WO2015011188A1

WO2015011188A1 - Fibroin-based pharmaceutical spray compositions for the treatment of skin lesions

Info

Publication number: WO2015011188A1
Application number: PCT/EP2014/065815
Authority: WO
Inventors: Piero Iamartino; Renzo Longhi; Marzio SORLINI; Giorgio NEMBRI; Silvio FARAGO'; Maria Luisa Torre; Francesco Gatti
Original assignee: Micro-Macinazione S.A.; Scuola Universitaria Professionale Della Svizzera Italiana; Nembri Industrie Tessili S.R.L.; Innovhub - Stazioni Sperimentali Per L'industria; Universita' Degli Studi Di Pavia; Centro Tessile Serico - Societa' Consortile Per Azioni
Priority date: 2013-07-26
Filing date: 2014-07-23
Publication date: 2015-01-29
Also published as: ITMI20131255A1; EP3024477A1

Abstract

The present invention relates to spray compositions comprising fibroin and a co-micronised alginic acid salt, a micronised calcium salt and a source of silver and/or copper, and the corresponding manufacturing processes.

Description

FIBROIN-BASED PHARMACEUTICAL SPRAY COMPOSITIONS FOR THE TREATMENT OF SKIN LESIONS

Prior art

Fibroin, the structural protein of silk, has found numerous applications in the medical field due to its characteristics of strength, rigidity, biocompatibility and workability.

Of its main applications, one of the most widely studied is in the dermatological field, in particular for promoting healing of wounds and ulcers.

For example, the use of fibroin has been proposed to promote regeneration of the epidermis for the treatment of skin wounds (Proc. Soc Exp Biol Med 225(1) 58-64, (2000)).

Biomaterials based on fibroin for covering/dressing wounds are also described in CN 102319448 and IN1998CH01330.

CN101450222 describes alginate microspheres containing silver which are useful to prepare wound-dressing fabrics.

Microspheres made of fibroin, alginate and colloidal silver suitable for topical spray administration for the treatment of skin wounds and ulcers were described by Bucco et al. in XVII^th Int. Conference on Bioencapsulation, Groningen, Netherlands; September 24-26, 2009, Poster P57. However, the preparation of said microspheres is difficult, and not easily applicable on an industrial scale because the spray-drying technique described is complex and expensive. Moreover, the spray formulation is only generically mentioned, but there is no complete, reproducible description of a method that allows the preparation of natural protein aerosols, a problem that is not easy to solve due to the need to guarantee the mechanical and functional characteristics of the protein, in particular maintaining its characteristics of stability, strength and flexibility.

Brief Description of the Figures

Figure 1. Size distribution of the mixture described in step 4 of Example 1 ; Figure 2. Image of Example of SEM-EDX mapping of silver (pale grey colour) in the mixture described in Example 1 ;

Figure 3. Size distribution of the mixture described in step 4 of Example 2;

Figure 4. Size distribution of the mixture described in step 4 of Example 3 (Batch I);

Figure 5. Size distribution of the mixture described in step 4 of Example 3

(Batch II).

Description of the invention

It has now been found that spray formulations containing fibroin, alginate, silver and/or copper can be obtained with co-grinding techniques that improve the characteristics of the occlusive film formed after spraying.

In particular, it has been found that fibroin can be advantageously co-micronised with an alginic acid salt after prior degumming and grinding treatment to give a composite which, when mixed with silver and/or copper and calcium salts, provides a composition that presents better characteristics than those obtainable with conventional techniques such as spray-drying.

A first aspect of the invention therefore relates to spray compositions containing fibroin and a co-micronised alginic acid salt, a micronised calcium salt and a source of silver and/or copper.

The invention also relates to a process for the preparation of said compositions, which comprises the following steps:

a) micronising previously degummed, destructured, ground fibroin;

b) co-micronising fibroin from step a) with an alginic acid salt;

c) adding to the co-micronised fibroin and alginate product a pre-micronised mixture of a calcium salt and a source of silver and/or copper;

d) adding optional excipients and distributing between pressurised canisters with a gaseous propellant.

