Method and Formulation for Inhalation

Abstract

This invention relates to drug delivery and in particular to the delivery of biologically active agents in the form of dry powders for inhalation. The invention also relates to methods for preparing such dry powder formulations and methods for their use.

Claims

1-21. canceled

22. A method for the treatment or prevention of post partum haemorrhage comprising administering to a subject in need thereof an inhalable dry powder, wherein said inhalable dry powder comprises spray dried particles comprising oxytocin and/or a derivative thereof, an amorphous glass matrix comprising one or more mono-, di- or polysaccharides and/or amino acids, and L-leucine and the L-leucine is in the amount of from 5 to 50% by weight based on the dry weight of said dry powder.

23. The method according to claim 22 wherein the inhalable dry powder is for nasal administration.

24. The method according to claim 22 wherein the inhalable dry powder is for inhalation into the pulmonary system via the mouth.

25. The method according to claim 22 wherein at least a portion of the L-leucine is located at the surface of the particles of the dry powder.

26. The method according to claim 22, wherein more than 40% of the particles of the dry powder upon inhalation have an aerodynamic diameter of less than 5 μm.

27. The method according to claim 22 wherein the inhalable dry powder is prepared by a process comprising: preparing an aqueous solution and/or suspension comprising oxytocin and/or a derivative thereof wherein the one or more mono-, di- or polysaccharides and/or amino acids capable of forming an amorphous glass matrix oxytocin and/or derivative thereof, to form an amorphous glass matrix, and L-leucine; and spray drying the aqueous solution or suspension to produce the inhalable dry powder.

28. The method according to claim 27 wherein the oxytocin is about 1.0% of the entire powder content.

29. The method according to claim 27 wherein the oxytocin is delivered at the required therapeutically active dose.

30. The method according to claim 27 wherein the L-leucine is present in an amount of from 10 to 40% by weight.

31. The method according to claim 27 wherein one or more mono-, di- or polysaccharides and/or amino acids capable of forming an amorphous glass matrix comprises D-mannitol and glycine.

32. The method according to claim 27 wherein the spray drying is carried out at a temperature below 80° C.

33. The method according to claim 22 wherein the dry powder comprises trehalose.

34. The method according to claim 22 wherein the onset of action following inhalation of the dry powder, as measured by uterine contraction, is achieved within 200 seconds of inhalation.

35. The method according to claim 34 wherein the onset of action is achieved within 150 seconds of inhalation.

36. The method according to claim 35 wherein the onset of action is achieved within 100 seconds of inhalation.

37. A dry powder comprising: an inhalable dry powder, wherein said inhalable dry powder comprises spray dried particles comprising oxytocin and/or a derivative thereof, an amorphous glass matrix comprising one or more mono-, di- or polysaccharides and/or amino acids, and L-leucine and the L-leucine is in the amount of from 5 to 50% by weight based on the dry weight of said dry powder.

38. The dry powder formulation of claim 37 wherein the oxytocin is about 1.0% of the amorphous glass matrix.

39. The dry powder formulation of claim 37 wherein the oxytocin is in the amount of the required therapeutically active dose.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0072] FIG. 1 is a schematic representation of an apparatus for spray drying.

[0073] FIG. 2 is a schematic representation of a Twin Stage Impinger for measuring the in-vitro aerosol deposition of dry powders.

[0074] FIG. 3 provides X-ray diffractograms of raw trehalose Trehalose (raw) and spray-dried trehalose (SD) after spray-drying under specified conditions.

[0075] FIG. 4 provides X-ray diffractograms of spray-dried trehalose and spray-dried trehalose with leucine 10% and 20% w/w after spray-drying under the specified conditions.

[0076] FIG. 5 is a scanning electronic microscope image of trehalose after spray-drying under specified conditions.

[0077] FIG. 6A provides scanning electronic microscope images of trehalose with leucine 10% w/w after spray-drying under the specified condition; FIG. 6B provides scanning electronic microscope images of trehalose with leucine 20% w/w after spray-drying under the specified condition.

[0078] FIG. 7 is an EMG trace showing uterine contraction following inhalation of oxytocin. The arrow denotes the delay between administration and contraction. The initial burst is circled with a dotted line. The black line represents a random thirty minute sample of oxytocin induced activity. The dashed line represents the total time oxytocin induced uterine activity lasted before returning to baseline.

[0079] FIGS. 8A-8D are a series of charts providing an analysis of Uterine EMG behaviour during the immediate post-partum period and following intrapulmonary (IP) and intramuscular (IM) oxytocin delivery. FIG. 8A shows the delay between oxytocin administration and EMU response; FIG. 8B shows the length of the first EMG burst; FIG. 8C shows the number of EMG burst in the first 30 minutes; and FIG. 8D shows the total duration of EMG activity. Data expressed as mean±SEM, P>0.05 RM ANOVA; n=5. Diagonal lined bars represent uterine activity immediately after delivery, white bars represent uterine activity after dry powder delivery and black bars represent uterine activity after intramuscular administration.

[0080] FIG. 9A shows bronchoscope images from a video of a sheep trachea before powder delivery; FIG. 9B shows bronchoscope images from a video of a sheep trachea after powder delivery.

