METHOD

TECHNICAL FIELD

This relates to the field of pharmaceutical formulation measurement. More particularly, this relates to measuring the size of particles in a nebulized pharmaceutical formulation and determining the particle size distribution and amount of the active pharmaceutical ingredients therein.

BACKGROUND

A common approach in the art for establishing Bioavailability (BA) and Bioequivalence (BE) of suspension formulations of locally acting nasal drug products for sprays is to conduct in vivo studies in addition to in vitro studies. In vivo studies are required since there is an inability to adequately

characterize drug particle size distribution (PSD) in suspension formulations of nasal sprays. Drug PSD has the potential to influence the rate and extent of drug availability to nasal sites of action and to the systemic circulation. Nevertheless, in vitro methods are less variable, easier to control and more likely to detect differences between products.

An important factor in the deposition of drug in the nasal passages is aerodynamic diameter of drug particles. It is known that the respirable mass of a drug dose is the amount of the administrated dose that comprises particles having a size of less than 5 microns. In the case of a nasal spray upon nasal administration, it will be the particles in this region that will be intended for local action in the nasal cavity and that has the potential to produce systemic activity. Consequently, the small particles or droplets (around 5 microns) and the Mass Median Aerodynamic Diameter (MMAD) that are typically measured by multistage cascade impactor (CI) are important characteristics of nasal suspensions.

Although there are several techniques that can measure the particles size of drug substance in drug product such as laser diffraction (Malvern Mastersizer); microscopy (Image Analyzer) or Raman spectroscopy combined with microscopy. Generally speaking, laser diffraction and microscopic methods are non-selective and unable to distinguish the drug particle from that of suspended cellulose. Raman Spectroscopy combined with microscopy can be selective to a certain extent but there are difficulties with data interpretation for the size of an "individual" particle as opposed to the size of agglomerates.

SUMMARY

The present invention is based, at least in part, on an apparatus that is capable of detecting the particle size of a drug substance suspended in other suspending agents. Such an apparatus may be used to determine an average amount of a first compound in a mixture that is nebulized to a particle having a particular size.

Illustrative embodiments of the present invention provide an apparatus for separating differently sized particles of a mixture, the apparatus comprising: a) a holding means for holding said mixture having a first holder opening and a second holder opening in fluid communication with the first holder opening; b) a nebulizing means in communication with the first holder opening and operable to nebulize the mixture thereby forming a nebulized mixture; c) a particle sizing means comprising: i) a first sizing means opening; ii) a second sizing means opening in fluid communication with the first sizing means opening; and iii) an at least one settling stage in fluid communication with the first sizing means opening and the second sizing means opening, the at least one settling stage operable to receive at least a portion of the nebulized mixture; d) a communicating means in fluid communication with the second holder opening and the first sizing means opening; e) a vacuum pump in communication with the second sizing means opening operable to control pressure in the particle sizing means; and f) a pressure control means operable to prevent said mixture from moving from the holding means to the communicating means until said mixture is nebulized by the nebulizing means into the nebulized mixture.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the at least one settling stage is a plurality of settling stages, each one of the plurality of settling stages in series fluid

communication with one another and operable to receive a portion of the nebulized mixture. Illustrative embodiments of the present invention provide an apparatus described herein wherein the plurality of settling stages comprises eight settling stages.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the particle sizing means is a cascade impactor that is USP<601 > compliant.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the nebulizing means is a jet nebulizer.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the nebulizing means is an electronic nebulizer.

Illustrative embodiments of the present invention provide an apparatus described herein wherein a flow rate of the nebulizing means is from about 2 L/minute to about 6 L/minute.

Illustrative embodiments of the present invention provide an apparatus described herein wherein a flow rate of the nebulizing means is from about 4 L/minute to about 5 L/minute.

Illustrative embodiments of the present invention provide an apparatus described herein wherein a flow rate of the vacuum pump is from about 26 L/minute to about 30 L/minute.