The compositions according to the invention are useful for the treatment of vascular ulcers, skin lesions, bedsores, psoriasis, dermatosis and burns.

Fibroin, when micronised and subsequently co-micronised with alginate and formulated as described above, forms a layer of powder on the skin lesion that absorbs the exudate and establishes optimum, balanced moisture levels, thus promoting wound healing. Silver or copper further promote the wound repair and healing processes by imparting antimicrobial and antifungal properties which are particularly desirable for the recommended applications.

The preferred size range for the silver and/or copper is nanometric. An alternative form to metallic silver could be silver phosphozirconate.

The calcium salt is preferably selected from gluconate, ascorbate, glucoheptonate, dobesilate, glubionate, levulinate, lactate, lactobionate, pantothenate, ketoglutarate and borogluconate, preferably gluconate.

The alginic acid salt is preferably selected from sodium and calcium alginate, preferably sodium alginate.

The fibroin-to-alginate weight ratio ranges between 1 :9 and 4: 1, preferably between 1 :2 and 2: 1, and even more preferably 1 : 1. The fibroin and alginate co-micronisate constitutes between 80 and 97.5%, preferably between 90 and 95% by weight of the total composition; the percentage weight of the calcium salt ranges between 2 and 20%, and the percentage of the silver or copper source ranges between 0.05 and 0.5% of the total weight of the composition.

If desired, other excipients, such as stabilisers, lubricants and anti-caking agents, in particular colloidal silicon dioxide, can be added to the compositions according to the invention before being used to fill spray cans containing suitable propellants (butane, chlorofluorocarbon, fluorocarbons, etc.). The qualitative and quantitative composition of a typical formulation according to the invention, to be introduced into a 125-150 ml canister containing n-butane as propellant, is set out below by way of example.

Table I. Typical formulation of the invention

The activation of the canister generates the deposit on the damaged skin of a layer of powder which, after absorbing the exudate, takes on the appearance of a flexible, strong, stable gel that protects the wound, sore, burn or skin ulcer for several hours, thus facilitating and promoting the wound-healing and tissue-repair processes.

The preparation process of the compositions according to the invention is based solely on physical processes (micronisation and mixing of ingredients).

The fibroin is first degummed to eliminate the sericin and then treated with alkali to destructure its protein structure^1"5 and facilitate the subsequent operation of grinding in a mill, typically a knife mill.

The use of a starting raw material (silkworm cocoons) originating from controlled cultivations according to a breeding protocol that does not involve the use of pesticides is preferred. The degummed, destructured, ground fibroin is then micronised in a jet-mill, the gas used typically being nitrogen, at a pressure ranging from 5 to 15 bars. The system for delivering the fibroin from the container to the jet-mill preferably comprises a special micro-delivery device, described in Italian patent application no. ΜΓ2012Α000635 filed on 17 April 2012, as an alternative to the current delivery systems based on a Venturi tube.

The micronised fibroin is then mixed with alginate in a mixer, such as a rotary powder mixer. The mixture is micronised in a jet-mill, preferably using nitrogen, at gas pressures ranging between 4 and 10 bars, to give a mixture of co-micronised alginate and fibroin (Mixture A).

Alternatively, the micronised fibroin and the alginate can be loaded directly into the loading hopper of the jet- mill. Loading is effected by two screw-feeding units suitably calibrated to load two products in the required proportions. Co-micronisation is then performed as described above.

The calcium salt and the silver and/or copper are mixed separately in a rotary powder mixer. The same quantities of silver and calcium salt are preferably loaded at the initial step; mixing continues for times ranging between about 10 and 20 min., gradually adding the desired quantity of calcium salt.

The mixture is then micronised in a jet- mill, preferably using nitrogen, at gas pressures ranging between 5 and 10 bars (Mixture B).

Mixture A is then added to Mixture B and, after the addition of any other excipients, proceeds to the mixing step and is then used to fill pressurised canisters.

Typically, 5 or 10 g of powder mixture are used to fill 125 ml canisters, with the addition of n-butane gas.