[0081] FIG. 10 shows non-polar surface energy distributions at finite dilution in Inverse gas chromatography.

[0082] FIG. 11 shows polar surface energy distributions at finite dilution in Inverse gas chromatography.

[0083] FIG. 12 shows total surface energy distributions at finite dilution in Inverse gas chromatography.

[0084] FIG. 13 shows work of cohesion surface energy distributions determined at finite dilution in Inverse gas chromatography.

[0085] FIG. 14 shows surface energy at infinite dilution in Inverse gas chromatography.

[0086] FIG. 15A is an EMG trace showing uterine contraction following inhalation of oxytocin delivered by example 10 formulation 1 containing mannitol; glycine and leucine in equal amounts. The dashed line shows the time of formulation delivery and the solid line the onset of uterine contraction. The x-axis is in hh:mm:ss and the y axis is in mV.

[0087] FIG. 15B is an EMG trace showing uterine contraction following inhalation of oxytocin delivered by example 10 formulation 2 containing 90% trehalose and 10% leucine. The dashed line shows the time of formulation delivery and the solid line the onset of uterine contraction. The x-axis is in hh:mm:ss and they axis is in mV.

[0088] FIG. 16A is an EMG trace showing uterine contraction following inhalation of oxytocin delivered by example 10 formulation 3 containing 90% PVP(30) and 10% leucine. The dashed line shows the time of formulation delivery and the solid line the onset of uterine contraction. The x-axis is in hh:mm:ss and they axis is in mV.

[0089] FIG. 16B is an EMG trace showing uterine contraction following intramuscular delivery of oxytocin (example 10 formulation 4). The dashed line shows the time of formulation delivery and the solid line the onset of uterine contraction. The x-axis is in hh:mm:ss and the y axis is in mV.

EXAMPLES

Example 1

Spray Drying

[0090] Spray drying is a one-step process that involves the formation of powders from a starting solution of the desired dissolved material. By definition, it is the transformation of feed from a fluid state into a dried form by spraying the liquid feed into a hot drying medium. Four keys stages in the spray drying process are: (i) atomisation of feed through the nozzle, (ii) spray-air contact between the liquid droplets and the drying gas, (iii) drying of particles via evaporation of liquid, and (iv) collection of the final powder.

[0091] Referring to the schematic in FIG. 1, the air is taken into the system 1 and heated by the supplied heater 2 prior to the measurement of the inlet temperature 3. The liquid teed is drawn up separately into the nozzle 4 where the droplets are formed and dispersed into the drying chamber 5 being mixed with the warm air. At this point, a dried particle is formed. The outlet temperature is measured 6 as the particles move into the cyclone 7 where the powder is separated from the air. The powder is trapped in the collection vessel 8 to be recovered whereas the air gets filtered from all the line particles that may have remained in the air stream at the bag filter 9. The circulation of air in the spray drying is continued by the work of the aspirator 10.

[0092] Atomisation is a very crucial part in defining the droplets, and hence the subsequent particle size and distribution. It involves forming a spray of droplets from the bulk liquid as the feed is pumped through to a small orifice in the nozzle. In the case of a two-fluid nozzle, the supplied gas impacts on the liquid bulk in the nozzle at high velocities. This high velocity gas creates high frictional forces over the liquid surfaces, causing the liquid to disintegrate and to form spray droplets, which project into the drying chamber.

[0093] The properties of the dissolved material and the drying conditions will influence the final powder characteristics. With evaporation of the liquid solvent from the droplet surface (water in this case) solute precipitation occurs. Often as the particle is forming, a crust may form and the crust may be porous, semi- porous or non porous allowing the removal of moisture at different rates and with varying effects. Particles of varying morphology can therefore form. Control of drying conditions is therefore an important consideration.

[0094] According to the experiments performed, formulations of the powders and their spray drying parameters were varied. In all the powder formulations, mannitol was used as the baseline material, with varying amino acids added. The spray drying parameters that remained constant throughout were the aspirator setting, set at full flow and the atomiser airflow rate (800 L/hour).

[0095] For each formulation, the mannitol, glycine and oxytocin were spray dried at fixed amounts with varying amounts of leucine. The spray drying conditions were fixed with the outlet temperature set at 70° C.

[0096] The parameters that were used arc shown in Table 1 below. The percentages shown of the amino acids were calculated to that of the mannitol amount only, not of the entire powder content. The percentage shown of oxytocin was that of the entire powder content.

TABLE-US-00001 TABLE 1 Trial 1 2 3 4 5 6 Mannitol (9) 5 5 5 5 5 5 Glycine (%) 30 30 30 30 30 30 Oxytocin (%) 1 1 1 1 1 1 Leucine (%) 15 50 15 50 32.5 32.5 Liquid feed rate 8 8 2 2 5 5 (mL/min)

[0097] The powders were weighed out and dissolved in the appropriate amount of Milli-Q water to achieve the desired feed concentration. The solutions were then spray dried to produce dry powders using the Buchi 190 Mini Spray Drier (Buchi, Switzerland).

