.630 and 0.690. Prior to assessing the test samples, 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 A260 (ΔA260) minutes was recorded over 10-12 minutes. An activity calibration curve was constructed by plotting the maximum ΔA260 vs. Kunitz units mg^"1 of the standard DNase I vials. The DNase I samples were diluted to attain a ΔA260 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.

Fourier Transform infrared spectroscopy

Fourier transform infrared spectroscopy was used to determine the secondary structure of the protein. 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.

RESULTS

Stability Study

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 addition of trehalose to the spray-dried DNase I microparticulate conserved the biological activity of the protein to a greater extent, compared to the protein spray- dried alone. The DT microparticulates appeared to have lost approximately between 2-20% of their original enzymatic activity after spray-drying. In addition to the small loss of activity in the microparticles, storage in HFA 134 propellant caused a further drop in biological activity. Figure 4 shows that whilst some of the DT samples were still within the product specification of ± 10%, the biological activity of the protein was becoming increasingly variable over the 24 weeks which resulted in a steady drop in the formulation's mean activity of CΩ.80%. Figure 5 shows the biological activity of DNase I formulated with trehalose and PVA. 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. However, in contrast to the DT formulation, the PVA containing particles recovered the majority of its lost activity (c.f. Figure 5) upon suspension within HFA 134a. Despite the reversal of the loss in biological activity within the DTPVA134a formulation, 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 formulation of DNase I within a microparticle containing PVP, PVA and trehalose was the most successful combination in terms of retaining the proteins biological activity over a 26 week period (Figure 6). This excipient blend not only protected the protein during the manufacture process, but after 26 weeks there was no significant difference (p > 0.05, ANOVA) between the lower boundary of the 10% specification employed for this assay and the activity of the protein and, therefore, the protein was deemed to have stayed within the stability limits set out in the study.

The MDI formulation containing microparticles of DNase I, spray-dried alone, emitted the highest fine particle fraction (FPF defined using the twin-stage impinger as particles < 6.4 μm and collected on stage 2 of the device) of any of the HFA formulations, depositing ca. 60% of its metered dose onto stage 2 of the BP twin-stage impinger. The high stage 2 deposition for the DNase I SD implies that the particles show some physical stability within the HFA propellant alone. Compared to any of the other microparticulates suspended in HFA, 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. However, Figure 7 illustrates that the linear trend fitted to the points in the stability work implies that the FPF is reducing over time.

Addition of trehalose to the DNase I microparticles decreased the FPF compared to the protein formulated alone. The DT formulation in HFA 134a deposited approximately 40% of the total dose in stage 2 of the twin-stage impinge device. Figure 8 shows that the FPF for this formulation remained unchanged throughout the time frame of the stability study although, even after 24 weeks, the stage 2 deposition was still significantly lower than the protein spray-dried alone.

Using PVA and trehalose, in combination, to stabilise the DNase I microparticles in HFA propellants, an FPF of ca. 40% was exhibited, which dropped to around 30% during the stability study (see Figure 9). However, the addition of PVP to the PVA, trehalose combination proved to provide the best combination of excipients with which to physically stabilise the DNase I in an MDI (see Figure 10). Although, initially, the DT PVAPVP formulation resulted in a significantly lower FPF compared to the DNase I SD alone (p < 0.05, ANOVA), after 24 weeks there was no significant difference (p > 0.05, ANOVA) between these two MDIs in terms of twin-stage impinger stage 2 deposition.

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.

Mechanisms of excipient mediated stabilisation

Five distinct secondary structures were identified in the FTIR spectra and these were identified using the assignments described in Dong et al. (1995). Compared to data from the fluorescence studies, there was a stronger correlation between the changes in secondary structure characterised using FTIR spectra and the biological activity of DNase I (an example is shown in Figure 12).

Whilst the intensity of the alpha helix and turn 1 in the secondary structure of DNase I was positively correlated to the activity of the protein, beta sheet intensity and turn 2 intensity was negatively correlated (c.f. Table 10). These trends infer that the loss of biological activity for DNase I may be as a result of a reduction in the alpha helix of the protein and. an increase in the formation of beta sheets and the turn observed at . 1683 nm.

