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Title:
MICRONEEDLE ARRAYS AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2019/227156
Kind Code:
A1
Abstract:
The present invention relates to microneedle arrays fabricated from materials that melt at or near body temperature to facilitate the delivery of agents (e.g. pharmaceuticals or cosmetics). Methods for the production of the microneedle arrays and their use in the delivery of agents into tissue are provided herein.

Inventors:
KANG LIFENG (AU)
JAMALUDIN MUHAMMAD NASHRUDIN BIN (AU)
Application Number:
PCT/AU2019/050539
Publication Date:
December 05, 2019
Filing Date:
May 30, 2019
Export Citation:
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Assignee:
UNIV SYDNEY (AU)
International Classes:
A61K47/10; A61K9/00; A61K31/352; A61K47/14; A61K47/26; A61P11/06; A61P27/10
Domestic Patent References:
WO2011071287A22011-06-16
Foreign References:
US20170189660A12017-07-06
US20110150765A12011-06-23
CN107875115A2018-04-06
JP2009254756A2009-11-05
Attorney, Agent or Firm:
SPRUSON & FERGUSON (AU)
Download PDF:
Claims:
Claims

1. A microneedle array comprising a plurality of microneedles capable of piercing tissue of a subject, wherein the microneedles are:

fabricated from a matrix material that exists in a solid state at temperatures below that of the tissue, and

capable of transitioning into a liquid state in a temperature-dependent manner at or above the temperature of the tissue.

2. The microneedle array according to claim 1, wherein the tissue is skin, a mucosal membrane, or muscle.

3. The microneedle array according to claim 1 or claim 2, wherein the matrix material:

(i) has a melting temperature point that is: less than l5°C, less than lO°C, less than 7°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than l°C, below the temperature of the tissue; and/or

(ii) has a melting temperature point that does not exceed the temperature of the tissue; and/or

(iii) has a melting temperature point above 30°C, above 35°C above 36°C, above 37°C, above 38°C, or above 39°C; and/or

(iv) exists in a solid state at or below a temperature of 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 37.5°C, or 38°C.

4. The microneedle array according to any one of claims 1 to 3, wherein the matrix material comprises any one or more of:

(i) substituted cyclohexanol-compounds and/or fatty acids;

(ii) substituted cyclohexanol-compounds selected from the group consisting of:

5-methyl-2-propan-2-ylcyclohexan- l-ol (menthol), 2-chlorocylohexanol, 2,6-dimethylcyclohexanol, 1 -ethylcyclohexanol, 2-phenylcyclohexanol, 3,3,5-trimethylcyclohexanol, and mixtures thereof.;

(iii) fatty acids comprising monoglycerides, diglycerides, triglycerides or mixtures thereof;

(iv) Witepsol H15 or menthol.

5. The microneedle array according to any one of claims 1 to 4, wherein the matrix material constitutes at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 95 wt%, or 100 wt% of microneedles within the array.

6. The microneedle array according to any one of claims 1 to 5, wherein the plurality of microneedles comprise one or more agents for delivery into tissue of a subject.

7. The microneedle array according to any one of claims 1 to 6, wherein the matrix material includes one or more agents for delivery into the tissue of the subject.

8. The microneedle array according to claim 6 or claim 7, wherein the agent for delivery is coated on an external surface of microneedles in the array.

9. The microneedle array according to any one of claims 6 to 8, wherein the agent for delivery is intermixed with the matrix material, and constitutes at least 0.5 wt%, at least 1 wt%, at least 2.5 wt%, at least 5 wt%, at least 7.5 wt%, at least 10 wt%, at least 12.5 wt%, at least 15.5 wt%, at least 17.5 wt%, or at least 20 wt%, of microneedles within the array.

10. The microneedle array according to any one of claims 6 to 9, wherein the agent for delivery is:

(i) a biological agent, therapeutic agent, diagnostic agent, or cosmetic agent; and/or

(ii) cromolyn or a salt thereof, or hydrochloride monohydrate.

11. The microneedle array according to any one of claims 6 to 10, wherein the one or more agents are retained in and/or on the microneedles:

(i) until the microneedles transition into said liquid state; and/or

(ii) at all temperatures more than l°C, 2°C, 3°C, 4°C 5°C, 6°C, 7°C, 8°C, 9°C, or l0°C under the temperature of the tissue to which the microneedles are to be applied until the microneedles transition into said liquid state.

12. The microneedle array according to any one of claims 1 to 11, wherein at least: 50%, 60%, 70%, 80%, 90%, 95%, of microneedles of the array, or all microneedles of the array, do not contain an internal cavity and/or does not contain a hole from which agent can be delivered into the tissue.

13. The microneedle array according to any one of claims 1 to 12, further comprising a bioadhesive film for application of the array to the tissue.

14. The microneedle array according to any one of claims 1 to 13, wherein the subject is:

(i) a mammalian or avian species subject; or

(ii) a human, a dog, a cat, a cow, a sheep, a pig, a horse, or poultry.

15. A method for delivering an agent into a tissue of a subject, the method comprising piercing the tissue with the microneedle array according to any one of claims 1 to 14, wherein the microneedles of the array comprise the agent and upon piercing the tissue the microneedles transition from a solid state to a liquid state in a temperature- dependent manner, thereby releasing the agent into the tissue of the subject.

Description:
Microneedle Arrays and Uses Thereof

Incorporation by Cross-Reference

The present application claims priority from Australian provisional patent application number 2018901919 filed on 30 May 3018, the entire contents of which are incorporated herein by cross-reference.

Technical Field

The present invention relates generally to the field of pharmaceutical sciences, and more specifically to microneedle arrays fabricated from materials that melt at or near body temperature to facilitate the delivery of agents (e.g. pharmaceuticals or cosmetics). Methods for the production of the microneedle arrays and their use in the delivery of agents into tissue are provided herein.

Background

The skin is a primary barrier defence protecting the body from penetration by extraneous substances. Consequently, it is often challenging to efficiently deliver agents through the skin and into the internal environment. Microneedle arrays are minimally invasive and painless devices that create micro-sized transport pathways to allow molecules of varied dimensions to penetrate through the skin. From there the molecules can enter into microcirculation and systemic delivery can be achieved by the transdermal route. Current microneedle arrays are fabricated from synthetic materials such as silicon, metals and polymer compounds. The dimensions and fabrication of microneedle arrays is important for achieving delivery of agents into tissue since microneedle arrays that are too short or narrow may stimulate dermal nerves or puncture dermal blood vessels.

Microneedle arrays can be pre-coated with an agent prior to insertion into tissue, or an agent-loaded patch can be applied to the tissue site following the application of the microneedle array. The former, involves delivery of agents via the“coat and poke” method and has been employed for the delivery of agents such as vaccines, proteins, peptides and DNA into the skin. The limitation of such an approach is the amount of drug that can be coated onto the finite surface of the microneedles.

