NON-NANOPARTICULATE APPLICATION FORMS OF MACROLIDES

Abstract

The present invention relates to a mucoadhesive layer, preferably mucoadhesive buccal layer, comprising a non-nanoparticulate macrolide. Furthermore, the present invention relates to a mucoadhesive film, preferably mucoadhesive buccal film, comprising a mucoadhesive layer comprising a non-nanoparticulate macrolide, and further comprising a backing layer. The present invention also relates to a mucoadhesive film, preferably mucoadhesive buccal film, comprising a mucoadhesive layer, a backing layer, and an intermediate layer. The present invention further relates to a mucoadhesive layer or mucoadhesive film for use as a medicament and for use in preventing and/or treating transplant rejection. Moreover, the present invention relates to a method of preparing a mucoadhesive film.

Claims

1. A mucoadhesive layer comprising a non-nanoparticulate macrolide.

2. The mucoadhesive layer according to claim 1, wherein the non-nanoparticulate macrolide is selected from macrolide immune suppressants, macrolide antibiotics, and macrolide antifungals.

3. The mucoadhesive layer according to claim 1, wherein said non-nanoparticulate macrolide is present in said layer in the form of micronized macrolide.

4. The mucoadhesive layer according to claim 3, wherein said micronized macrolide has an average particle size from 1 to 100 μm.

5. The mucoadhesive layer according to claim 1, wherein the mucoadhesive layer comprises a mucoadhesive polymer selected from a) amphiphilic polymers and hydrophilic polymers, b) poly(methacrylates), c) crosslinked polyacrylic acid polymers, d) copolymers of methyl vinyl ether and maleic anhydride, e) polymers comprising polyvinyl acetate and/or polyvinylpyrrolidone, and combinations thereof; and wherein the mucoadhesive layer further comprises: a cellulose derivative selected from hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose (HEC), methyl cellulose (MC), hydroxyethyl methyl cellulose (HEMC), ethyl cellulose (EC), and a combination thereof; a plasticizer; optionally a colorant; and the non-nanoparticulate macrolide.

6. The mucoadhesive layer according to claim 3, wherein said non-nanoparticulate macrolide is present in said layer in the molecularly dissolved non-nanoparticulate form; and wherein said mucoadhesive polymer and/or said cellulose derivative is a polymer soluble in an organic solvent.

7. The mucoadhesive layer according to claim 1, wherein the mucoadhesive layer comprises: a) a mucoadhesive polymer; hydroxypropyl cellulose (HPC); carboxymethyl cellulose (CMC); a plasticizer; and optionally a colorant; or b) a mucoadhesive polymer; hydroxypropyl cellulose (HPC); ethyl cellulose (EC); a plasticizer; preferably 2 wt %-17 wt % of mucoadhesive polymer; 30 wt %-70 wt % of hydroxypropyl cellulose (HPC); 5 wt %-40 wt % of said ethyl cellulose (EC); 3 wt %-20 wt % of said plasticizer; and optionally 1 wt % 5 wt % of said colorant.

8. The mucoadhesive layer according to claim 1, wherein said macrolide is present in said mucoadhesive layer at a concentration of at least 2 wt %; and/or in an amount in the range of from approximately 0.01 mg to approximately 10 mg, with reference to 1 cm.sup.2 of said mucoadhesive layer.

9. A mucoadhesive film comprising a mucoadhesive layer comprising a non-nanoparticulate macrolide as defined in claim 1; and further comprising a backing layer impermeable for the macrolide and/or impermeable for water.

10. The mucoadhesive film according to claim 9, wherein the backing layer comprises a plasticizer selected from glycerol, polyethylene glycol, propylene glycol, sorbitol, triacetin, and tributylcitrate; and/or comprises a water-insoluble polymer and/or a cellulose derivative.

11. The mucoadhesive film according to claim 9, wherein the mucoadhesive film further comprises an intermediate layer arranged between the mucoadhesive layer and the backing layer; wherein said intermediate layer facilitates adhesion between the mucoadhesive layer and the backing layer; wherein, optionally, said intermediate layer comprises a plasticizer.