The product in the pressurised canisters is then sterilised by known techniques, such as gamma-ray radiation.

The product sprayed by the canister forms a layer of biodegradable, allergen-free, non-cytotoxic, well-tolerated powder on the damaged skin, which is useful for the treatment of bedsores, skin lesions of various origins, dermatosis, psoriasis and the like. The invention is illustrated in greater detail in the examples below.

Example 1

Step 1. Fibroin, previously degummed and ground, was micronised using a jet-mill {model: MCI 00) input pressure: 6 bars; grinding pressure: 8 bars; flow rate: lKg/h).

Step 2. Fibroin produced in Step 1 was co-micronised with sodium alginate using a jet-mill {model: MCI 00; input pressure: 10 bars; grinding pressure: 8 bars).

Fibroin and sodium alginate were loaded simultaneously into the jet-mill with two screw feeders calibrated to a total flow rate of 1 Kg/h.

Step 3. Calcium gluconate was gradually mixed with the nanometric silver for 12 min, and the resulting mixture was subsequently micronised with a jet-mill {model: MCI 00; input pressure: 10 bars; grinding pressure: 8 bars; flow rate: 0.8 Kg/h).

Step 4. The mixtures produced in steps 2 and 3 and the colloidal silicon dioxide were mixed in a planetary mixer (Turbula T-2C- WAB) for 15 min.

Step 5. 5 g of the mixture from step 4 was distributed between 125 ml pressurised canisters (propellant: n-butane).

Table II. Formulation used in the process described in Example 1.

The size distribution of the mixture of step 4 was obtained with a laser diffractometer (Laser Diffraction Sensor: Helos H0990; Dispersing Unit: Sucell, Sympatec GmbH) using heptane as dispersing agent (Figure 1).

The distribution of the silver in the mixture was established by SEM-EDX mapping (Figure 2) of various portions of a sample of the mixture of step 4. Silver, shown in red, proved to be evenly distributed in the mixture.

Example 2

Step 1. Fibroin, previously degummed and ground, was micronised with a jet-mill {model: MC50; input pressure: 6 bars; grinding pressure: 10 bars; flow rate: 0.5 Kg/h).

Step 2. Fibroin produced in Step 1 was mixed with sodium alginate in a planetary mixer (Turbula T-2C- WAB) for 15 min.

Step 3. The mixture produced in Step 2 was micronised with a jet-mill {model: MC50; input pressure: 8.5 bars; grinding pressure: 8 bars; flow rate: 0.5 Kg/h).

Step 4. The calcium gluconate was gradually mixed with the nanometric silver for 12 min, and the resulting mixture was subsequently micronised with a jet-mill {model: MC50; input pressure: 8.5 bars; grinding pressure: 8 bars; flow rate: 0.3 Kg/h).

Step 5. Mixtures produced in steps 3 and 4 and the colloidal silicon dioxide were mixed in a planetary mixer (Turbula T-2C- WAB) for 15 min.

Step 6. 5 g (batch I) or 10 g (batch II) of the mixture of step 5 was distributed between 125 ml pressurised canisters (propellant: n-butane). Table III. Formulation used in the process described in Example 2

The size distribution of the mixture of step 5 was obtained with a laser diffractometer (Laser Diffraction Sensor: Helos H0990; Dispersing Unit: Sucell, Sympatec GmbH) using heptane as dispersing agent (Figure 3).

The following tests were used to test the delivery of batches I and II by the pressurised canisters:

• Test A: 6 consecutive 2-sec deliveries, each separated by a 2-sec pause

• Test B: 2 consecutive 10-sec deliveries, each separated by a 2-sec pause Table IV. Results of delivery test on batches I and II

As summarised in Table IV, delivery was possible from the canisters containing both batch I and batch II. The delivery rates recorded were 25 mg/sec and 130 mg/sec respectively.

Example 3

Step 1. Fibroin, previously degummed and ground, was micronised with a jet-mill {model: MCI 00; input pressure: 6 bars; grinding pressure: 8 bars; flow rate: 1 Kg/h).