Some of the first microneedle arrays for drug delivery were based on silicon. This was due at least in part to silicon being a favourable material in manufacturing. However, silicon-based microneedle arrays are of higher cost, and involve multi-stage, labour intensive fabrication. Typically, silicon based microneedle arrays deliver their agent via the “poke and patch” approach, wherein the microneedle array is firstly used to penetrate the skin followed by a patch containing the agent of interest being applied. Alternatively, silicon based microneedle arrays have been coated with agents and then applied to the skin to facilitate delivery of the agent. Recently, there have been some health concerns with silicon-based microneedle arrays due to brittleness and reports of microneedles fracturing within skin leading to granulomas.

Microneedle arrays may also be manufactured from materials that degrade by enzymatic means upon contact with, for example, interstitial fluid to thereby release the agent from the microneedles. The drug delivery rate of these microneedle arrays is dependent on the material used to fabricate the microneedles and the rate at which it can be degraded by the enzyme. Polymeric compounds have been used to fabricate microneedles reliant on enzymatic degradation for agent delivery into tissue, but many involve heating of the polymers to high temperatures in order to facilitate fabrication and moulding. This in turn makes the addition of agents to the material at the time of moulding difficult, and sometimes degrades the agent for delivery or renders it inactive. Additionally, the high temperatures required in this moulding process produce“hot-melts” which are highly viscous and resistant to flow making moulding very labour intensive. Microneedle arrays reliant on enzymatic degradation can also be made from carbohydrates, however, carbohydrates react with many agents commonly used for delivery by the microneedles. Additionally, during the agent release process partially-dissolved sugar can act to seal the microneedle-induced holes. Furthermore, complex storage procedures are necessary for many of the polymeric- or carbohydrate -based microneedle arrays, with humidity and storage for longer periods a recurring concern.

Microneedle arrays made from common sugars such as maltose, trehalose, sucrose, mannitol, xylitol and galactose have been described. However, microneedle arrays fabricated from sugars such as maltose require thermal treatment of powdered forms at elevated temperatures (e.g. 140 degrees) over extended time periods. Following this, the agent for delivery may be added and the mixture casted into a micromould. This excludes many agents that require lower temperatures during microneedle fabrication to maintain their conformation/integrity (e.g. many protein based vaccines), and the high viscosity of the hot-melts can cause a loss of the loaded active pharmaceutical ingredient (API) . Further, these microneedle arrays usually need to be stored at controlled humidity levels (less than 50%) and stable temperatures.

It is evident that microneedle arrays present in the art are associated with various drawbacks including any one or more of complex and/or costly fabrication techniques, incompatibility of fabrication techniques with agents for delivery, and/or instability during storage. Other key issues include whether the physiochemical properties of the matrix materials used are suitable for straightforward fabrication, whether the microneedle array once fabricated is mechanically strong enough to pierce the target tissue (e.g. skin), and how effectively the microneedle array is able to deliver agents into tissue during use.

A need exists for microneedle arrays capable of convenient and/or effective delivery of agents into tissue. Microneedle arrays generated by cost-effective and/or simplified fabrication processes are also desirable.

Summary of the Invention

The present invention alleviates at least one of the shortcomings of existing microneedle arrays and/or methods for their production.

The microneedle arrays of the present invention are fabricated using temperature- sensitive matrix materials facilitating the efficient delivery of agents into tissue via a temperature-dependent mechanism. The microneedle arrays of the present invention can provide any one or more advantages over prior art microneedle arrays, including but not limited to: (i) simplified fabrication using non-laborious techniques, (ii) stability at room temperature and/or at temperatures above room temperature, (iii) mechanical strength to enable penetration of tissues (e.g. skin) to facilitate delivery of agents, (iv) an efficient delivery mechanism relying on the temperature-sensitive disintegration of the microneedle material at the temperature of the tissue to facilitate delivery of the agent, (v) the absence of a requirement for enzymes and/or solvent to facilitate delivery of the agent from the microneedles into the tissue.

By way of non-limiting example, the present invention relates at least in part to the following embodiments:

Embodiment 1. A microneedle array comprising a plurality of microneedles capable of piercing tissue of a subject, wherein the microneedles are: fabricated from a matrix material that exists in a solid state at temperatures below that of the tissue, and

capable of transitioning into a liquid state in a temperature-dependent manner at or above the temperature of the tissue.

Embodiment 2. The microneedle array according to embodiment 1 , wherein the tissue is skin or muscle.

Embodiment 3. The microneedle array according to embodiment 1 or embodiment 2, wherein the matrix material has a melting temperature point that is: less than l5°C, less than lO°C, less than 7°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than l°C, below the temperature of the tissue.

Embodiment 4. The microneedle array according to any one of embodiments 1 to 3, wherein the matrix material has a melting temperature point that does not exceed the temperature of the tissue.

Embodiment 5. The microneedle array according to any one of embodiments 1 to 4, wherein the matrix material has a melting temperature point above 30°C, above 35°C above 36°C, above 37°C, above 38°C, or above 39°C.

Embodiment 6. The microneedle array according to any one of embodiments 1 to 5, wherein the matrix material exists in a solid state at or below a temperature of 20°C, 25 °C, 30°C, 35°C, 36°C, 37°C, 37.5°C, or 38°C.

Embodiment 7. The microneedle array according to any one of embodiments 1 to 6, wherein the matrix material comprises substituted cyclohexanol-compounds and/or fatty acids.

Embodiment 8. The microneedle array according to embodiment 7, wherein the substituted cyclohexanol-compounds are selected from the group consisting of: 5-methyl- 2-propan-2-ylcyclohexan-l-ol (menthol), 2-chlorocylohexanol, 2,6-dimethylcyclohexanol, l-ethylcyclohexanol, 2-phenylcyclohexanol, 3,3,5-trimethylcyclohexanol, and mixtures thereof.

Embodiment 9. The microneedle array according to embodiment 7 or embodiment 8, wherein the fatty acids comprise monoglycerides, diglycerides, triglycerides or mixtures thereof.

Embodiment 10. The microneedle array according to any one of embodiments 7 to 9, wherein the matrix material comprises Witepsol H15 or menthol.

Embodiment 11. The microneedle array according to any one of embodiments 1 to 10, wherein the matrix material constitutes at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 95 wt%, or 100 wt% of microneedles within the array.

Embodiment 12. The microneedle array according to any one of embodiments 1 to

11, wherein the plurality of microneedles comprise one or more agents for delivery into tissue of a subject.

Embodiment 13. The microneedle array according to any one of embodiments 1 to

12, wherein the matrix material includes one or more agents for delivery into the tissue of the subject.

Embodiment 14. The microneedle array according to embodiment 13, wherein the agent for delivery is coated on an external surface of microneedles in the array.

Embodiment 15. The microneedle array according to embodiment 13, wherein the agent for delivery is intermixed with the matrix material, and constitutes at least 0.5 wt%, at least 1 wt%, at least 2.5 wt%, at least 5 wt%, at least 7.5 wt%, at least 10 wt%, at least 12.5 wt%, at least 15.5 wt%, at least 17.5 wt%, or at least 20 wt%, of microneedles within the array.