Example 2

In-Vitro Aerosol Deposition

[0098] The in-vitro aerosol deposition of the powders was measured using the Twin Stage Impinger (™) (Copley Scientific Ltd., Nottingham, UK). The TSI methods and set up was done so according to the British Pharmacopeia 2011 as shown in FIG. 2. In the glassware part D and part H, 7 mL and 30 mL of water respectively was added during the assembly of the TSI.

[0099] The TSI is a simple model of the respiratory tract; with the upper (stage 1) and lower (stage 2) chambers representing the upper and lower airways respectively. The cut off aerodynamic diameter at the first stage is 6.4 μm. Particles larger than 6.4 μm should ideally be collected in the 7 mL liquid; smaller particles (<6.4 μm) that are not collected will proceed to the lower stage, which contains 30 mL of liquid. Most particles will he collected in the lower stage due to the excess of liquid, however if particle size is too small for collection in the lower stage, they will be emitted at the exit.

[0100] Measurements for each powder sample were done in four replicates. For each replicate, five size 3 HPMC capsules were manually filled with 20.4±0.24 mg with the sample powder and placed in five Monodose inhalers (Miat, Italy). A vacuum pump was attached to part F and the airflow rate was calibrated to 60L/min and was set to 5 seconds. The capsule was pierced in the device and placed on the adapter (pan A) ready to be activated by the vacuum pump. When the pump was turned on, the powder was carried from the inhalation device into the TSI apparatus.

[0101] All five capsules were activated into the same TSI. The used capsules and the inhalers were then washed with Milli-Q water into a 100 mL volumetric flask and made up to volume. This was called the ‘residual’ stage. The parts that made up Stage 1 (parts A, B, C and D) were washed into a 200 mL volumetric flask and the parts that made up Stage 2 (parts E, F, G and H) were washed into a 50mL volumetric flask with Milli-Q water and called ‘stage 1’ and ‘stage 2’ respectively. The amounts of oxytocin in each stage of the TSI were determined by LC/MS assay. The fine particle fraction (FPF) was calculated as the amount of powder that had reached stage 2 of the TSI apparatus divided by total amount of drug that was assayed. This test was the most important measure as it can determine whether a powder containing oxytocin can be formulated with suitable aerosol deposition, and consequent absorption from the lung.

TABLE-US-00002 TABLE 2 Trial Number Fine Particle Fraction (%) 1 57 2 70 3 67 4 73 5 64

[0102] Particles passing to the lower portion of the TSI device i.e. stage 2, were considered to be respirable, therefore the higher the fine particle fraction (FPF), the higher the chance of the drug reaching the alveoli and getting absorbed into the bloodstream, which is ideal in a DPI. The FPF of the five trials shown in Table 2 were high compared to an average FPF from traditional carrier formulation powders (˜10-20%).

[0103] Results showed that the FPF could reach between 55 and 75% which means that very efficient levels of aerosolisation were achieved and high amounts of oxytocin in the formulations were delivered as the required therapeutically active dose.

[0104] Oxytocin Stability

[0105] Peptides can potentially be denatured due to extreme heat. From the tests that were conducted in this study, the only indication as to oxytocin stability was the LC/MS assay content following the TSI experiments. When oxytocin content was assayed from all the stages of the TSI apparatus, capsules and the inhalation devices, on average 90.23±5.41% of the initial capsule's dose was recovered, suggesting that oxytocin was not degraded from the temperatures used in the spray drying process or from the handling processes.

Example 3

Trehalose/Leucine

[0106] Trehalose is a non-reducing sugar with high glass transition temperature (T.sub.g of 117° C. that has been used as an excipient in various studies for stabilisation of protein in dry solid slate formulations. Sugar molecules are generally used as stabilising excipients in this context as they contain carboxyl groups that are able to form hydrogen bonds with the protein of interest and therefore stabilise the bio-macromolecule with hydrogen bond replacement in dry solid state. Spray-drying has been successfully used in various studies for the manufacturing of inhalable dry powder formulations as the process is able to produce line particles with particle size range that is suitable for pulmonary delivery.

[0107] In an attempt to formulate inhalable protein pharmaceuticals for pulmonary delivery, spray-dried trehalose is produced at relatively low outlet temperature of 70° C. in order to minimise the impact of heat stress on processing stability of the relevant protein. Conditions were otherwise the same as the examples described with mannitol and amino acids.

[0108] While trehalose is relatively crystalline as a raw material, spray-dried trehalose under the specified spray-dried conditions appears to be fully amorphous (see FIG. 3). This resultant formulation may further stabilise the protein of interest with glassy state stabilisation by providing an amorphous matrix which reduces molecular mobility of the bio-macromolecule in the formulation.

[0109] The resultant formulation of trehalose only is however, composed of fused primary structures with large particle size that is unlikely to be suitable for pulmonary delivery (FIG. 5). The incorporation of leucine in the formulation from a concentration of 10% w/w substantially improves particle size and morphology of the spray-dried formulation under the specified conditions (FIG. 6). The incorporation of leucine appears from this to be able to prevent fusion of the otherwise highly hygroscopic primary particle structures of trehalose from the spray drying process, and therefore retains the fine particle size range suitable for pulmonary delivery. In addition, the presence of leucine is also able to improve these particle properties over longer term storage, by providing a moisture protection of the internal amorphous matrix of the spray-dried trehalose formulations (FIG. 4).