Table 10 The correlation of secondary structure changes in DNase I compared to the biological activity of the enzyme. R represents linearity, r is correlation coefficient between the two data sets

Secondary FTIR band R² r² Gradient Structure (nm)

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

There are, at present, two main barriers to the successful formulation of proteins in MDIs, first, finding an industrially relevant method to micronise the proteins to the correct particle size and, second, protecting these structurally complex therapeutics within the hydrophobic environment of the HFA propellant. In this respect, whilst all three excipients selected conferred an element of protection both during spray-drying and formulation, only the combination of all three excipients produced an MDI that was both biologically and physically stable over the 24 weeks of the stability study. DNase I, alone, lost 40% of its biological activity after spray-drying and, although it delivered a high fine particle fraction from the MDI, it was not physically stable. DNase I formulated with trehalose, PVA and PVP was found to remain within the predetermined specification for biological activity and consistently delivered 50% of its dose to the second stage of the twin-stage impinger.

Variations in the secondary structure of the DNase I formulations observed using FTIR appeared to have a good correlation to the functional activity of the molecule, compared to fluorescence spectroscopy. Dong et al, 1992 postulated that protein denaturation in the solid state is often a result of a loss of alpha helix and the formation of intra-molecular beta-sheets. This appeared to be the case here. The intensity of the alpha helix was greatly reduced when DNase I was formulated alone, compared to the protein stabilised with the stabilising excipients, thus suggesting that the preservation of the higher order structure of the molecule by the excipients was indeed the method by which they conferred their protection.

REFERENCES

Allison, S.D., B. Chang, T.W. Randolph, J.F. Carpenter, 1999, Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding: Archives of Biochemistry and Biophysics 365 pp. 289-298.

Aoudia, M., R. Zana, 1998a, Aggregation behavior of sugar surfactants in aqueous solutions: Effects of temperature and the addition of nonionic polymers: Journal of Colloid and Interface Science 206 pp. 158-167.

Aoudia, M., R. Zana, 1998b, Aggregation Behavior of Sugar Surfactants in Aqueous Solutions: Effects of Temperature and the Addition of Nonionic Polymers: Journal of Colloid and Interface Science 206 pp. 158-167.

Berggren, J.A.G., 2003, Effect of polymer content and molecular weight on the morphology and eat- and moisture-induced transformations of spray dried composite particles of amorphous lactose and poly(vinylpyrrolidone): Pharmaceutical Research 20 pp. 1039-1046.

Blondino, F. E., P. R. Byron. Drug stability in non-aqeous solutions-influence of surfactant concentration. V, pp 125-131. 1998.

Byron, P. R., V. Naini, E. M. Phillips. Drug carrier selection-important physicochemical characteristics. V, pp 103-113. 1996.

Chan, H.K., A. Clark, I. Gonda, M. Mumenthaler, C. Hsu, 1997, Spray dried powders and powder blends of recombinant human deoxyribonuclease (rhDNase) for aerosol delivery: Pharmaceutical Research 14 pp. 431-437.

Clarke, J.G., S.R. Wicks, SJ. Farr, 1993, Surfactant-Mediated Effects in Pressurized Metered-Dose Inhalers Formulated As Suspensions .1. Drug Surfactant Interactions in A Model Propellant System: International Journal of Pharmaceutics 93 pp. 221-231.

Dong, A.C., S . Prestrelski, SD. Allison, J.F. Carpenter, 1995, Infrared spectroscopic studies of lyophilization-induced and temperature-induced protein aggregation: Journal of Pharmaceutical Sciences 84 pp. 415-424.

Gonda,1, 1992, Targeting by deposition, in AJ Hickey (ed), Pharmaceutical inhalation aerosol technology: New York, Marcel Dekker, p. 61-82.

Gonda, I., 1996, Inhalation therapy with recombinant human deoxyribonuclease I: Advanced Drug Delivery Reviews 19 pp. 37-46.