12. The mucoadhesive film according to claim 9, wherein said mucoadhesive layer has an area weight in the range of from 70 g/m.sup.2 to 180 g/m.sup.2l; and/or wherein said backing layer has an area weight in the range of from 40 g/m.sup.2 to 100 g/m.sup.2; and/or wherein, said intermediate layer, if present, has an area weight in the range of from 40 g/m.sup.2 to 100 g/m.sup.2.

13. A mucoadhesive film comprising a mucoadhesive layer, a backing layer, and an intermediate layer, wherein said intermediate layer is arranged between the mucoadhesive layer and the backing layer, and wherein said mucoadhesive layer comprises tacrolimus; wherein said intermediate layer facilitates adhesion between the mucoadhesive layer and the backing layer; wherein, optionally, said intermediate layer comprises a plasticizer; wherein, optionally, said intermediate layer comprises a polymer.

14. (canceled)

15. A method for preventing and/or treating transplant rejection wherein said method comprise the use of the mucoadhesive layer according to claim 1; wherein, optionally, said use involves an attachment of said film or layer in the body cavity of a patient.

16. A method of preparing a mucoadhesive film, comprising: i) providing a micronized macrolide; and a polymer mixture, wherein said polymer mixture comprises a mucoadhesive polymer selected from a) amphiphilic polymers and hydrophilic polymers, b) poly(methacrylates), c) crosslinked polyacrylic acid polymers, d) copolymers of methyl vinyl ether and maleic anhydride, e) polymers comprising polyvinyl acetate and/or polyvinylpyrrolidone, and combinations thereof; a cellulose derivative selected from hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose (HEC), methyl cellulose (MC), hydroxyethyl methyl cellulose (HEMC), ethyl cellulose (EC), and a combination thereof; optionally, a plasticizer; optionally a colorant; ii) optionally, dispersing the micronized macrolide in a liquid medium to obtain a micronized macrolide dispersion and/or dissolving said micronized macrolide in an organic solvent to obtain a micronized macrolide solution; iii) mixing the micronized macrolide, optionally micronized macrolide dispersion or micronized macrolide solution, with the polymer mixture to obtain a mucoadhesive layer composition; iv) preparing a mucoadhesive layer using the mucoadhesive layer composition; optionally by coating the mucoadhesive layer composition on a surface; v) optionally, drying said mucoadhesive layer, thereby obtaining the mucoadhesive film comprising said mucoadhesive layer.

17. The method according to claim 16, further comprising preparing a backing layer, comprising: a) providing a backing layer polymer mixture; optionally comprising a plasticizer; optionally comprising a water-insoluble polymer and/or a cellulose derivative; b) preparing a backing layer using the backing layer polymer mixture, optionally on a surface of said mucoadhesive layer; c) optionally, drying the backing layer.

18. The method according to claim 16, further comprising preparing an intermediate layer, comprising: 1) providing an intermediate layer polymer mixture; optionally comprising a plasticizer and a polymer; 2) preparing an intermediate layer using the polymer mixture, optionally on a surface of said mucoadhesive layer; 3) optionally, drying the intermediate layer.

19. The mucoadhesive layer according to claim 1, which is a buccal layer.

20. The mucoadhesive layer according to claim 2, wherein the macrolide immune suppressant is selected from tacrolimus, sirolimus, everolimus, and pimecrolimus; the macrolide antibiotic is selected from erythromycin, clarithromycin, azithromycin, roxithromycin, josamycin, spiramycin, telithromycin, tylosin, and fidaxomicin; and/or the macrolide antifungal is selected from polyenes.

21. The mucoadhesive layer according to claim 21, wherein said non-nanoparticulate macrolide is non-nanoparticulate tacrolimus or sirolimus.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0121] The present invention is now further described by reference to the following figures.

[0122] All methods mentioned in the figure descriptions below were carried out as described in detail in the examples.

[0123] FIG. 1 shows a schematic representation of the method of producing a mucoadhesive layer and/or mucoadhesive film of the invention.

[0124] FIG. 2 shows the release and unidirectional release of the film comprising micronized crystalline macrolide (MFK10) and a film having the same composition but without Soluplus®.