Example 4

Materials

[0110] D-Mannitol was obtained from VWR International Ltd. (Poole, BH15 1TD, England). L-leucine (LEU), glycine (GLY) and L-alanine (ALA) were obtained from Sigma-Alrich Chemicals (Castle Hill, NSW, Australia).

[0111] Preparation of Spray-Dried Powders

[0112] Aqueous solutions containing mannitol and selected amino acids (LEU, GLY, ALA) in various compositions as shown in Table I were dissolved in 200 mL of Milli-Q water. A small amount of methylene blue (10 mg) was incorporated in each formulation to allow a simple quantification of powder by UV-VIS spectrophotometric analysis as described below. The prepared formulations were subsequently spray-dried using a Buchi 190 mini spray-dryer with a 0.5 mm two-fluid nozzle, using the following standard operating conditions: airflow rate, 800 L/h; pump setting, 5 (6.67 mL/min); aspirator setting, 20; outlet temperature, 75° C.

[0113] Particle Size Distribution Analysis

[0114] The particle size distribution of the powders was determined by laser-light scattering using the Malvern Mastersizer 2000 (Malvern Instruments Ltd, Worcestershire, UK) equipped with a Scirocco cell and a Scirocco 2000 dry powder dispersion unit. The powders were dispersed in air at a shear pressure of 3.0 to 4.0 bar, which was selected to achieve suitable de-agglomeration. The average particle size was measured in three replicates for each sample. The volume median diameter (D50) was derived from the diffraction data using the in-built software for each sample.

[0115] In vitro Powder Aerosolisation and Particle Deposition

[0116] The in vitro powder aerosolisation performance and particle deposition was assessed using a twin stage impinger (TSI, Apparatus, A; British Pharmacopoeia, 2000) with the Monodose inhaler (Miat S.p.A., Milan, Italy) as the aerosol dispersion device. The flow rate was adjusted to 60 L/min using a Critical Flow Controller Model TPK 2000 & Flow meter model DFM 2000 (Copley Scientific Limited, Nottingham, UK). Approximately 20 mg of each powder was filled into size 3 1-IPMC capsules (Capsugel, Peapack, N.J., USA) for the tests which were performed at an air-conditioned laboratory (20±2° C., 50±5% relative humidity). Each capsule was actuated from the inhaler over 4 seconds for each measurement (n=5). The amount of powder deposited at different stages was determined using a UV-VIS light spectrophotometer as described below. The cut-off diameter for the TSI at 60 L/min is approximately 6.3 μm

(Hallworth and Westmoreland. 1987).

[0117] The total amount of powder deposited in the inhaler, stage 1 (S.sub.1) and stage 2 (S.sub.2) was the recovered dose (RD). The amount of powder deposited in stage 1 and 2 was the emitted dose (ED) and it was calculated as the percentage of the RD (Eq. 1). The fine particle fraction (FPF) was defined as the percentage of RD deposited in stage 2 (Eq. 2).

[00002]$\begin{array}{cc}\mathrm{ED}.Math..Math.\%=\frac{\left(S_1+S_2\right)\times 100}{\mathrm{RD}}& \left(1\right)\\ \mathrm{FPF}.Math..Math.\%=\frac{S_3\times 100}{\mathrm{RD}}.& \left(2\right)\end{array}$

[0118] Scanning-Electronic Microscopy (SEM)

[0119] The morphology of the particles was visualised under a scanning electron microscope (Phenom™, FEI company, USA). Powder samples were gently poured onto a double-sided carbon tape mounted on a sample holder for examination under the SEM. Excessive powder was removed to leave a fine layer of particles on the surface of the tape. The samples were sputter coated with gold using an electrical potential of 2.0 kV at 25 mA for 6 minutes with a sputter coater (K550X, EMITECH). SEM micrographs were captured using the in-built image capturing software.

[0120] Results

[0121] The volume median particle size (D50) of all the formulations measured using Mastersizer 2000 are listed in Table 4. Spray drying mannitol alone produced small particles with D.sub.50 of 1.87 μm. However, this powder was fully crystalline and did not have the amorphous glass structure required to stabilise bio-molecules.

[0122] Leucine is an excipient that can be used to improve aerosolisation of spray-dried particles, but also leucine assists in the formation of suitable small-sized particles. However glycine and alanine, though being structurally similar to leucine, were not able to achieve similar effects as they significantly increase the particle size of the formulations. It is worth noting that while initial concentration in feed solution is a known determinant of particle size, the range of solid loading used within the study design space did not appear to have a strong influence on geometric particle size as measured by laser diffraction. The total solid loading in the feed solution ranged from 2.50% to 3.72% in the present study. It is proposed that the change in particle size within this relatively small range of solid loading was negligible compared to the effects of the formulation excipients on cohesion and shape. Furthermore, considering the particle sizes produced from the mixed amino acids, it is possible that the combination use of these amino acids with leucine at appropriate concentrations may also influence particle size contrasting to that achieved by leucine alone.