Embodiment 16. The microneedle array according to any one of embodiments 12 to 15, wherein the agent for delivery is a biological agent, therapeutic agent, diagnostic agent, or cosmetic agent.

Embodiment 17. The microneedle array according to embodiment 16, wherein the agent for delivery is cromolyn or a salt thereof, or hydrochloride monohydrate.

Embodiment 18. The microneedle array according to any one of embodiments 1 to

17, wherein the subject is a mammalian or avian species subject.

Embodiment 19. The microneedle array according to any one of embodiments 1 to

18, wherein the subject is a human, a dog, a cat, a cow, a sheep, a pig, a horse, or poultry.

Embodiment 20. The microneedle array according to any one of embodiments 1 to

19, further comprising a bioadhesive film for application of the array to the tissue.

Embodiment 21. The microneedle array according to any one of embodiments 12 to

20, wherein the one or more agents are retained in and/or on the microneedles until the microneedles transition into said liquid state.

Embodiment 22. The microneedle array according to any one of embodiments 12 to

21, wherein the one or more agents are retained in and/or on the microneedles at all temperatures more than l°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or l0°C under the temperature of the tissue to which the microneedles are to be applied. Embodiment 23: The microneedle array according to any one of embodiments 1 to

22, wherein at least: 50%, 60%, 70%, 80%, 90%, 95%, of microneedles of the array, or all microneedles of the array, do not contain an internal cavity.

Embodiment 24: The microneedle array according to any one of embodiments 1 to

23, wherein at least: 50%, 60%, 70%, 80%, 90%, 95%, of microneedles of the array, or all microneedles of the array, do not contain a hole from which agent can be delivered into the tissue.

Embodiment 25. A method for manufacturing the microneedle array of any one of embodiments 1 to 24, the method comprising:

I) providing the matrix material in liquid form at a temperature above its melting point;

II) pouring the liquid matrix material into cavities of a microneedle array mould;

III) solidifying the liquid matrix material poured into the cavities by reducing the temperature of the liquid matrix material to a level that is below its melting temperature, thereby forming the microneedle array; and

IV) removing the microneedle array from the mould.

Embodiment 26. The method according to embodiment 25, further comprising compressing the liquid matrix material as it solidifies in the cavities and pouring additional liquefied matrix material into the cavity following the compression.

Embodiment 27. The method according to embodiment 25 or embodiment 26, wherein the microneedle array mould comprises or consists of polydimethylsiloxane (PDMS).

Embodiment 28. A method for delivering an agent into a tissue of a subject, the method comprising piercing the tissue with the microneedle array according to any one of embodiments 1 to 24, wherein the microneedles of the array comprise the agent and upon piercing the tissue the microneedles transition from a solid state to a liquid state in a temperature-dependent manner, thereby releasing the agent into the tissue of the subject.

Embodiment 29. Use of a microneedle array of any one of embodiments 1 to 24 for the delivery of an agent into the tissue of a subject.

Embodiment 30. The method of embodiment 28 or the use of embodiment 29, wherein the agent is intermixed with matrix material of microneedles in the array.

Embodiment 31. The method of embodiment 28 or embodiment 30, or the use of embodiment 29 or embodiment 30, wherein the tissue is skin or muscle, and the subject is mammalian or avian. Embodiment 32 /The method of any one of embodiments 28, 30 or 31, or the use of any one of embodiments 29 to 31, wherein the agent to is retained in and/or on the microneedles until the microneedles transition into said liquid state.

Embodiment 33. The method of any one of embodiments 28 or 30 to 32, or the use of any one of embodiments 29 to 32, wherein the agent is retained in and/or on the microneedles at all temperatures more than l°C, 2°C, 3°C, 4°C , 5°C, 6°C, 7°C, 8°C, 9°C, or l0°C under the temperature of the tissue to which the microneedles are to be applied.

Embodiment 34: The method of any one of embodiments 28 or 30 to 33, or the use of any one of embodiments 29 to 33, wherein at least: 50%, 60%, 70%, 80%, 90%, 95%, of microneedles of the array, or all microneedles of the array, do not contain an internal cavity.

Embodiment 35: The method of any one of embodiments 28 or 30 to 34, or the use of any one of embodiments 29 to 34, wherein at least: 50%, 60%, 70%, 80%, 90%, 95%, of microneedles of the array, or all microneedles of the array, do not contain a hole from which agent can be delivered into the tissue.

Definitions

As used in this application, the singular form“a”,“an” and“the” include plural references unless the context clearly dictates otherwise. For example, the term “microneedle” also includes a plurality of microneedles.

As used herein, the term“comprising” means“including.” Variations of the word “comprising”, such as “comprise” and “comprises,” have correspondingly varied meanings. Thus, for example, a microneedle“comprising” a given agent‘A’ may contain only agent A or may contain one or more additional agents (e.g. agent B, agent C and so on).

As used herein, the term“subject” includes any animal of economic, social or research importance including bovine, equine, ovine, primate, avian and rodent species. Hence, a“subject” may be a mammal such as, for example, a human or a non -human mammal.

As used herein, the term“about” when used in reference to a recited numerical value includes the recited numerical value and numerical values within plus or minus ten percent of the recited value. As used herein, the term“between” when used in reference to a range of numerical values encompasses the numerical values at each endpoint of the range. For example, a temperature of between 30°C and 35°C is inclusive of a temperature of 30°C and a temperature of 35°C.

Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art.

For the purposes of description, all documents referred to herein are hereby incorporated by reference in their entirety unless otherwise stated.

Brief Description of the Figures

Preferred embodiments of the present invention will now be described by way of example only, with reference to the accompanying figures wherein:

Figure 1 depicts a schematic of the steps employed in fabricating microneedle arrays according to embodiments of the present invention, using an exemplary polydimethylsiloxane (PDMS) mould and menthol as the microneedle array matrix material. A: Material. B: Material melted at 65°C. C: Drug. D: Filled mould placed onto heating plate (hot-plate method). E: Filled mould allowed to rest at room temperature to solidify. F: Mixture compressed into cavity using spatula (compression method). G: Demoulded MN array.