[0125] FIG. 3 shows an overlay of XRPD spectra of and MFK10 (3), TiO.sub.2 (2) and pure crystalline Tacrolimus (1).

[0126] FIG. 4 shows the in vivo macrolide blood level after administration of the mucoadhesive film.

[0127] FIG. 5 shows a schematic representation of the method of producing a mucoadhesive film of the invention which comprises a mucoadhesive layer, an intermediate layer (adhesive layer), and a backing layer.

[0128] FIG. 6 shows pictures of a film comprising micronized crystalline macrolide (MFK10) adhered onto the buccal mucosa during 30 min of treatment (the particular experiment shown being designated as “T2_No.3_xx min”, with xx designating the number of treatment minutes for which the film adhered to the buccal mucosa). Successful adhesion and treatment were also observed with the film comprising molecularly dissolved non-nanoparticulate macrolide (FK1).

[0129] FIG. 7 shows the mucosa of the animal from FIG. 6 after treatment and after removal of the film MFK10. No irritation of the mucosa is visible.

[0130] In the following, reference is made to the examples, which are given to illustrate, not to limit the present invention.

EXAMPLES

Example 1: Preparation of a Mucoadhesive Film with Micronized Crystalline Macrolide

[0131] A polymer mixture was prepared by mixing water with acetone, and by dispersing titanium dioxide in the mixture while stirring. Glycerol and Soluplus® were added. Hydroxypropyl cellulose (HPC) powder was then slowly added, and sodium carboxymethyl cellulose (CMC) powder was added after complete dissolution of HPC. The viscosity of the mixture increased with dissolution of CMC. To avoid air bubbles in the polymer mixture, the stirring speed was reduced.

[0132] The micronized macrolide was predispersed in 50 mM sodium acetate buffer with pH 4, and then added to the polymer mixture. The crystals of the micronized macrolide had D.sub.10 2 μm; D.sub.50 10 μm; D.sub.90 25 μm.

[0133] 300 g of the mixture were prepared. The concentration of solids in the mixture was 15% by weight.

TABLE-US-00001 TABLE 1 Weighed portions for the production of the mucoadhesive layer (MFK11). Weighed MFK11 portion [g] Solid [wt.-%] Tacrolimus 3.68644  8.0% Buffer 106.8 TiO.sub.2 1.14903  2.5% Glycerol 5.53318 12.1% Soluplus ® 11.12 24.2% HPC 14.59 31.8% CMC 9.81 21.4% Acetone 10.75 Water 136.7 Total 300  100% Solid content 15%

[0134] The obtained mixture was coated onto a process liner FL2000 in a thickness of 850 μm (thickness in undried form) at a speed of 5 mm/s with a double skrew coating knife and then dried at 60° C. for 40 minutes (FIG. 1). Several sheets were produced. The targeted area weight of the film was about 104 g/m.sup.2. Die-cuts of 6 cm.sup.2 size were prepared, and the area weight of the die-cuts was determined:

TABLE-US-00002 TABLE 2 Weight per unit area of the mucoadhesive layer. Weight per unit area Sheet mucoadhesive layer MFK11-1 106.7 g/m.sup.2 (RSD 3.32%) MFK11-2 108.3 g/m.sup.2 (RSD 2.74%) MFK11-3 110.2 g/m.sup.2 (RSD 3.49%) RSD: relative standard deviation of 10 samples

Example 2: Sealing of the Mucoadhesive Layer with a Backing Layer MFK12

[0135] The polymer mixture for sealing was prepared. Ethanol was mixed with glycerol (as a plasticizer) under agitation. Ethyl cellulose powder was then slowly added and the mixture was stirred until homogenization.

[0136] 300 g of the mixture were prepared. The concentration of solids in the mixture was 17.5% by weight.