[0123] Powder Dispersibility and De-Agglomeration

[0124] Spray-dried mannitol produced particles with D.sub.50 of 2.83 μm which appears to indicate a satisfactory dispersibility for inhalable dry powder formulations, but it also produces the lowest emitted dose (ED). The retention of powder in the device after the experiment was visually evident, and suggests a more cohesive powder than other formulations here. The presence of amino acids in all combinations resulted in improved ED (Table 4). The beneficial effect of leucine was evident in its capacity to offset the effect of the other two amino acids on D.sub.50 and of improving both de-agglomeration and ED.

[0125] In vitro Aerosolisation and Particle Deposition

[0126] The TSI was used as a preliminary screen of this range of formulations to provide aerodynamic aerosol information.

[0127] Fine particle fraction (FPF) results show the formulations containing leucine, with D.sub.50 below 5 μm demonstrate the highest FPF of greater than 68% (Table 4). Powders containing amino acids without leucine, with D.sub.50 above 5 μm show significantly lower FPF as demonstrated by formulations containing glycine/alanine 30/30%, alanine 30% and glycine 30%, with FPF of 2.96%, 9.11% and 34.62%, respectively. While mannitol alone shows reasonable FPF of 66.20%, this formulation also demonstrates the lowest ED.). The combined amino acids at 15% were more effective at improving FPF (Table 4). These results suggest that the inclusion of glycine and alanine with leucine at the appropriate concentrations may improve formulation aerosolisation performance.

[0128] Surface Morphology

[0129] Spray-dried mannitol as a foundation material alone was observed to form small spherical particles that are heavily agglomerated. The result is consistent with the particle size distribution data from the Mastersizer. Upon addition of amino acids, spherical particles were preserved in all formulations containing leucine regardless of the presence of glycine and/or alanine. Other formulations containing glycine and/or alanine without the addition of leucine formed much larger particles of irregular shape with rough surfaces.

[0130] The result suggests that the presence of leucine assists in the formation of spherical particles by coating the drying particle surface, and therefore providing a protective shell which preserves the individual particles as they collect, preventing any fusion, while the presence of glycine and alanine did not prevent this effect. Previous results indicated that a relatively high concentration of leucine (i.e. >5% w/w) tends to lead to corrugated particles. The morphology of leucine-containing particles in the present study appears to behave differently. The concentrations of leucine used within the study design space (15 to 30 molar %), which corresponds to roughly 10 to 18% w/w, did not form corrugated particles. It is therefore speculated that the presence of glycine and/or alanine altered the core structure of the spherical drying particles, while leucine tended to reside on the particle surface, providing a coating to reduce surface cohesiveness and prevent fusion in the drying process.

[0131] In the present study, leucine was able to enhance aerosolisation performance of the mannitol formulations without necessitating the formation of corrugated particles. Furthermore, the FPF results suggest that this may be advantageous.

TABLE-US-00003 TABLE 3 Levels of factors used in the formulation Factors −1 0 +1 X.sub.1 = leucine (molar %) 0 15 30 X.sub.2 = glycine (molar %) 0 15 30 X.sub.3 = alanine (molar %) 0 15 30 Process and formulation Responses parameters kept constant Y.sub.1 = Mastersizer D.sub.50 (μm) Mannitol content: 5 g Y.sub.2 = Spraytec D.sub.50 (μm) Feed solution volume: 200 mL Y.sub.3 = Spraytec ED (mg) Aspirator setting: 20 Y.sub.4 = TSI fine particle Pump setting: 5 (6.67 mL/min) fraction (%) Y.sub.5 = TSI ED (%) Airflow: 800 L/h Y.sub.6 = cohesion value (kPa) Outlet temperature: 75° C. Abbreviation: ED, emitted dose; SD, spray-drying; TSI, twing-stage impinger.

TABLE-US-00004 TABLE 4 Batch X.sub.1 X.sub.2 X.sub.3 Y.sub.1 Y.sub.2 Y.sub.3 Y.sub.4 Y.sub.5 Y.sub.6 1 0 0 0 1.87 2.83 15.80 66.20 78.04 4.53 2 30 0 0 1.75 2.70 17.50 80.10 91.11 2.21 3 0 30 0 3.75 5.52 19.20 34.62 89.59 2.39 4 30 30 0 2.05 3.50 18.00 72.62 88.00 1.04 5 0 0 30 6.69 12.02 18.90 9.11 92.17 0.71 6 30 0 30 2.27 3.24 17.50 68.64 89.99 1.21 7 0 30 30 13.97 28.55 19.20 2.96 85.90 0.43 8 30 30 30 1.97 3.58 17.20 69.13 87.40 1.32  9.sup.a 15 15 15 2.05 2.58 16.60 76.84 88.79 1.82 10.sup.a 15 15 15 1.95 2.49 17.40 76.40 86.97 1.79 11.sup.a 15 15 15 1.99 2.28 16.40 74.27 88.82 2.11 12.sup.a 15 15 15 n/a 2.40 17.0 n/a n/a 1.32 .sup.aIndicates the centre point of the design. Abbreviation: n/a, not available.