Figure 2 provides images of droplet solutions of water (Figure 2A, left) and menthol (Figure 2A, right) and water (Figure 2B, left) with Witepsol H15 (Figure 2B, right) applied to the surface of a PDMS mould. The contact angles of the various solutions with the mould surface are contrasted with both menthol and Witepsol H15 showing lower contact angles than water;

Figure 3 shows images of exemplary microneedle arrays according to embodiments of the present invention fabricated from mixtures of matrix material and agents for delivery by the microneedle array into tissue. Figure 3A shows a mixture of menthol matrix material with eflomithine hydrochloride monohydrate at a 15 wt% concentration. Figure 3B shows a mixture of menthol matrix material with cromolyn sodium at a 10 wt% concentration (bottom);

Figure 4 depicts images of erythema reaction on human skin following the addition of menthol based microneedle arrays according to embodiments of the present invention. - Si -

Fabricated microneedle arrays before application (Figure 4A) and after application on the skin (Figure 4B) are shown. The microneedle arrays are applied to human skin, where (Figure 4C) depicts the skin before the application and (Figure 4D) shows skin during application of the microneedle array. The skin is monitored for the appearance of erythema reaction at 0, 5, 20 and 60 minutes after removal of the microneedle array (Figure 4E, Figure 4F, Figure 4G and Figure 4H, respectively); and

Figure 5 shows an image of pre-treated pig skin following application of the menthol microneedle arrays according to embodiments of the present invention. Sections of pig skin (4 cm x 4 cm) pretreated with trypan blue were photographed prior to (left) and following application (right) of the menthol based microneedle array, demonstrating that the microneedle array had sufficient mechanical strength to penetrate the epidermis of the pig skin.

Figure 6 is a graph showing recovery rates of cromolyn from microneedle arrays of the present invention.

Figure 7 is a graph showing cromolyn content of microneedle arrays of the present invention.

Figure 8 is a schematic representation of an in vitro skin permeation study using Franz diffusion cells according to prophetic Example 5. A: Infusion pump system fitted with a syringe containing receptor solution. B: Water bath. C: Sampling tube.

Detailed Description

The present invention provides microneedle arrays fabricated from temperature- sensitive matrix materials combined with deliverable agents, wherein the microneedle arrays are capable of facilitating delivery of the agents into tissue in a temperature- dependent manner. To achieve this, the microneedle arrays of the present invention are fabricated using temperature- sensitive matrix materials which disintegrate at the temperature of the target tissue, thereby delivering the agents.

The microneedle arrays described herein can facilitate the delivery of agents into tissues in a manner that is superior to equivalent known microneedle arrays due to factors including any one or more of: (i) the relative simplicity and accessibility of the fabrication method used; (ii) the temperature- sensitive matrix materials used which allow for simplified fabrication via moulding; (iii) reduced temperature for fabrication via moulding providing a lower propensity to damage deliverable agents incorporated in the microneedles; (iv) the microneedle arrays have sufficient strength to penetrate the skin and deliver the agents in a temperature-dependent mechanism; (v) the microneedle arrays do not have a requirement for enzymes or solvents to enable delivery once they have penetrated the skin, unlike soluble or polymer based microneedle arrays.

The prevent invention thus relates, among other things, to microneedle arrays fabricated using temperature- sensitive matrix materials, methods for their use, and methods for their fabrication.

The various features of the invention described below should not be considered limiting unless the context clearly indicates that to be the case.

Microneedle arrays

The present invention provides microneedle arrays for delivering agents into tissue which are fabricated from temperature-sensitive substances. The temperature-sensitive substances may be bio-compatible materials available widely in pharmacy

Without any particular limitation, the microneedle arrays of the present invention may have properties including biocompatibility (e.g. non-toxic), being biodegradable, having sufficient mechanical strength to penetrate tissues such as skin or mucosal membranes, and being compatible with a variety of agents for delivery during fabrication processes.

Individual microneedles of the microneedle arrays are fabricated with temperature- sensitive matrix materials. These matrix materials are temperature-sensitive insofar as they remain solid below a certain target temperature at which they disintegrate (i.e. melt) in response to a threshold level of heat. While there is no specific limitation as to the threshold temperature at which the microneedles disintegrate, in some embodiments the threshold temperature is above ambient temperature. For example, the temperature at which the microneedles disintegrate may be at or above 25°C, at or above 27.5°C, at or above 30°C, at or above 32°C, at or above 33°C, at or above 34°C, at or above 35°C, at or above 36°C, at or above 36.5°C, at or above 37°C, at or above 37.5°C, at or above 38°C, at or above 38.5°C, at or above 39°C, at or above 39.5°C, or at or above 40°C.

In some embodiments, the temperature at which the microneedles disintegrate may be between 25°C and 36°C, between 25°C and 36.5°C, between 25°C and 37°C, between 25°C and 37.5°C, between 25°C and 38°C, between 25°C and 38.5°C, between 25°C and 39°C, between 25°C and 39.5°C, between 25°C and 40°C, between 30°C and 36°C, between 30°C and 36.5°C, between 30°C and 37°C, between 30°C and 37.5°C, between 30°C and 38°C, between 30°C and 38.5°C, between 30°C and 39°C, between 30°C and 39.5°C, between 30°C and 40°C, between 32.5°C and 36°C, between 32.5°C and 36.5°C, between 32.5°C and 37°C, between 32.5°C and 37.5°C, between 32.5°C and 38°C, between 32.5°C and 38.5°C, between 32.5°C and 39°C, between 32.5°C and 39.5°C, between 32.5°C and 40°C, between 35°C and 36°C, between 35°C and 36.5°C, between 35°C and 37°C, between 35°C and 37.5°C, between 35°C and 38°C, between 35°C and 38.5°C, between 35°C and 39°C, between 35°C and 39.5°C, or between 35°C and 40°C.

The skilled addressee will recognise that the specific temperature at which the matrix material used to fabricate the microneedles disintegrates is not limited to those outlined above which are exemplary only, and that a specific temperature or temperature range can be targeted by selecting an appropriate matrix material or mixture thereof to fabricate the microneedles.

In some embodiments, the microneedles are fabricated from a mixture of different matrix materials. Additionally or alternatively, individual microneedles within the microneedle array may be fabricated from different matrix materials. Microneedles made from different matrix materials may disintegrate at the same, similar or different temperatures.

The temperature- sensitive microneedles of the present invention can be included in any microneedle array format known in the art. Non-limiting examples include those described by Larraneta el al. (Materials Science and Engineering: R: Reports Volume 104, June 2016, Pages 1-32), van der Maasden et al. (Drug Deliv Transl Res. 2015; 5(4): 397- 406), Ogundele and Okafor (Current trends in Biomedical Engineering & Biosciences, Volume 10 Issue 1 - October 2017) and Donnelly etal (Drug Deliv. 2010 May ; 17(4): 187- 207).

By way of non-limiting example only, the microneedles of the arrays may be provided at various lengths as suitable for application to the target tissue in question (e.g. any length between 50 pm and 900 pm, such as between 100 pm and 800 pm in length, between 200 pm and 700 pm in length, between 300 pm and 500 pm in length, about 100 pm length, about 200 pm length, about 250 pm length, about 300 pm length, about 350 pm length or about 400 pm length).

The base diameter of individual microneedles may be of any suitable diameter, such as, for example, between 100 pm base diameter and 500 pm base diameter, between 200 pm base diameter and 400 pm base diameter, between 200 pm base diameter and 200 pm base diameter, about 200 pm base diameter, 250 pm base diameter or about 300 pm base diameter. In one non-limiting embodiment, a microneedle array of the present invention may be fabricated using a suitable mould (e.g. a polydimethylsiloxane (PDMS) mould of suitable dimensions (e.g. about 15 x 15 microneedles/cm 2 , about 750 mhi height, about 250 mhi base diameter, and a base up to 2000 cm 2 ).