TABLE-US-00003 TABLE 3 Weighed portions for the production of the backing layer (MFK12). Weighed MFK12 portion [g] Solid [wt.-%] EC 47.69 90.1% Glycerol 5.2431  9.9% Ethanol 247.09 Total 300  100% Solid content 17.6%

[0137] After drying of the mucoadhesive layer, the mucoadhesive layer sheets were coated with a solution of ethyl cellulose in ethanol in a thickness of 425 μm at a speed of 5 mm/s using a double skrew coating knife and then dried for 10 minutes at 40° C. (FIG. 1). The targeted area weight of the film was about 50 g/m.sup.2. Die-cuts of 6 cm.sup.2 size were prepared, and the area weight of the die-cuts was determined:

TABLE-US-00004 TABLE 4 Weight per unit area of the mucoadhesive film. Area weight Area weight mucoadhesive layer backing layer Area weight Sheet Sheet MFK11 MFK12 film MFK10 MFK10-4 MFK11-1 106.7 g/m.sup.2 (RSD 3,32%) 43.9 g/m.sup.2 150.6 g/m.sup.2 (RSD 1.22%) MFK10-5 43.6 g/m.sup.2 150.3 g/m.sup.2 (RSD 2.09%) MFK10-2 MFK11-2 108.3 g/m.sup.2 (RSD 2,74%) 47.4 g/m.sup.2 155-7 g/m.sup.2 (RSD 1.30%) MFK10-3 MFK11-3 110.2 g/m.sup.2 (RSD 3,49%) 44.0 g/m.sup.2 154.2 g/m.sup.2 (RSD 1.34%) RSD: relative standard deviation of 10 samples

[0138] Square films with rounded corners of 6 cm.sup.2 area and 2.48 cm edge length were punched out of the two-layer laminate comprising the mucoadhesive layer and the backing layer. The process liner was removed and the film was packed in air- and moisture protected individual sachets of size 8 cm×9.6 cm. Approximately 70 laminates were produced.

Example 3: Preparation of a Mucoadhesive Film with Solid Solution of Macrolide

[0139] Solubility of Tacrolimus

[0140] Tacrolimus was dissolved in an organic solvent. Particularly, the saturation solubility of tacrolimus in different organic solvents was tested. Crystalline active ingredient was added to a defined amount of solvent at room temperature while stirring until an insoluble sediment was formed. After filtration, the concentration of active ingredient was quantified by HPLC in the supernatant.

[0141] Tacrolimus was soluble in all tested organic solvents. For the further experiments relating to a solvent-based mucoadhesive film, ethanol was chosen as organic solvent. Non-toxicity and low boiling point—and thus the possibility of complete removal by drying at low temperatures—characterize the solvent ethanol.

TABLE-US-00005 TABLE 5 saturation solubility of tacrolimus. Solubility/ Boiling point/ mg/ml ° C. Water 0.00076 100 Acetic Acid 545 118 Ethanol 389  78 iso-Propanol 243 82,5 DMSO 582 189

[0142] Polymer Matrix for Solid Solution

[0143] For developing a mucoadhesive film comprising macrolide as solid solution, polymers were chosen that are soluble in organic solvents. Thus, it was possible to work in a water-free system. Sample films with an area of 6 cm.sup.2 were prepared from each of the matrices and tested for water solubility and adhesion.

[0144] For example, polymers soluble in ethanol having film-forming and mucoadhesive characteristics are crosslinked polyacrylic acid polymers, e.g. Carbopol®; copolymers of methyl vinyl ether and maleic anhydride, e.g. Gantrez™; poly(methacrylates), e.g. Eudragit®; polymers comprising polyvinyl acetate and/or polyvinylpyrrolidone, e.g. Kollidon® SR (8:2 mixture of poly(vinylacetate) and poly(vinyl pyrrolidone)).

[0145] Various combinations of polymers were tested:

TABLE-US-00006 TABLE 6 Development of an ethanol-based mucoadhesive layer. Ingredient/Excipient FK2 FK3 FK4 FK5 FK6 FK7 FK1 HPC Klucel EF 54.9% 56.2% 56.2% 51.7% 42.7% 42.7% 42.7% HPC Klucel GF  7.0%  7.0%  7.0% 10.0% 10.0% 10.0% 10.0% Carbopol 934 15.0%  7.5% Gantrez AN 169 15.0%  7.5%  8.5%  8.5% Gantrez MS-955  4.0%  4.0% EC (Aqualon N50NF) 10.0% 30.0% 34.5% 34.5% Glycerol 18.0% 18.0% 18.0% 16.0%  5.0%  5.0%  5.0% Titandioxid  3.8%  3.8%  3.8%  3.8%  3.8%  3.8%  3.8% NaCl  1.3% SUM  100%  100%  100%  100%  100%  100%  100% Solid content 20.0% Area weight/g/m.sup.2 104 150

[0146] The tested combinations successfully showed mucosa adhesion. Furthermore, the tested polymer combinations provided low water solubility.