Illustrative embodiments of the present invention provide an apparatus described herein wherein a flow rate of the vacuum pump is from about 26.9 L/minute to about 29.7 L/minute.

Illustrative embodiments of the present invention provide an apparatus described herein wherein a flow rate of the vacuum pump is from about 28.3 L/minute.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the pressure control means is a valve operable to be moved from a closed position to an open position wherein the open position allows the nebulized mixture to flow from the holding means to the communicating means and the closed position allows nebulization of said mixture without said mixture entering the communicating means.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the pressure control means is a gap between the communicating means and the second holder opening. Illustrative embodiments of the present invention provide an apparatus described herein wherein the holding means comprises a sample cup and a mouthpiece.

Illustrative embodiments of the present invention provide an apparatus described herein wherein communicating means is a USP<601 > compliant induction port.

Illustrative embodiments of the present invention provide an apparatus described herein wherein mixture comprises an active pharmaceutical ingredient.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the mixture is a suspension.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the suspension is suitable for nasal administration.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the suspension is suitable for oral administration.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the suspension is suitable for inhalation.

Illustrative embodiments of the present invention provide an apparatus described herein wherein the suspension is suitable for ophthalmic

administration.

Illustrative embodiments of the present invention provide a method for determining an average amount of a first compound in a mixture that is nebulized to a particle having a particular size, the method comprising:

nebulizing a mixture comprising said first compound thereby forming a plurality of nebulized mixture particles in a holding means; moving the nebulized mixture particles from the holding means to a particle sizing means thereby forming an at least one stage deposit comprising nebulized mixture particles of a known average size; and analyzing the at least one stage deposit comprising nebulized mixture particles of a known average size to determine the amount of said first compound in the at least one stage deposit.

Illustrative embodiments of the present invention provide a method described herein wherein said first compound is an active pharmaceutical ingredient. Illustrative embodiments of the present invention provide a method described herein wherein the mixture is a suspension.

Illustrative embodiments of the present invention provide a method described herein wherein the suspension is suitable for nasal administration.

Illustrative embodiments of the present invention provide a method described herein wherein the suspension is suitable for oral administration.

Illustrative embodiments of the present invention provide a method described herein wherein the suspension is suitable for inhalation.

Illustrative embodiments of the present invention provide a method described herein wherein the suspension is suitable for ophthalmic

administration.

Illustrative embodiments of the present invention provide a method described herein wherein the at least one stage deposit comprising nebulized mixture particles of a known average size is a plurality of stage deposits comprising nebulized mixture particles of a known average size, each stage deposit of the plurality of stage deposits comprising nebulized mixture particles of a single and different known average size.

Illustrative embodiments of the present invention provide a method described herein wherein the plurality of stage deposits comprising nebulized mixture particles of a known average size comprises eight settling stages.

Illustrative embodiments of the present invention provide a method described herein wherein the particle sizing means is a cascade impactor that is USP<601> compliant.

Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a flow diagram illustrating a typical communication between various possible components of the present invention.

Figure 2 is a photograph showing an illustrative assembly for an apparatus for determination of drug particle size in a nasal suspension Figure 3 is a photograph showing an example of the various stages within a cascade impactor as well as a typical deposition of both drug particles and other suspending agents on the various settling stages within the cascade impactor.

DETAILED DESCRIPTION

The present invention is based, in part, on an apparatus that is capable of detecting the particle size of a drug substance suspended in other suspending agents. Such an apparatus may be used to determine an average amount of a first compound in a mixture that is nebulized to a particle having a particular size.

As used herein, the term "USP" refers to the United States

Pharmacopeia, which is a drug regulating agency that sets standards for various routine measurements, apparatuses and other factors related to pharmaceuticals.

As used here, the term "about" means that that precise adherence to the exact numerical value following the term 'about" is not absolutely required or essential and that some minor deviation from the exact value is

permissible. In many circumstances a deviation of ±5% is acceptable.