The microneedles of the arrays will generally possess mechanical characteristics facilitating their intended function of piercing tissue (e.g. skin) to which they are applied, with minimal breakage or without breakage. Accordingly, the microneedles may have any one or more of sufficient: yield strength, tensile strength, fatigue strength, crack resistance, and/or other characteristics, allowing them to pierce a tissue to which they are applied. For example, the fracture force (N) of the microneedles may be approximately 100 N, as compared to the force of a thumb (approximately 10 N). In some embodiments, the fracture force of the microneedles may be at least: 40 N, 50 N, 60 N, 70 N, 80 N, 90 N or 100 N.

The microneedles of the arrays may be fabricated without any internal cavity and/or hole/s.

The microneedles of the arrays described herein may be fabricated from a variety of matrix materials or mixtures of matrix materials. The matrix materials selected may be based on properties including mechanical strength and the capacity to pierce target tissues without breakage (or without substantial breakage), biological compatibility (e.g. toxicity), and/or ease of fabrication and/or storage. A primary consideration in selecting the matrix material/s used to fabricate the microneedles will be the temperature at which the matrix material/s disintegrate (i.e. melt) and consequently change from solid to liquid form. This in turn will depend at least in part on the tissue to which the microneedles are intended for application.

The skilled addressee is capable of testing the relevant characteristics of matrix materials potentially suitable to fabricate into the microneedle arrays of the present invention.

By way of non-limiting example only, the microneedles may be fabricated using substituted cyclohexanol-compound/s. Without limitation, such compounds may include menthol (5-methyl-2-propan-2-ylcyclohexan-l-ol), 2-chlorocylohexanol, 2,6- dimethylcyclohexanol, l-ethylcyclohexanol, 2-phenylcyclohexanol, and 3,3,5- trimethylcyclohexanol.

In some embodiments the microneedles are fabricated from menthol (5-methyl-2- propan-2-ylcyclohexan-l-ol; CioFhoO). Any menthol stereoisomer or mixture of menthol stereoisomers may be used (i.e. any one or more of: (+)-Menthol, (-)-Mentho!, (+)- Isomenthol, (-)-Isomenthol, (+)-Neomenthol, ( ) Neomenthol, (-h)-Neoisomenthol, (-)- Neoisomenthol). The menthol may be synthetic and/or isolated from natural sources (e.g. mint oils, peppermint and the like). By way of further non-limiting example, the microneedles may be fabricated from fatty acids or mixtures thereof. For example, the microneedles may be fabricated from monoglycerides, diglycerides, triglycerides or mixtures thereof. In some embodiments, the microneedles may be fabricated from a mixture of monoglycerides, diglycerides, and triglycerides. In some embodiments, the microneedles may be fabricated from Witespol ® , such as any one or more of Witespol H 12 ® , Witespol H 15 ® , Witespol H 19 ® , Witespol H 32 ® , Witespol H 35 ® , Witespol H 37 ® , Witespol W 25 ® , Witespol W 31 ® , Witespol W 32 ® , Witespol W 35 ® , Witespol W 45 ® , Witespol S 51 ® , Witespol S 55 ® , Witespol S 58 ® , Witespol S 75 ® , Witespol S 76 ® , Witespol S 85 ® . In still other embodiments the microneedles may be fabricated from camphor or related materials.

According to certain embodiments of the present invention, the microneedles may be fabricated using a first matrix material having a higher melting temperature than is desirable for application to a given target tissue to which the array is intended for application. For example, the first matrix material may have a melting temperature significantly above a target tissue to which the microneedles will be applied. The first matrix material may be combined with a second matrix material to form a mixture with an overall lower melting temperature than the first matrix material and/or the second matrix material. The second matrix material of the mixture may, for example, be a substituted cyclohexanol-compound selected from menthol (5-Methyl-2-(propan-2-yl)cyclohexan-l-ol), 2-chlorocylohexanol, 2,6-dimethylcyclohexanol, l-ethylcyclohexanol, 2-phenylcyclohexanol, and 3,3,5- trimethylcyclohexanol.

It is generally preferred that any temperature- sensitive substance utilised in the microneedle arrays is non-toxic to the subject, and/or capable of clearance from the body of the subject over time.

In some embodiments, temperature- sensitive substances utilised to make the microneedle arrays may have therapeutic properties.

Microneedle arrays according to the present invention are provided for the purpose of delivering one or more agent/s into tissue.

The agent/s to be delivered can be mixed with the matrix material/s used to fabricate the microneedles, such that the microneedles are fabricated from a mixture of the matrix material/s and agent.

Additionally or alternatively, the agent/s to be delivered can be maintained in an internal reservoir or cavity within the fabricated microneedles.

Additionally or alternatively, the agent/s to be delivered may be coated onto the external surface of the fabricated microneedles. The outer coating may be antiseptic, assist in facilitating entry into the skin, act as an analgesic, and/or reinforce the tensile strength of the microneedle.

Regardless of how the agent/s is/are associated with the microneedles, temperature- dependent disintegration of the microneedles upon piercing the tissue may facilitate release of the agent.

Accordingly, the agent/s to be delivered may be retained in and/or on the microneedles until the microneedles disintegrate (i.e. melt) in response to a threshold level of heat (e.g. a threshold temperature arising from application of the microneedles to a tissue).

By way of non-limiting example, the agent/s may be retained in and/or on the microneedles at all temperatures below the temperature of the tissue to which the microneedles are to be applied.

In some embodiments, the agent/s may be retained in and/or on the microneedles at all temperatures more than l°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or lO°C under the temperature of the tissue to which the microneedles are to be applied.

By way of non-limiting example, exposure of the microneedles to a temperature: at or above 25°C, at or above 27.5°C, at or above 30°C, at or above 32°C, at or above 33°C, at or above 34°C, at or above 35°C, at or above 36°C, at or above 36.5°C, at or above 37°C, at or above 37.5°C, at or above 38°C, at or above 38.5°C, at or above 39°C, at or above 39.5°C, or at or above 40°C, causes release of the agent/s from the microneedles.

In some embodiments, exposure of the microneedles to a temperature: between 25°C and 36°C, between 25°C and 36.5°C, between 25°C and 37°C, between 25°C and 37.5°C, between 25°C and 38°C, between 25°C and 38.5°C, between 25°C and 39°C, between 25°C and 39.5°C, between 25°C and 40°C, between 30°C and 36°C, between 30°C and 36.5°C, between 30°C and 37°C, between 30°C and 37.5°C, between 30°C and 38°C, between 30°C and 38.5°C, between 30°C and 39°C, between 30°C and 39.5°C, between 30°C and 40°C, between 32.5°C and 36°C, between 32.5°C and 36.5°C, between 32.5°C and 37°C, between 32.5°C and 37.5°C, between 32.5°C and 38°C, between 32.5°C and 38.5°C, between 32.5°C and 39°C, between 32.5°C and 39.5°C, between 32.5°C and 40°C, between 35°C and 36°C, between 35°C and 36.5°C, between 35°C and 37°C, between 35°C and 37.5°C, between 35°C and 38°C, between 35°C and 38.5°C, between 35°C and 39°C, between 35°C and 39.5°C, or between 35°C and 40°C, causes release of the agent/s from the microneedles.