Example 5

In vivo testing

[0132] On day 135 gestational age, pregnant ewes (n=5) were anaesthetised with thiopentone in preparation for surgery. Isuflorane (2.5% in oxygen) was used to maintain anaesthesia and depomycin, procaine penicillin and dihydrostreptomycin were given for pain relief and to reduce the risk of infection. Each ewe was shaved and a 10 cm incision was made in the abdominal skin at the midline below the navel to expose the uterine wall, with care taken to avoid large blood vessels.

[0133] Three sterile stainless steel wires for measuring electromyographical (EMG) activity (0.07 mm diameter, inside a 2 mm catheter) were embedded in the smooth muscle layer of the myometrium surrounding the womb and held there by two stitches. The electrodes were passed through a catheter and out of the ewe via a small incision (2 cm) through the right flank. A catheter was inserted in the right jugular to allow for blood samples and to induce labour. Ewes were returned to metabolic cages and given 3-5 days to recover from surgery.

[0134] Labour was induced with two 5 ml intravenous injections of dexamethasone (consisting of 5 mg of dexamethasone phosphate and 10 mg of dexamethasone phenylpropionate) 24 hours apart. Labour occurred 54±2 hours after the first dexamethasone injection.

[0135] Oxytocin administrations, as detailed above, were performed within 15 hours of delivery. Each sheep received an intra-tracheal dose of dry powder oxytocin formulation, an intra-tracheal instillation of oxytocin in solution and an intramuscular injection of oxytocin. There was at least a one and a half hour washout period between each treatment.

[0136] For intra-tracheal administration, an endoscope was passed through the nasal passage into the trachea and positioned near the first bronchial bifurcation, where either a 1 mL aliquot of oxytocin in solution (10 IU) was released or 10 mg (average) of dry powder was delivered through a modified PennCentury powder delivery device.

[0137] The dry powder formulation comprised a spray dried composition, as described in example 1. This powder contained 13 units of oxytocin per mg by mass, co-spray dried with equal proportions by mass of mannitol, glycine and leucine.

[0138] During this procedure, bronchoscope video images were captured using a Linvatec IM3301 Pal Video Camera attached to an endoscope (Pentax FG-16X), which was saved as a digital file onto a computer using Video Capture Software. Examples of the images are provided in FIGS. 9a and 9b. The image in FIG. 9a shows the sheep trachea before powder delivery, and the FIG. 9b shows the image approximately 30 seconds from delivery. FIG. 9b shows clear evidence of white undissolved powder, as white patches not present before delivery, indicating that immediate dissolution had not occurred within the pulmonary system.

[0139] A cyberamp 380 in conjunction with MACLAB hardware (400 Hz Sample rate) and Chart 4 software (10V Input range) was used to display and record action potential originating from smooth muscle cells within the uterus. The cyberamp 380 used the AI401 probe with positive input set at AC and negative input set at Ground. The AC cut off was 10 Hz and the prefilter gain set at 100 mV. The low pass filter was set at 300 Hz, the notch filter set to off, the output gain set to 5 and the total gain left at 500. Two way repeated measure ANOVA was performed to determine the statistical significance of our data. See FIG. 7.

[0140] A number of properties of recorded EMG activity were analysed. With regard to elapsed time (delay) from delivery of oxytocin to the initial burst of EMG activity, delivery via the lungs results in a faster time of onset for the first contraction in contrast to IM delivery (FIG. 8a). No differences were seen in the length of the initial burst of EMG activity (FIG. 8b) nor was there a difference in the number of bursts occurred over the first 30 minutes following the initial burst of activity (FIG. 8). However, the total duration of EMG activity was significantly longer for IM of oxytocin compared to airway delivery and the normal activity observed immediately post-partum (FIG. 8).

[0141] These in vivo studies demonstrate that uterine contractile responses to oxytocin administered via pulmonary delivery occur on average after approximately 120 seconds in contrast to the IM delivery which occurred on average after approximately 250 seconds. Surprisingly, the onset of action from the powder pulmonary delivery was significantly more rapid compared to intramuscular delivery, and was also consistent with the plasma versus time profiles. The average onset of action was approximately 50% less than for IM. This is despite the fact that the dry powder particles contain approximately 30% leucine which is a poorly soluble and hydrophobic amino acid, which could be expected to delay dissolution. Furthermore, it is expected that a substantial proportion of the leucine will be present at the surface of the powder. The image 9b supports this concept that rapid dissolution of such powder is not expected in this environment. The data also demonstrates that uterine contractile responses to pulmonary dry powder oxytocin mimics the activity seen naturally in the immediate post-partum period, as observed with the length of the initial burst of uterine activity and the total number of EMG activity bursts recorded over the following thirty minutes.