Guiavarc'h, Y., D. Sila, T. Duvetter, A. Van Loey, M. Hendrickx, Influence of sugars and polyols on the thermal stability of purified tomato and cucumber pectinmethylesterases: a basis for TTI development: Enzyme and Microbial Technology In Press, Corrected Proof.

Harlow, 1993, Spray drying handbook, UK longman scientific and technical. Hochhaus, G., S. Suarez, R. J. Gonzalez-Rothi, H. Schreier. Pulmonary targeting of inhaled glucocorticoids: How is it influenced by formulation. VI, pp 45-52. 1998.

Imamura, K., T. Ogawa, T. Sakiyama, K. Nakanishi, 2003, Effects of types of sugar on the stabilization of protein in the dried state: Journal of Pharmaceutical Sciences 92 pp. 266-274.

Jones, S. A., Y. H. Liao, G. P. Martin, M. B. Brown. Metered dose inhaler preparations. PCT/GB2003/004836 [WO 2004/043442]. 2003.

Leach, C.L., P.J. Davidson, B.E. Hasselquist, RJ. Boudreau, 2002, Lung deposition of hydrofluoroalkane-134a beclomethasone is greater than that of chlorofluorocarbon fluticasone and chlorofluorocarbon beclomethasone - A cross-over study in healthy volunteers: Chest 122 pp. 510-516.

LeBelle, M., R.K. Pike, S J. Graham, E . Ormsby, H.A. Bogard, 1996, Metered-dose inhalers .1. Drug content and particle size distribution of beclomethasone dipropionate: Journal of Pharmaceutical and Biomedical Analysis 14 pp. 793-800.

Lewis, D., S. Johnson, B. J. Meakin, D. Ganderton, G. Brambilla, R. Garzia, P. Ventura. Effects of Actuator Orifice diameter on beclomethasone dipropionate delivery from a pMDI HFA solution formulation. VI, pp 363-364. 1998.

Prime, D., P.J. Atkins, A. Slater, B. Sumby, 1997, Review of dry powder inhalers: Advanced Drug Delivery Reviews 26 pp. 51-58.

Prinn, K.B., H.R. Costantino, M. Tracey, 2002, Statistical Modeling of Protein Spray Drying at the Lab Scale: AAPS PharmSciTech 3 pp. 1-8.

Quan, C.P., S. Wu, N. Dasovich, C. Hsu, T. Patapoff, E. Canova-Davis, 1999, Susceptibility of rhDNase I to glycation in the dry-powder state: Analytical Chemistry 71 pp. 4445-4454.

Quinn, E.A., R.T. Forbes, A.C. Williams, M.J. Oliver, L. McKenzie, T.S. Purewal, 1999, Protein conformational stability in the hydrofluoroalkane propellants tetrafluoroethane and heptafluoropropane analysed by Fourier transform Raman spectroscopy: International Journal of Pharmaceutics 186 pp. 31-41.

Sonie, W.H., F.E. Blondino, P.R. Byron, 1992, Chemical Stabiliy pressurised inhalers formulated as solutions: Journal of Biological and Pharmaceutical Science 3 pp. 41-47.

Stahl, K., M. Claesson, P. Lilliehorn, H. Linden, K. Backstrom, 2002, The effect of process variables on the degradation and physical properties of spray dried insulin intended for inhalation: International Journal of Pharmaceutics 233 pp. 227-237.

Stein, S.W., 1999, Size distribution measurements of metered dose inhalers using Andersen Mark II cascade impactors: International Journal of Pharmaceutics 186 pp. 43-52. Terzano, C, 2001, Pressurized metered dose inhalers and add-on devices: Pulmonary Pharmacology & Therapeutics 14 pp. 351-366.

Vervaet, C, P.R. Byron, 1999, Drug-surfactant-propellant interactions in HFA- formulations: International Journal of Pharmaceutics 186 pp. 13-30.

Yamashita, C, T. Nishibayashi, S. Akashi, H. Toguchi, M. Odomi, 1998, A novel formulation of dry powder for inhalation of peptides and proteins: Respiratory Drug Delivery VI pp. 483-485.