[0147] Preparation of the Mucoadhesive Layer FK1

[0148] An ethanol based polymer mixture was prepared. Titanium dioxide was dispersed in the ethanol under agitation by stirring. Glycerol was added. The powdered polymers were slowly added, and stirring continued until complete dissolution. The active ingredient was dissolved in ethanol and stirred into the prepared polymer mixture to obtain a layer composition.

[0149] The layer composition was coated on a process liner (FO Scotchpak 9755) and subsequently dried. The targeted are weight of the film was about 150 g/m.sup.2. 500 g of the mixture were prepared. The concentration of solids in the mixture was 20% by weight.

[0150] Preparation of the Backing Layer

[0151] The polymer mixture for sealing was prepared as described above. 1000 g of the mixture were prepared. The concentration of solids in the mixture was 15% by weight.

[0152] The mixture was coated on a process liner and subsequently dried. The targeted are weight of the film was about 50 g/m.sup.2.

TABLE-US-00007 TABLE 7 Weighed portions for the production of the backing layer FK8. Weighed FK8 portion [g] Solid [wt.-%] EC 135 90% Glycerol 15 10% Ethanol 850 Total 1000 100% Solid content 15%

[0153] The backing layer and the mucoadhesive layer were joined via an adhesive intermediate layer.

Example 4: Preparation of an Intermediate Layer FK9

[0154] A mixture was prepared, coated on a process liner (FO Scotchpak 9755), and then dried. The targeted are weight of the film was about 60 g/m.sup.2. 1000 g of the mixture were prepared. The concentration of solids in the mixture was 55% by weight.

TABLE-US-00008 TABLE 8 Weighed portions for the production of the intermediate layer FK9. Weighed FK9 portion [g] Solid [wt.-%] Kollidon VA64 440 80% Glycerol 110 20% Ethanol 550 Total 1000 100% Solid content 55%

[0155] Alternatively, the intermediate layer comprised a blend of polyvinyl acetate and povidone (K 30) in the ratio 8:2, namely Kollidon® SR, instead of the vinylpyrrolidone-vinyl acetate copolymer Kollidon® VA64.

[0156] The 3 layers, namely mucoadhesive layer, intermediate layer, and backing layer, were laminated on top of each other by pressure. Rectangular films with 2×3 cm edge length were punched out of the finished three-layer laminate, the film was packed in individual sachets (Hutamaki COC) to protect it from air and moisture. About 150 laminates were produced.

Example 5: Composition of the Layers

[0157] The composition of the layers is shown below:

TABLE-US-00009 TABLE 9 Characteristics and composition of the layers Batch no MFK10 FK1 Function Appearance Square. Rectangular shaped with rounded corners Size 6 cm.sup.2 6 cm.sup.2 Length × width 2.48 cm × 3 cm × 2 cm 2.48 cm Target Area weight 154 g/m.sup.2 260 g/m.sup.3 Target Weight 92.4 156 mg/film mg/film Target macrolide content 5 mg/film 5 mg/film Mucoadhesive layer [mg/film] [mg/film] Tacrolimus 5.0 5.0 Active Substance Soluplus ® 150 — Solubilizer Ethyl cellulose (Aquaion N50NF) — 29.3 Matrix polymer Hydropropyl cellulose EF (Klucel EF) 19.9 36.3 Matrix polymer Hydropropyl cellulose GF (Klucel GF) — 8.5 Matrix polymer Gantrez MS.955 — 3.4 Mucoadhesive Sodium Carboxymethyl cellulose (Blanose 7LP) 13.3 — Matrix polymer Glycerol 7.7 4.3 Softening agent Titanium dioxide 1.6 3.2 Colorant Ethanol — / Process solvent Water / — Process solvent Target Area weight (dry) 104 g/m.sup.2 150 g/m.sup.2 Adhesive interlayer Kollidon VA64 — 2.88 Matrix polymer Glycerol — 0.72 Softening agent Ethanol — / Process solvent Area weight (dry) — 60 g/m.sup.2 Backing layer Ethyl cellulose (Aquaion N50NF) 2.7 2.697 Matrix polymer Glycerol 0.3 0.3 Softening agent Brilliant Blue q.s. 0.003 Colorant Ethanol / / Process solvent Target Area weight (dry) 50 g/m.sup.2 50 g/m.sup.2