There is no particular limitation placed on the agents that may be delivered using microneedle arrays of the present invention. The agents may, for example, be biological agents, therapeutic agents, diagnostic agents, cosmetic agents, and the like. Non-limiting examples of agents exemplified in the Examples of the present application include eflomithine hydrochloride monohydrate, and cromolyn (e.g. cromolyn sodium). The skilled address will recognise that the microneedle arrays of the present invention can be fabricated to include any one or more of a wide variety of different agents. The microneedles of the arrays may comprise stabilising substance/s which facilitate stability of the agent for delivery by the array, which facilitate stability of the agent for delivery post-delivery into the tissue, and/or which facilitate stability of the agent for delivery during fabrication.

The skilled addressee will recognise that the various exemplary embodiments of the microneedle arrays discussed above are not intended to be limiting, and that other alternative forms of the temperature-dependent microneedle array devices provided herein are also within the scope of the invention as described.

Methods for Fabricating Microneedle arrays

The present invention provides methods for fabricating the microneedle arrays described herein. In general, there is no particular limitation on the fabrication method utilised which may be selected at least in part on the basis of the matrix material/s to be used in the fabrication method and/or the agents to be delivered by the microneedle array.

By way of non-limiting example, the microneedle arrays may be fabricated using any one or more of casting, hot embossing, injection moulding, investment casting, drawing lithography, laser micromachining, particle replication in non-wetting templates (PRINT), X-ray based methods, compression methods and hot plate methods. Non-limiting examples of suitable techniques include those described by Larraneta el al Materials Science and Engineering: R: Reports Volume 104, June 2016, Pages 1-32) and Perry et al. (Acc Chem Res. 2011 Oct 18;44(10):990-998).

In some embodiments, a compression fabrication method may be used to produce microneedle arrays of the present invention. For example, a master mould made from a suitable material (e.g. a negative polydimethylsiloxane (PDMS) master mould) can be heated to an appropriate temperature above the melting temperature of the matrix material to be used to fabricate the microneedles. The matrix material to be used to fabricate the microneedles may also be heated to a temperature above its melting point so as to provide it in liquid form, and it may then be poured into the mould cavities. The melted matrix material poured into the cavity may be compressed by any appropriate means, and further melted matrix material then poured into the cavities. Further compression/pouring cycles may be carried out until the cavities are full of compressed matrix material. The compressed matrix material may be allowed to solidify by reducing its temperature to a level below that of its melting point. The solidified matrix material may then be removed from the mould, providing the microneedle array. Agent/s to be delivered by the microneedle arrays can be mixed in with the liquid form of the matrix material used to fabricate the microneedles at any time prior to that matrix material solidifying in the mould. Additionally or alternatively, agents to be delivered by the microneedles may be encapsulated within the solidified matrix material used for fabrication (e.g. in a cavity). Additionally or alternatively, agents to be delivered by the microneedles may be coated on the external surface of the microneedles.

In other embodiments, the microneedle arrays may be fabricated using a hot plate method employing the same principles as the compression method, without compression upon pouring the liquefied matrix material into the cavities of the mould.

In embodiments where an agent for delivery is mixed in with the liquid form of the matrix material used to fabricate the microneedles, the amount of the agent for delivery in the resulting mixture utilised to fabricate the microneedles may be at least 5 wt%, at least 8 wt%, at least 10 wt%, at least 12 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, less than 5 wt%, less than 8 wt%, less than 10 wt%, less than 12 wt%, less than 15 wt%, less than 20 wt%, or less than 25 wt%.

Microneedle array fabrication processes relying on the application of liquefied matrix material/s to moulds will preferably utilise the liquefied matrix material/s at temperatures which assist or maximise their capacity to spread over the surface of the mould upon contacting it, thereby facilitating efficient movement into cavities of the mould. For example, a favourable contact angle between the mould surface and the liquefied matrix material once applied to the mould surface may be less than about 75°, less than about 60°, less than about 55°, less than about 50°, less than about 45°, less than about 40°, less than about 35°, or less than about 30°.

Applications of Microneedle arrays

The microneedle arrays of the present invention may be used for the purpose of delivering agents into the tissue of a subject.

Without any particular limitation, the microneedle arrays may be used for delivery of agents into tissues including skin, mucosa, and/or internal tissues including those exposed by surgery.

In some embodiments, the microneedle arrays may be used to deliver agents into or through the skin. In some embodiments, the microneedles of the arrays are of a sufficient length to pierce through the stratum corneum layer of the skin, the epidermal layer of the skin, the dermal layer of the skin, or beyond the dermal layer of the skin (e.g. into or through the hypodermal layer of the skin). In some embodiments, the microneedles of the arrays may be of sufficient length to deliver agents into the stratus corneum of the skin, into the epidermis of the skin, into the dermis of the skin, or beyond the dermis of the skin (e.g. into or through the hypodermis). For example, the microneedles of the array may be between about 600 pm and 800 pm (e.g. about 700 pm) in length to deliver agents into the epidermal and/or dermal layers of skin. Alternatively, the microneedles of the array may be of sufficient length to reach the hypodermis, or to reach tissue underlying the hypodermis (e.g. muscle tissue).

In other embodiments, the microneedle arrays may be used to deliver agents into or through mucosal surfaces. For example, the microneedle arrays may be used to deliver agents into or through the mucosa of the nose, mouth, eye, ear, respiratory system, vagina or rectum.

In other embodiments, the microneedle arrays may be used to deliver agents into internal tissues of a subject. The internal tissues may be exposed by surgery prior to using the microneedle arrays to deliver the agents. Non-limiting examples of suitable internal tissues include the gastrointestinal tract, bone, blood, lymph, epithelial tissue, muscle tissue, internal organs, connective tissue, and nerve tissue.