Example 6

Dry Powder Influenza Antigen Formulation

[0142] The powder sample of influenza antigen, haemagglutinin (HA), was first dissolved with other excipients (i.e. mannitol 45% w/w, glycine 45% w/w and leucine 10% w/w) into an aqueous solution to produce a final formulation with an antigen loading of 5 μg HA per mg of powder. This solution was then spray-dried in a Buchi 190 laboratory spray dryer, at a relatively low temperature i.e. 70 degrees C. outlet temperature to minimise the effect of heat stress on the integrity of the antigen at the following spray-drying conditions: pump setting 6.7 mL/min; aspirator, 20 (100%); airflow, 800 1/hr. The spray-dried influenza antigen formulation was then collected from the collecting vessel for storage.

Example 7

Method for Testing Biological Activity of the Dry Powder Influenza Antigen Formulation

[0143] The following describes the method for the testing of biological activity of the spray dried dry powder influenza antigen formulation containing haemagglutinin (HA) as the active protein. The haemagglutination assay (HA assay) is used to test the integrity of the HA protein in the dry powder formulation. The dry powder influenza antigen formulation of interest is first reconstituted with phosphate buffered saline (PBS) immediately before the test into a solution of standard HA protein concentration. A small amount of this reconstituted solution is then place into the first column of a 96-well plate. The solution is then diluted 1:2 with PBS across the 96-well plate by serial dilution. A standard amount of chicken red blood cell solution with a standard red blood cell concentration (i.e. 1%) is then added to each well of the plate. The plate is incubated at room temperature for 30 minutes immediately after addition of the red blood cells. Since intact HA protein will cause haemagglutination of red blood cells, the level of dilution that the HA containing solution is able to sustain before it is no longer capable of causing haemagglutination at 30 minutes will indicate the amount of intact HA protein in the formulation. The antigen was found to be in excess or 95% active, within the limits of this procedure.

Example 8

Cohesion Measurement of Particles and Preferred Cohesion Values

Apparatus and Materials

[0144] The apparatus used were the 1 mL shear cell module, and vented piston, as part of the FT4 FREEMAN Rheometer unit (Freeman Technology, UK) and computer user interface and 1 mL shear cell conditioning module. The materials used were spray dried powders of 1:99% w/w_leucine/mannitol, 3:97% w/w_leucine/mannitol, 5:95% w/w_leucine/mannitol, and 10:90% w/w_leu/mannitol. These powders were produced following conditions of Example 1 above.

[0145] A powder sample was loaded into the cell and conditioned. During conditioning the 1 mL shear cell conditioning module was employed to gently disturb the powder as it moved throughout the whole sample. The purpose of this was to homogenise the powder by removing excess air and isolated pre-compacted powder particles. After conditioning, the powder was compressed. This was executed by the flat-surface vented piston in order to ensure uniform particle-particle interactions. Compression was followed by shearing. During shearing, a 24 mm shear cell (a unit component of: base, slide, splitting shim and shear cell module) was employed. The shear head comprising 18 blades, moved vertically downwards inducing normal shear stress while the shear head blades pierced the powder surface. The shear stress was then measured and was at maximum when the powder failed to resist the shear stress. The graph of shear stress against normal stress was generated by the FT4 FREEMAN integrated software. From the graph, the extrapolated y-intercept provides the cohesiveness of the powder at zero consolidation. The ffc [the ratio of the major principal stress (consolidation stress), σ1, to the unconfined yield strength, σ1 data was also recorded.

TABLE-US-00005 TABLE 5 Sample Mean Cohesion (kPa) Flowability (ffc) 1% Leucine 3.4 1.4 3% Leucine 2.4 2 5% Leucine 2.3 2 10% Leucine 1.4 2.9

[0146] From 1 to 10% leucine content, an increase in leucine content decreases cohesion and improves flowability parameter (ffc).

[0147] This experiment was than repeated using spray dried leucine and PV1), produced and tested under similar conditions. The results were as follows:

TABLE-US-00006 TABLE 6 Sample Mean Cohesion (kPa) 0% Leucine 4.0 2% Leucine 3.5 4% Leucine 3.4 8% Leucine 1.2 10% Leucine 1.2 20% Leucine 0.7

Example 9

Surface Energy Measurements

Samples

[0148] Two batches of powder were spray dried from water using the conditions as described in example 1, but where the powders comprised compositions of pure mannitol or mannitol with 10% w/w L-Leucine added.

[0149] Surface Energy Determination by Inverse Gas Chromatography

[0150] Surface energies of these powders were determined using Inverse Gas Chromatography (IGC, Surface Measurement Systems Ltd, and London, UK). Approximately 0.33 g of each powder was packed into prc-silanised glass columns (300 mm×3 mm internal diameter) which were loosely stoppered with silanised glass wool in both ends. The powder filled columns were conditioned for 2 h at 303 K before each measurement in order to remove impurities of surface. Probes were carried into the column by helium with a gas flow rate of 10 sccm (standard cubic centimetre per minute) and the retention times were detected by a flame ionization detector. The dead volume was calculated based on the elution time of methane which was run at a concentration of 0.1 p/p.sup.0 (where p denotes the partial pressure and p.sup.0 the vapour pressure).