In illustrative embodiments, there is provided an apparatus for separating differently sized particles of a mixture. The apparatus comprises a holding means for holding the mixture, a nebulizing means operable to nebulize the mixture, a particle sizing means operable to receive at least a portion of the nebulized mixture, a communicating means in fluid

communication with the holding means and with the particle sizing means, a vaccuum pump operable to control pressure in the particle sizing means, and a pressure control means operable to prevent the mixture from moving from the holding means to the communicating means until the mixture is nebulized.

The holding means has a first holder opening and a second holder opening that are in fluid communication with one another. The holding means is operable to hold the mixture so that it may be nebulized into the nebulized mixture. Examples of suitable holding means include a variety of containers known to a person of skill in the art, including a sample cup typically used with a nebulizer and other similar structures commonly associated with nebulizers. The holding means optionally has an additional resealable opening that may be used to introduce material into the holding means. Such material may include a mixture to be tested or part thereof, such as a pharmaceutical formulation and/or excipient or diluent.

The nebulizing means is in communication with the first holder opening and is operable to nebulize the mixture in the holding means thereby forming a nebulized mixture. Examples of suitable nebulizing means include a variety of nebulizers known to a person of skill in the art, including a jet nebulizer, an electronic nebulizer and compact electronic nebulizers. Some suitable nebulizers use vibration of membranes or meshes to produce a nebulized mixture rather than compressed air, as is common with jet nebulizers.

Ultrasonic nebulizers are another type of suitable nebulizer. Some examples of specific and suitable nebulizers include Pari ProNeb Ultra™, Pari eFlow™, Respironics i-Neb™, Omron MicroAir™ series and Aerogen Aeroneb™.

The particle sizing means comprises a first sizing means opening; a second sizing means opening in fluid communication with the first sizing means opening; and at least one settling stage in fluid communication with the first sizing means opening and the second sizing means opening, the at least one settling stage operable to receive at least a portion of the nebulized mixture. Examples of suitable particle sizing means include a variety of cascade impactors known to a person of skill in the art, including USP<601 > compliant cascade impactors, such as USP apparatus 1 , 2, 3, 4, 5 and 6.

The first sizing means opening is defined by a body in the particle sizing means. The first sizing means opening is operable to allow the passage of the nebulized mixture from the communicating means into the particle sizing means.

The second sizing means opening is defined by a body in the particle sizing means, which body may be the same body or a different body that defines the opening of the first sizing means. The second sizing means opening is operable to allow the vacuum pump to control the pressure inside the particle sizing means. A settling stage is a body that defines a plurality of openings that are operable to permit a particle having a particular particle size to pass through the settling stage while the body collects particles having a particle size greater than the particular particles size. For example, a settling stage may have a body that defines a plurality of openings that are sized to permit a particle having a particle size of 5 microns or less through the settling stage and the body will collect particles having a particle size of greater than 5 microns. It is understood that some particles having a particle size that is small enough to pass through the settling stage may, by chance, be collected on the body. However, a settling stage, when in use in apparatuses of the present invention, may not allow a particle having a particle size larger than the particular particle size to pass though the settling stage. Examples of suitable settling stages include a variety of settling stages that are sold separately or in combination with cascade impactors known to a person of skill in the art as well as a variety of filters and filter papers, including, but not limited to, stage collection cups or liquid impingers.

A plurality of settling stages, each settling stage operable to permit a particle having a different particular particle size to pass though, may be set up in series so as to separate particles of different sizes and collect the individual sizes on a separate settling stage. Generally, settling stages should be set up so that fluid traveling through a plurality of settling stages travels so that the fluid first passes thought the settling stage operable to permit passage of the largest particles followed by the settling stage operable to permit passage of the next largest particles and so on so that the last settling stage that the fluid passes through is operable to permit passage of particles having the smallest size. For example, if two settling stages are placed in series and one settling stage is operable to permit the passage of particles having a size of 5 microns or less and the other settling stage is operable to permit the passage of particles having a size of greater than 5 microns, then fluid should be passed through the settling stage operable to permit the passage of particles having a size of greater than 5 microns first and then passed through the settling stage operable to permit the passage of 5 microns or less. It is to be noted that apparatuses of the present invention will work functionally if such an organization of settling stages is not used, but careful consideration as to the size of drops collected on each settling stage will need to be made in order to obtain meaningful results. It is within the

understanding of a person of skill in the art to interpret the results from a particular organization of settling stages.