Step 2. Fibroin produced in Step 1 was co-micronised with sodium alginate using a jet-mill {model: MCI 00). The following parameters were used for Batch I: input pressure: 10 bars; grinding pressure: 8 bars; while the following parameters were used for Batch II: input pressure: 8 bars; grinding pressure: 6 bars.

For both Batch I and Batch II the fibroin and sodium alginate were loaded simultaneously into the jet- mill with two screw feeders calibrated to a total flow rate of 1 Kg/h.

Step 3. Calcium gluconate was gradually mixed with the nanometric silver for 12 min, and the resulting mixture was subsequently micronised with a jet- mill {model: MCI 00; input pressure: 10 bars; grinding pressure: 8 bars; flow rate: 0.8 Kg/h).

Table V. Formulation used in the process described in Example 3

The size distribution of the mixture of step 4, for Batch I (Figure 4) and Batch II (Figure 5), was obtained with a laser diffractometer (Laser Diffraction Sensor: Helos H0990; Dispersing Unit: Sucell, Sympatec GmbH) using heptane as dispersing agent. REFERENCES

1 Roy H. Walters, O.A. Hougen - Silk Degumming: I. - Degradation of Silk Sericin by Alkalies, Textile Research Journal December, 1934 5: 92-104.

2 Roy H. Walters, O.A. Hougen - Silk Degumming: II. - The Rate of Degumming Silk., Textile Research Journal January, 1935 5: 134-148.

3 S. Chopra, R. Chattopadhyay, M. L. Gulrajani - Low Stress Mechanical Properties of Silk Fabric Degummed by Different Methods, Journal of The Textile Institute, 1996 87, 3: 542-553.

4 Gulrajani, M. L. - Silk Dyeing, Printing, and Finishing. Department of Textile Technology, Indian Institute of Technology, 1988.

5 Gulrajani, M. L. - Degumming of silk. Review of Progress in Coloration and Related Topics, 1992 22: 79-89.

Claims

1. A composition designed for spraying from pressurized canisters, comprising fibroin co-micronized with an alginic acid salt, a micronized calcium salt and a silver and/or copper source.

2. A composition according to claim 1 wherein the alginic acid salt is selected from sodium alginate and calcium alginate, preferably sodium alginate.

3. A composition according to claims 1 and 2 wherein the calcium salt is selected from gluconate, ascorbate, glucoheptonate, dobesilate, glubionate, levulinate, lactate, lactobionate, pantothenate, ketoglutarate and borogluconate, preferably calcium gluconate.

4. A composition according to one or more of claims 1 to 3 wherein the preferred dimensional range for silver and/or copper is nanometric.

5. A composition according to one or more of claims 1 to 4, wherein the silver source is metal silver or silver phosphozirconate.

6. A composition according to one or more of claims 1 to 5 wherein the fibroin to alginic acid salt weight ratio ranges from 1 :9 to 4: 1.

7. A composition according to one or more of claims 1 to 6 wherein the co-micronized fibroin and alginic acid salt amounts to 80 to 97.5%, preferably 90 to 95% of the total weight of the composition, the calcium salt weight percentage ranges from 2 to 20% and the silver or copper source weight percentage ranges from 0.05 to 0.5% of the total weight of the composition.

8. A composition according to the above claims for use in the treatment of vascular ulcers, skin lesions, bedsores, psoriasis, dermatosis and burns.

9. A process for the preparation of the compositions according to the above claims, which comprises the following steps:

a) micronizing fibroin previously degummed, destructured and ground; b) co-micronizing the fibroin from step a) with an alginic acid salt;

c) adding a calcium salt and a previously micronised mixture of a silver and/or copper source to the co-micronized fibroin and alginate product;

d) adding optional excipients and distributing between pressurized canisters with a gas propellant.

10. A process according to claim 9 wherein step a) is effected in a jet mill, wherein the gas is typically nitrogen, at a pressure of 5 to 15 bars.

1 1. A process according to claim 9 or 10 wherein step b) is effected in a jet mill wherein the gas is typically nitrogen, at a pressure of 5 to 15 bars.