Example 6: Characterisation of the Films

[0158] The prepared films were analyzed for content, release, unidirectional release, water content and solvent content. The analytical methods used are given in Table 10, the results of the surveys are given in Table 11.

[0159] To determine the macrolide release, the film was placed with the backing layer on a rotating cylinder (with the mucoadhesive layer facing the medium) and the amount of drug released into the medium at the indicated time points was quantified by HPLC. The medium used was 500 ml of phosphate buffer having pH 7 with 0.1% sodium dodecyl sulfate. The values are given as mean values; the minimum and maximum values of the n individual determinations are given in parentheses.

[0160] To determine the unidirectional release, the film was placed with the mucoadhesive layer on a rotating cylinder (with the backing layer facing the medium) and the amount of drug released into the medium was quantified after 10 mill, 20 min and 30 min as described above. The medium was the same as described above.

TABLE-US-00010 TABLE 10 Analytical methods used to characterize the films. Test Analytical method API content HPLC method gradient according to USP41: C18 RP HPLC column UV detection 30 or 60 min run time Dissolution Apparatus: Rotating cylinder or Paddle over sinker (Ph.Eur. 2.9.3) Strirring speed: 50 rpm, Medium: sodium hydrogen phosphate buffer pH 7, 0.1% SDS Volume: 500 ml medium HPLC method: isocratic, C18 RP HPLC column, UV detection Inverse dissolution MBF attached to the cylinder with the backing layer facing the dissolution medium Method see above Water content Karl Fischer oven method Ph.Eur. Residual solvents GC.FID method

[0161] Both films, i.e. the film comprising micronized crystalline macrolide and the film comprising molecularly dissolved macrolide (MFK10 and FK1, respectively), successfully released the macrolide almost completely. The macrolide release of MFK10 (FIG. 2), was not caused by the presence of Soluplus® (3-fold excess to the active ingredient).

[0162] Only a small amount of tacrolimus (max. 5%) was released via the edges of the film. The backing layer efficiently prevented the release of tacrolimus. Accordingly, a release of the active ingredient into the oral cavity—and thus a potential swallowing of the active ingredient—can be efficiently prevented by the backing layer.

[0163] A small amount of ethanol was detected in both films. The value determined was below the permissible amount defined by the ICH for ethanol and is therefore suitable for in vivo application. Acetone was not detected in either of the films.

[0164] The water content of the film comprising micronized crystalline macrolide, e.g. MFK10, was less than 3%; the film comprising molecularly dissolved macrolide, e.g. FK1, was produced without the use of water and thus the water content was not analyzed.

[0165] Low water content and low residual solvent content of both films prove efficient drying of the laminate.

TABLE-US-00011 TABLE 11 Results of the characterization of the films. Batch no MFK10 FK1 Area weight [g/m.sup.2] 150.3.155.7 API content [mg/film, %] 4.35 mg/film 4.7 mg/film 84.4% 94% (RSD 1.18%; n = 3) Dissolution [%] n = 6 (Min.Max) 10 min 64% (60.67%) 14% (11.20%) 20 min 59% (54.64%) 44% (39.54%) 30 min 62% (57.69%) 57% (51.64%) 60 min   69% (67.72%) 120 min   69% (66.73%) Inverse dissolution [%] n = 6 (Min . Max) 10 min 2.4% (1.8.2.9%) not tested 20 min 4.2% (3.1.6.1%) not tested 30 min 4.4% (3.6.64%) not tested Water content [%] 2.9% not tested Residual solvents [%] Ethanol 1.9% 5.2% Acetone     RSD: relative standard deviation of n die.cuts

Example 7: Stability of the Laminates

[0166] Both laminates, i.e. the film comprising micronized crystalline macrolide and the film comprising molecularly dissolved macrolide (MFK10 and FK1, respectively), were stored under temperature and humidity control and analytically characterized after the indicated time points. The results are reported in Table 12 and Table 13.