The temperature of tissue to which the microneedle arrays of the present invention may be applied will vary depending on the subject to which it belongs {in vivo use) or the conditions that it is being maintained under {ex vivo and in vitro use). By way of non limiting example only, the temperature of the tissue may at or above 35°C, at or above 36°C, at or above 36.5°C, at or above 37°C, at or above 37.5°C, at or above 38°C, at or above 38.5°C, at or above 39°C, at or above 39.5°C, or at or above 40°C, between 35°C and 36°C, between 35°C and 36.5°C, between 35°C and 37°C, between 35°C and 37.5°C, between 35°C and 38°C, between 35°C and 38.5°C, between 35°C and 39°C, between 35°C and 39.5°C, or between 35°C and 40°C, between 36°C and 36.5°C, between 36°C and 37°C, between 36°C and 37.5°C, between 36°C and 38°C, between 36°C and 38.5°C, between 36°C and 39°C, between 36°C and 39.5°C, between 36°C and 40°C, between 37°C and 37.5°C, between 37°C and 38°C, between 37°C and 38.5°C, between 37°C and 39°C, between 37°C and 39.5°C, between 37°C and 40°C, between 38°C and 38.5°C, between 38°C and 39°C, between 38°C and 39.5°C, or between 38°C and 40°C.

In some embodiments the microneedles of the arrays may include or be utilised in combination with analgesic agent/s. In other embodiments, a“hot-patch” may be utilised to heat a surface to which the microneedles of the array are to be applied. This may in turn enhance the capacity of the microneedles to pierce the tissue and/or deliver agents upon doing so.

In still other embodiments, iontophoresis and/or sonophoresis may be used to permeate tissue to which the microneedle arrays are applied (before, during and/or after application of the microneedles to the tissue). This may in turn enhance the capacity of the microneedles to pierce the tissue and/or deliver agents upon doing so.

The microneedle arrays of the present invention can be used to deliver agents into or though tissue in vivo, ex vivo, or in vitro.

The microneedle arrays described herein may be used to deliver agents into or through the tissue of a subject. The subject may be any animal of economic, social or research importance including bovine, equine, ovine, primate, avian and rodent species. In some embodiments of the present invention the subject is poultry. In other embodiments the subject is a mammalian subject (e.g. a human, dog, cat, cow, sheep, pig, or horse). The microneedle arrays may thus be used, for example, to deliver agents into the tissue of humans, poultry and/or livestock.

The subject may be in need of or desire therapeutic treatment, anaesthesia, immunisation/vaccination, and/or cosmetic treatment. Agents delivered by the microneedle arrays of the present invention may facilitate or assist one or more of these outcomes, and/or be applied to the tissue of the subject for other purpose/s.

It will be appreciated by persons of ordinary skill in the art that numerous variations and/or modifications can be made to the present invention as disclosed in the specific embodiments without departing from the spirit or scope of the present invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Examples

The present invention will now be described with reference to specific Examples, which should not be construed as in any way limiting. Example One: fabrication of Temperature-Sensitive Microneedle (MN) arrays

Microneedle (MN) arrays were fabricated using either menthol, Witespol H15 or maltose as a base/matrix material.

Matrix materials for MN fabrication

MN arrays were fabricated using either menthol, Witespol H15, camphor or maltose as a base/matrix material.

Menthol is a naturally occurring compound extracted from plants and has been used in pharmaceutical applications in combination with other ingredients for its anti-pruritic and analgesic properties. Menthol exists as a white crystalline solid (at room temperature) with peppermint odour and racemic mixture of L(-) and D (+) -menthol. It has an experimental melting point of 38°C and the liquid phase possess favourable low viscosity.

Witepsol H15 is a synthetic triglyceride containing hydrogenated vegetable oils and is used as a drug carrier in the fabrication of suppositories. It has an experimental melting point of 33.5°C-35.5°C and the liquid phase also has a favourable low viscosity. Witepsol H15 contract significantly on cooling, and thus mould lubrication is not necessary.

Camphor is an active ingredient found in Vicks Vapour rub and possess analgesic properties similar to that of menthol.

Maltose was used as a matrix for fabricating dissolving MN. Agents for delivery by menthol-based MN arrays

Cromolyn was incorporated into the microneedles during fabrication. Cromolyn solution is a mast cell stabilizer and comes in various formulations such as an inhalant and as eye drops to treat asthma and allergic conjunctivitis respectively. Topical cromolyn sodium was tested in uremic nephrogenic patients who developed puritus. The study results showed that using cromolyn sodium as a 4% cream was effective in reducing pruritus in patients compared to placebo in the third and fourth week of the study. Hence, in some cases 4-10 wt% cromolyn sodium was incorporated in the liquid mixture used to fabricate the microneedles. In other cases, hydrochloride monohydrate was incorporated into the microneedles during fabrication. A total of 15 wt% hydrochloride monohydrate was incorporated into the liquid mixture used to fabricate the microneedles. - Methods of fabricating menthol based MN arrays

Compression and hot-plate methods were used to fabricate the MN arrays. These methods are based on principles that underpin the hot embossing and PRINT methods.

The compression method involved using a negatively charged polydimethylsiloxane (PDMS) master mould which was heated on a hot-plate at 65 °C for approximately 10 minutes. While heating the PDMS mould, the material was melted in a micro-centrifuge tube at 65°C using a MD-MINI heating block (Major Science) till all contents had melted.

The PDMS mould was then removed from the hot-plate and the melted materials poured into the cavity. Compression was delivered manually using a dispensing spatula after which, more melted material was poured into the same cavity due to overflow of material as a result of pressure exerted during compression. The mould was allowed to rest at room temperature and subsequently de-moulded when the material had solidified (Figure 1).

Under the hot-plate method, a PDMS master mould was heated on a hot-plate at 65°C for approximately 10 minutes. While heating the PDMS mould, the material was melted in a microcentrifuge tube at 65°C, using a MD-MINI heating block (Major Science) till all contents have melted.

The PDMS mould was then removed from the hot-plate and the melted materials poured into the cavity. The filled mould was then placed onto a hot-plate for 15 minutes and subsequently removed from the hot-plate. The mould was allowed to rest at room temperature and subsequently de-moulded when the material had solidified (Figure 1).

Surface tension and contact angles

Observed contact angles between the surface of the PDMS mould and water (control), menthol and Witepsol H15 were approximately l20°C, 44°C and 54°C respectively. Therefore, wetting of the surface of PDMS by menthol and Witepsol H15 was found to be favourable and thus led to spreading over a larger surface. This means that the menthol and Witepsol H15 were observed to move easily into the cavities of the PDMS MN array mould and fill it (Figure 2). Demoulding of MN arrays fabricated from menthol versus Witepsol HI 5

The menthol array was easy to demould compared to the array made up of Witepsol H15. This is most likely due to the chemical structure of the two materials differing. Menthol, solidified as a crystal. In contrast, Witepsol H15 has a structure that consists of elongated carbon chains which does not have as much mechanical strength as compared to menthol.

Example Two: properties of fabricated microneedle (MN) arrays

Camphor possesses analgesic properties similar to those of menthol. However, due to its high experimental melting point of l80°C, it could not fill the cavity of the PDMS mould as the liquid phase solidified almost instantaneously upon contact with air at room temperature. Also, using camphor in a eutectic mixture with menthol was found to be unviable as the melting temperature was too low and MNs made from it melted too quickly upon touch. Additionally, the solidified mixture did not possess strong enough mechanical structure to form MN arrays.