A communicating means is a body that defines a passage for fluid communication of the nebulized mixture from holding means, more particularly from the second holder opening, to the particle sizing means, more particularly to the first sizing means opening. Examples of suitable communicating means include a variety of conduits, pipes and other hollow elongated bodies known to a person of skill in the art, including an induction port. The communicating means can be quite varied in structure including being made of different materials and differences in sizes, but changes in the structure of the communicating means may result in a change to the amount and type of nebulized mixture that is ultimately exposed to the particle sizing means. A person of skill in the art is able to understand such changes and can select a communicating means together with selecting the suitable calculations that need to be made so that a particular communicating means may be used to provide the desired information. A specific example of a specific communicating means is an induction port for apparatus 1 in the USP.

A vacuum pump is well understood in the art and any of a variety of vacuum pumps known to the skilled person may be used, including any pump that can run at a flow rate of 10-100 L/minute. Section of the flow rate is well understood to a person of skill in the art and is typically within the range provided for a particular apparatus (e.g. USP apparatus 1 ) by the USP. For example, for USP apparatus 1 , the flow rate is typically 28.3 L/minute ± 5%.

The pressure control means operable to prevent said mixture from moving from the holding means to the communicating means until said mixture is nebulized by the nebulizing means into the nebulized mixture. The pressure control means may be any sort of a valve or a gap that permits pressure from the holding means to escape from the holding means so that mixture from the holding means does not move into the communicating means before it has a chance to nebulize. Some specific, but non-limiting examples of suitable pressure control means include a clamp, a flow adjuster a gap and/or a valve.

For example, a suitable pressure control means may be a gap between the second holder opening and the communicating means. The gap typically ranges from a few millimeters wide to as large as 1 cm wide. The gap may be merely the distance between an inner surface of the communications means and an outer surface of the holding means. Often, but not always, the holding means is positioned such that the second holder opening is surrounded by an inner surface of the communicating means. It is possible that some of the nebulized mixture may be expelled from the system via this gap, but such loss of nebulized mixture is acceptable if enough of the nebulized mixture is collected in a settling stage. The amount of nebulized mixture lost may be reduced if the second holder opening is positioned within the hollow portion of the communicating means.

Alternatively, if the holding means is suitably selected to withstand higher pressures, the pressure control means may be a valve that is operable to contain the pressure in the holding means until nebulization of the mixture occurs and after nebulization has occurred, the valve is operable to permit the pressure in the holding means to be released, together with the nebulized mixture, into the communicating means. Another alternative is to carefully control the pressure in the communicating means so that the pressure in the communicating means is insufficient to draw mixture from the holding means into the communicating means.

Further still, the pressure control means may be a valve or a gap in the communicating means that is operable to prevent the pressure effect of the vacuum being exerted on the holding means until such time as nebulization has occurred when the valve or gap may be switched or plugged to permit the pressure effect of the vacuum pump to be exerted on the holding means.

A flow diagram generally illustrating a typical operation of an apparatus of the invention is shown in Figure 1. Referring to Figure 1 , the hollow, block arrows indicate the flow of material and/or mixture through an illustrative embodiment of the present invention. The solid line arrows indicate an interaction between two elements that does not also necessarily involve the flow of mixture to be tested between the two elements. Referring now to Figure 2, an illustrative embodiment is shown generally at 10. In this particular illustrative embodiment, the holding means is a sample cup 20. The sample cup 20 has a mouthpiece 22 and is in communication with a nebulizer compressor 24 (Pari ProNeb Ultra™

Nebulizer) via a conduction tube 26 which is attached to the sample cup 20 at a first holder opening 28. The nebulizer compressor 24 is operable, together with the conduction tube 26 and sample cup 20, to convert mixture in the sample cup 20, such as a nasal suspension for example, into a nebulized mixture. A nebulized mixture is often a fine mist and may be composed of drug particles and particles of other suspending reagent such as cellulose or other excipients.