Claims

CLAIMS:

1. A formulation of a therapeutic protein or peptide suitable for delivery to a patient by a metered dose inhalation device, the formulation comprising a substantially dry powder preparation of the protein or peptide in association with a stabilising amount of a glycoside, PVP, and a polyhydroxylated polyalkene, in combination with one or more propellants therefor.

2. A formulation according to claim 1, wherein the PVP has a K value of no greater than 50.

3. A formulation according to claim 2, wherein the PVP has a K value of no greater than 30.

4. A formulation according to claim 3, wherein the protein or peptide is selected from antibodies, interferons, enzymes, hormones, euprolide acetate, CFTR, and αl-antitrypsin.

5. A formulation according to claim 4, wherein the protein or peptide is a hormone selected from insulin, LHRH, granulocyte-colony stimulating factor, calcitonin, heparin, human growth hormone, and parathyroid hormone.

6. A formulation according to claim 3, wherein the protein or peptide is DNase I.

7. A formulation according to any preceding claim, which is non-immunogenic.

8. A formulation according to any preceding claim which is capable of being stored at room temperature without losing more than 50% biological activity of the protein or peptide after two months.

9. A formulation according to any preceding claim, wherein the glycoside comprises at least one oligosaccharide.

10. A formulation according to claim 9, wherein the glycoside comprises at least one disaccharide.

11. A formulation according to claim 10, wherein the disaccharide is selected from trehalose, mannitol, sucrose, and mixtures thereof.

12. A formulation according to any preceding claim, wherein the glycoside constitutes between about 30% and 400% by weight of the protein or peptide.

13. A formulation according to any preceding claim, wherein the propellant is alkane based.

14. A formulation according to claim 13, wherein the propellant is at least one haloalkane.

15. A formulation according to claim 14, wherein the propellant is selected from HFA- 134a and HFA-227.

16. A formulation according to any preceding claim, wherein at least one polyhydroxylated polyalkene has the general structure

-(CH₂-CHOR)_n-

where 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.

17. A formulation according to claim 16, wherein, when R is not hydrogen, the number of carbon atoms, excluding any -CO- group, is between 1 and 6, inclusive.

18. A formulation according to claim 16 or 17, wherein the polyhydroxylated polyalkene is selected from polyvmylalcohol, polyvinylacetate, polyvinyl alcohol-co- vinyl acetate, poly( vinyl butyral), poly(vinyl alcohol-cø-ethylene), and mixtures thereof.

19. A formulation according to claim 18, wherein the polyhydroxylated polyalkene is PVA.

20. A formulation according to claim 18 or 19, wherein the PVA is a hydrolysate of PVAc, the level of hydrolysis being between 40% and 100%.

21. A formulation according to claim 18 or 19, wherein the PVA a hydrolysate of PVAc, the level of hydrolysis being between 50 and 90%.

22. A formulation according to any of claims 18 to 21, wherein the PVA has a molecular weight of between about 9kDa and 50kDa.

23. A formulation according to any preceding claim, wherein the polyhydroxylated polyalkenes are present in an amount of from about 5% to about 200% by weight of the therapeutic substance.

24. A formulation according to claim 23, wherein the polyhydroxylated polyalkene is present between about 10% and about 50% by weight of the substance.

25. A method for the preparation of a formulation as defined in any preceding claim, comprising blending the protein or peptide with the glycoside, PVP, and polyhydroxylated polyalkene substances in an aqueous vehicle, drying the resulting blend to a powder, and then formulating with propellant.

26. A method according to claim 25, wherein the aqueous vehicle is selected from saline, a suitable buffer, and deionised water.

27. A method according to claim 25 or 26, which comprises spray-drying the blend.

28. A powdered formulation of a therapeutic agent, a glycoside, PVP, and a polyhydroxylated polyalkene, as defined in any of claims 1 to 24, which is suitable for incorporation with a haloalkane propellant for dispensing from a metered dose inhaler.

29. A powdered formulation according to claim 28, wherein the powder particles have an aerodynamic diameter of between about 1 μm and 50 μm.

30. A metered dose inhalation device provided with a reservoir comprising a formulation according to any of claims 1 to 24.