Maltose was used as a matrix for fabricating dissolving MN as the viscosity and triple state of maltose was easily regulated. Additionally, maltose has an experimental melting point of l02°C and forms a hard, crystalline mass when solidified. The principle underpinning using maltose as a structural matrix is that it dissolves readily because of the hydrolytic cleavage of maltase-glucomylase in skin.

The menthol MN arrays were observed to demould effectively following solidification in PDMS moulds, including those fabricated with eflomithine hydrochloride monohydrate (Figure 3, top) and cromolyn sodium (Figure 3, bottom) in 15 wt% and 10 wt% mixtures, respectively. Menthol, when solidified, has a structure that consists of cyclohexane conformations while ring itself is in a chair conformation.

Witepsol H15 MN arrays were also observed to demould effectively following solidification in PDMS moulds, and have a structure that consists of elongated carbon chains which has less mechanical strength compared to menthol. Example Three: Application of the microneedle arrays to the skin of humans and pigs A proof-of-concept test was conduct on human skin, whereby the menthol MN arrays described in Examples 1 and 2 above (including those with incorporated comprising eflomithine hydrochloride monohydrate (Figure 3, top) and cromolyn sodium (Figure 3, bottom) displayed that they are mechanically stable enough to penetrate skin. The menthol based arrays were applied to the skin (Figure 4A) using a finger-thumb grip using an average force of 98 N to the MN array for 20 seconds (Figure 4B, showing MN array following pressure). The weight of the arrays was 0.4845g before the application and 0.4754g after the application. The deficient 0.009lg arose from microneedles melting and absorbing through the skin via micropores created by the array. The skin was monitored for the appearance of erythema, a reaction of the skin to menthol. Figure 4C, shows the skin before application and Figure 4D shows the skin during the application. After 30 minutes, the arrays were removed and the skin monitored at 0, 5, 20 and 60 minutes (shown by Figures 4E, 4F, 4G and 4H, respectively).

A simple ex vivo test was contacted on pig skin (Coles“Australian Pork Crackling”) A 4 cm by 4 cm pig skin was pre-treated for 60 minutes on a hot plate at 37 °C followed by application of the menthol MN array using a finger-thumb grip for 20 seconds (as described above). The arrays were left on the skin for 30 minutes before removal. The area was then stained with a 1:1 ratio of trypan blue to water solution followed by washing in water to remove the excess stain. The pre-application and post-application weight of the MN arrays were 0.5l05g and 0.500g respectively, and hence assumed that 0.0l05g of material had melted into the pig skin. Results showed that the arrays had sufficient mechanical strength to at least penetrate the epidermis of the pig skin (Figure 5).

MN arrays fabricated by either the compression method or hot-plate method using menthol thus possess the necessary mechanical strength to penetrate the epidermis of the skin. Therefore, menthol based MN arrays can be used as a carrier to transport drug into the skin via micropores created during penetration.

It is envisaged that matrix materials that have similar properties to menthol can be used to create MN arrays with similar properties to those observed. Example Four: Stability studies of microneedle patches

Materials and Methods

Standard samples of cromolyn sodium (>99.86%) were provided by Quimica Sintetica, Spain. Phosphoric acid solution (85wt. in FU O) was purchased from SAFC, USA. Acetonitrile was obtained from RCI labscan, Thailand. All materials were used as supplied without further purification.

Methods

( i ) HPLC Analysis of Cromolyn

A calibration curve of cromolyn sodium was done with Shimadzu HPLC system using a Agilent ODS C18 column (3.0 mm x 150 mm, 5 pm). UV detection was performed at a wavelength of 220 nm. The mobile phase consisted of phosphoric acid aqueous solution which pH = 2 (solvent A) and acetonitrile (solvent B) with a gradient elution program of solvents A and B as follows: 15 - 50% B for 0 - 10 min and 50% for 10-11 min. The flow rate was set at 0.6 ml/min and 20 pl of the samples was loaded.

To prepare standards for the calibration curve and assessment of validation, a stock solution of cromolyn was prepared with a concentration of 1 mg/ml. This solution was diluted with distilled water to yield 1 pg/ml to 10 pg/ml working standard solutions for preparation of calibration curve. A 20 mΐ aliquot was injected (3 replicated) and the calibration curves were con- structed by plotting the peak area of cromolyn (y) against concentration of cromolyn (x), using simple linear regression analysis.

(ii) Recovery rate determination

The stock solution was diluted with distilled water to yield 3, 5 and 7 pg/ml solutions for preparation of recovery rate determination. Analyse the cromolyn content under the same HPLC conditions. Calculate the cromolyn content of 3 different concentrations according to the linear equation and then calculate the recoveries.

( Hi ) Stability Study

One microneedle patch was placed at 40°C for 1 hr and its physical appearance then observed.

Three microneedle patches were placed at 25 °C for 7 days, and their physical appearance on day 2, day 4 and day 7 then observed.

Eighteen microneedle patches were placed at 5 ° C for 80 days and their physical appearance then observed.

The drug content of the microneedle patches placed at 5 ° C for 80 days was analysed on day 0 (patch numbers 1-3), day 20 (patch numbers 4-6, and on day 40 (patch numbers 7-9). The physical appearance of patch numbers 16-18 was observed every 10 days.

Results

(i) Calibration curve of cromolyn sodium

The linear equation of cromolyn sodium was observed to be: y= 52992 c-5612.2 (R 2 =0.9998). The result showed a good linearity within 1 pg/ml to 10 pg/ml. (ii) Recovery rate determination

The recovery rate was studied under 3 different concentrations and was between 94.42% and 98.84% (Figure 6).

(Hi) Stability study

Since the melting point of menthol is 38 °C, the microneedle patch completely melted under 40°C.

The needle part of the patches placed under 25 °C was observed to collapse and fracture after 7 days.

The physical appearance of the microneedle patches placed under 5°C proved to be relatively stable, and the drug content analysis showed no significant degradation of cromolyn (Figure 7).

Example Five: Microneedle delivery to human skin samples

Example five is prophetic.

( i ) Objective

To evaluate cromolyn in Franz diffusion cell and quantify the amounts of actives accumulated-in and permeated-through dermatomed human skin samples. (Figure 8). ( ii ) Experimental

The following approach will be undertaken to detect penetration of molecules in skin. At least 3 replicates per sample will be run to check for repeatability.

1. Solutions of cromolyn will be prepared in the solvent/vehicle isopropyl myristate at the appropriate concentrations that will allow for quantitative measurement.

2. Microneedles containing cromolyn will be prepared at the appropriate concentrations that will allow for quantitative measurement.

3. Franz diffusion cells will be used to assess permeation of cromolyn into the receptor chambers at different time points. At the end of the run, the tissue will be homogenized, and samples will be collected for testing using HPFC to determine the amount peptides inside the skin samples.

( iii ) Projected Results

It is expected the amount of cromolyn delivered through skin by using microneedles will be 3-5 times higher than that using solutions.




 
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