A gap occurs between the outside edge of the mouthpiece 22 and the inside surface of an induction port 30. This gap is an illustrative pressure control means and is hidden from view in Figure 2 by the exterior of the induction port. Also hidden from view in Figure 2 is a second holder opening which is an opening in the mouthpiece 22. By inserting the mouthpiece 22 into the induction port 30 a reduction in the loss of nebulized mixture may be achieved while at the same time altering the negative pressure applied to the sample cup 20 via the induction port 30. By altering the negative pressure applied to the sample cup 20 via the induction port 30, it is meant that the pressure at the second holder opening is permitted to be higher than if no gap existed. When a gap is used as a pressure control means, this pressure alteration is achieved by permitting atmospheric pressure to enter the system via the gap. Such an alteration in pressure must be sufficient to permit at least a part of the mixture to nebulize in the holding means.

Once the nebulized mixture is formed in the sample cup 20, the nebulized mixture passes from the sample cup 20 into the communicating means, which in this illustrative embodiment is an induction port 30. The nebulized mixture passes through the induction port 30 into a multistage cascade impactor 32 (Anderson Multistage Cascade Impactor). The multistage cascade impactor 32 is an illustrative embodiment of a particle sizing means. The multistage cascade impactor 32 comprises a plurality of settling stages 34, the first sizing means opening (obscured by induction port 30) and second sizing means opening. The second sizing means opening is obscured by a rubber tube 40, which connects a vacuum pump 42 to the second sizing means opening. The purpose of the vacuum pump 42 is to provide a negative pressure inside the multistage cascade impactor 32 and the induction port 30 so as to draw the nebulized mixture into the multistage cascade impactor 32.

Inside each settling stage 34 of cascade impactor 32 there is a collection body. Illustrative examples of collection bodies are shown in Figure 3. Each collection body is designed so that it is operable to collect droplets having a particular average size and to permit particles having a smaller average size than the particular average size to pass through an opening in the collection body. The collection body collects particles of a particular average size and the collected material can then be analyzed to identify relative composition of the mixture based on particle size. The nebulized mixture deposits on the various collection bodies depending on the size of the nebulized particle of the nebulized mixture. Consequently, it is known that particles of a known average size are more likely to have deposited on one collection body rather than another. Based on this distribution of particle size on the collection bodies, the collected mixture on each collection body is able to be analyzed so as to determine the relative quantities of each component of the mixture in a particle of a given average size.

For example, if a nasal suspension is used as an illustrative example of a mixture, then the nasal suspension may be used 'as is' or suitably diluted using a suitable surfactant reagent. Such a dilution may enhance the deliverable amount to the particle sizing means. Selection of surfactants or suspending agent to be used for dilution and their preparation should be done with care in order to ensure the representative portion of the suspended drug particles to be nebulized. For example and without limitation, the diluent may be ionic or nonionic and may be selected depending on the nature of the material to be tested. The diluents preparation (i.e. concentration of the diluents) may be dependent on the nature of the material to be tested also. A person of skill in the art will understand the sorts of diluents and materials that are suitable for use with each other and those that are not. One to two grams of the undiluted or diluted nasal suspension may be placed into the sample cup 20 via a resealable opening 44. Once the diluted nasal suspension is in the sample cup 20, vacuum pump 42 may be turned on with a flow rate of about 26L/minute to about 30L/minute. Often the flow rate of the vacuum pump is about 26.9L/minute to about 29.7L/minute. In many circumstances the flow rate of the vacuum pump is about 28.3Uminute. The nebulizer compressor 24 may then be turned on with a flow rate of about 2L/minute to about 6L/minute. Often the nebulizing means is switched on with a flow rate of about 4L/minute to about 5 L/minute. Keep the vacuum pump 42 and nebulizer compressor 24 turned on continuously untill no more diluted nasal suspension is in the sample cup 20. During nebulisation, the sample cup 20 may need to be lightly tapped to aid uniform nebulisation. Once the diluted nasal suspension is no longer in the cup, the nebulizer compressor 24 and the vacuum pump 42 may be switched off.