[0151] Surface Energy Determination at Infinite Dilution:

[0152] GC grade hexane, heptane, octane, nonane and decane (all from Sigma-Aldrich GmbH, Steinheim, Germany) for non-polar surface energy (γ.sup.NP), and two polar probes (i.e., dichloromethane and ethyl acetate) for polar surface energy (γ.sup.P) were used at a concentration of 0.03 p/p.sup.0. The detailed of γ.sup.P calculation was described elsewhere (Thielmann et al., Investigation of the acid-base properties of an MCM-supported ruthenium oxide catalyst by inverse gas chromatography and dynamic vapour sorption. Jackson, S. D., Hargreaves, J. S. J., Lennon, D., editors. Catalysis in application Great Britain, Royal Soc. Chem., p 237 (2003), and Traini et al., Drug Development and Industrial Pharmacy 34: 992-1001 (2008). FIG. 1 shows the surface energy contributions at infinite dilution:

[0153] The total surface energy (γ.sup.T) was the additive result of non-polar (γ.sup.NP) and polar contributions (γ.sup.P) (Grimsey et al., Journal of Pharmaceutical Sciences 91: 571-583 (2002). The work of cohesion (W.sub.∞) was calculated (see Vanoss et al., Langmuir 4: 884-891 (1988) and Tay, et al., International Journal of Pharmaceutics (Kidlington) 383: 62-69 (2010). These experiments were run in triplicate.

[0154] Surface Energy Distributions and Surface Area Determination at Finite Dilution:

[0155] The distribution profiles of non-polar surface energy (γ.sup.NP profile) were determined according to the method described elsewhere (F. Thielmann et al., Drug Development and Industrial Pharmacy 33: 1240-1253 (2007) and Yla-Maihaniemi, et al., Langmuir 24: 9551-9557 (2008)). This is shown in FIG. 10. The non-polar surface energies at all coverages are clearly reduced for the leucine containing powder, and in this case shows a reduction by more than 30% at a 1% coverage level. The polar surface energy distribution is shown in FIG. 11.

[0156] The Brunauer-Emmet-Teller (BET) surface area was calculated from hexane adsorption isotherms. Dividing the adsorbed amount (n) by the monolayer capacity (n.sub.m, the number of moles of the probe adsorbed for monolayer coverage), the surface coverage (n/n.sub.m) was calculated. At each surface coverage, the net retention volume (V.sub.N) was calculated for each probe. The non-polar surface energy (γ.sup.NP) was calculated from the slope (2 N.sub.A √γ.sup.NP) of a plot of RT1nV.sub.N against a √γ.sup.NP of alkanes. The γ.sup.P and γ.sup.T were calculated at each surface coverage and their distribution profiles were then constructed (as described in Das et al. Langmuir 27: 521-523 (2011a)).

[0157] FIGS. 12 and 13 show that the calculated total surface energy distributions and the work of cohesion are also substantially reduced in the case where leucine is added, for example by 20% or more at a surface coverage of approximately 1%.

Example 10

In vivo Testing of Other Formulations

[0158] As per example 5 a single pregnant ewe was prepared by surgery to implant the EMG electrodes and a catheter to allow for blood collection. The induction of labour commenced on the same day as surgery also as per the method set out in example 5. Labour and delivery occurred 2 days after the beginning of the induction period.

[0159] Oxytocin administrations, as detailed below, were begun within 22 hours of delivery. There was a one and a half hour washout period between each treatment.

[0160] For intra-tracheal administration, an endoscope was passed through the nasal passage into the trachea and positioned near the first bronchial bifurcation, where a dose of dry powder (as detailed below) was delivered through a modified PennCentury powder delivery device. In addition to the dry powder delivery to the lung an intramuscular injection of oxytocin (as per example 5) was also delivered.

[0161] The dry powder formulations comprised spray dried compositions made as described in example 1. The compositions of these powders along with the nominal delivered dose are shown in Table 7, elapsed time (delay) from delivery of oxytocin to the initial burst of EMU activity. The delay, elapsed time from delivery of oxytocin to the initial burst of EMG activity is also shown in Table 7. EMG traces for these four doses arc shown in FIGS. 15 and 16.

TABLE-US-00007 TABLE 7 Dose Oxytocin Mannitol Glycine Trehalose PVP(30) Leucine Delay No Dose % w/w % w/w % w/w % w/w % w/w (s) 1 200 IU 33.3 33.3 33.3 181 2 200 IU 90 10 65 3 200 IU 90 10 150 4 10 IU 1 ml intramuscular injection 232

[0162] Surprisingly, the onset of action from the powder pulmonary delivery was significantly more rapid compared to intramuscular delivery, and was also consistent with the plasma versus time profiles, the data also shows that the rapid response can be attained with dry powder formulations made with a range of excipients such as polyols, sugars, amino acids and polymers. This is despite the fact that the dry powder particles contain between 10-30% leucine which is a poorly soluble and hydrophobic amino acid, which could be expected to delay dissolution. Furthermore, it is expected that a substantial proportion of the leucine will be present at the surface of the powder.

[0163] The invention has been described by way of non-limiting example only and many modifications and variations may be made thereto without departing from the spirit and scope of the invention described.

[0164] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0165] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.