Nebulisation conditions, including the set up of the apparatus, including the cascade impactor 32, the amount of mixture in the sample cup 20, flow control of nebulizer compressor 24, flow control of vacuum pump 42, nebulization time and sample cup handling procedure during nebulization should be carefully considered. For example, selecting a sample amount for inclusion in the holding means will affect a rate of nebulization, which in turn may affect the operation of the pressure control means. A person of skill in the art will be able to make the necessary calculations and adaptations to correctly operate the apparatus depending on the amount of time require for nebulization of a given amount of a given material in the holding means and other factors.

Certain materials may be able to be nebulized very quickly and/or easily and others may take a longer time and/or be harder to nebulize.

Apparatuses described herein are suitable for materials that nebulize quickly and/or easily as well as materials that nebulize slowly and/or more difficultly. Apparatuses described herein are particularly suitable for materials that take longer to nebulize or are more difficult to nebulize. In particular, those sorts of materials that are not normally nebulized quickly, such as suspensions and non-aerosol spray formulations (for example some nasal sprays, some oral suspensions, some ophthalmic suspensions, etc.), may be tested using apparatuses described herein to give consistent and accurate results. Such non-aerosol spray formulations are difficult, if not impossible, to test using previously known technology.

Once the nebulizer compressor 24 and the vacuum pump 42 are switched off, the cascade impactor 32 may be disassembled and separated from induction port 30 and rubber tube 40. Also separate sample cup 20 from induction port 30 and set sample cup 20 and induction port 30 aside with the collection bodies from each settling stage 34 from cascade impactor 32 for further analysis.

Using a suitable diluting solvent (could be the same or different from the solvent used to dilute the mixture in the first place) rinse sample cup 20 to a known volume and precipitate any cellulose by adding salt. Then filter off the precipitate or directly filter off cellulose by special filter. Keep the filtrate (liquid portion). Using chromatography or other suitable analytical techniques available to the person of skill in the art (e.g. selective HPLC), it is possible to identify the relative quantities of individual elements that remained in the sample cup 20. Repeat this analytical approach for each of the induction port 30 and collection bodies in each of the settling stages 34.

A statistical analysis may be performed on the results of the analytical approach used to identify the relative quantities of individual elements that appeared in the various sections of the illustrative apparatus of the invention. Such analyses are known to a person of skill in the art and include, for example USP methods including USP method <601 >. Such an analysis may yield information such as the deposited mass of the fine mist, the median aerodynamic diameter (MMAD) of the particles, the fine particle dose, the geometric standard deviation (GSD) of the particles, the relative material balance for particles having a particular size and/or for each size and other particle size distribution parameters. USP <601 > provides guidance to a person of skill in the art as to what such parameters may be measured and what such measurements may mean. It is possible to calculate, based on the detected deposited mass for each constituent part of the apparatus,

MMAD, GSD, a total % of particles of up to a particular desired size, including but not limited to a total % of particles of up to 5 microns in size that accumulate and/or a total % of particles of up to 10 microns in size that accumulate, material balance, quantity and composition of mixture delivered from the mouthpiece, quantity and composition of the respirable fraction (e.g. those portions collected by the collection bodies that correspond to a particular size of particle that would be respired by a typical subject) as well as the fine particle dose.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. Furthermore, numeric ranges are provided so that the range of values is recited in addition to the individual values within the recited range being specifically recited in the absence of the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing.

Citation of references herein is not an admission that such references are prior art to the present invention. Furthermore, material appearing in the background section of the specification is not an admission that such material is prior art to the invention. Any priority document(s) are incorporated herein by reference as if each individual priority document were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.