TABLE-US-00012 TABLE 12 Stability of the laminate MFK10 when stored at room temperature. MFK10 MFK10 Batch no After production 6 weeks at 21° C. Weight 91.4 mg/film 90.5 mg/film (RSD 0.23%, n = 3) (RSD 0.32%, n = 3) API content 4.35 mg/film 4.16 mg/film (95.8% of initial value) Dissolution n = 6 (Min.Max) 10 min 64% (60.67%) 44% (29.57%) 20 min 59% (54.64%) 57% (51.60%) 30 min 62% (57.69%) 55% (52.58%)

[0167] The macrolide continent of the laminate MFK10 decreased by only 4.2% within 6 weeks when stored at room temperature due to degradation of the active ingredient.

TABLE-US-00013 TABLE 13 Stability of the laminate FK1 when stored under conditions of accelerated aging. After 2 weeks at 1 month at FK1 production 25° C. 25° C. Sum of degradation products 0.4% Not tested 0.8% Difference to initial value 0 Not tested 0.4% After 2 weeks at . 1 month at FK1 production 40° C 40° C. Sum of degradation products 0.4% 1.5% 3.8% Difference to initial value 0 1.1% 34%

[0168] After two weeks of storage of the film comprising molecularly dissolved macrolide (laminate FK1) at 40° C., 1.5% of degradation products were detected. After production, this batch contained 0.4% degradation products. Thus, 1.1% of degradation products were formed by 2 weeks of storage at 40° C., and 3.4% of degradation products after 4 weeks of storage at 40° C.

TABLE-US-00014 TABLE 14 Comparison of the stability of the laminates MFK10 and FK1. Batch no Macrolide degradation MFK10 −4.2% in 6 weeks at room temperature FK1 −0.4% in 4 weeks at room temperature −3.4% in 4 weeks at 40° C.

[0169] Accordingly, the water free formulation of the film comprising molecularly dissolved macrolide (laminate FK1) even further increases the chemical stability of the film. Copolymers of methyl vinyl ether and maleic anhydride, e.g. Gantrez™, even further enhances the chemical stability of the film due to its complexing and stabilizing characteristics.

Example 8: Morphology of the Active Agent

[0170] The film MFK10 was characterized radiographically. Samples of the active ingredient, micronized Tacrolimus, and samples of the colorant titanium dioxide served as controls.

[0171] The XRPD image (FIG. 3) of the film comprising micronized crystalline tacrolimus (MFK10; red, 3) shows the characteristic reflections of the crystalline tacrolimus (blue, 1) and the excipient TiO.sub.2 (orange, 2). The reflection detected at about 28° can be assigned to silicon, which was used as substrate for the XRPD measurement.

[0172] As shown in FIG. 3, the morphology of the micronized crystalline macrolide did not change by the incorporation of the micronized crystalline macrolide into the film (MFK10).

Example 9: In Vivo Application (First Study)

[0173] The pig was chosen as animal model, since this animal model is very similar to the functional-anatomical relationships of humans. All animals were 6 to 8 months old at the time of treatment with a body weight between 14 and 16 kg. Animals were fasting (overnight) for at least 16 hours before treatment and anesthetized during treatment. The film was placed with the mucoadhesive layer side on the mucosa in the animal's mouth and lightly pressed until the film adhered. The film remained on the buccal mucosa for 30 min and was then removed. Blood samples were collected via a venous catheter. For determination of blood levels of tacrolimus, 2 ml of blood was collected from all animals into K2EDTA-coated tubes. Samples were collected at the following time points: immediately before administration (o min), then 15, 30, 60, 90, 120, 180, and 240 min, and 6, 8, 12, 18, and 24 h after administration of the film. After blood collection, the blood tubes were cooled on ice and stored in a freezer (at a temperature in the range of −30° C. to −15° C.) until analysis. The concentration of tacrolimus was determined by a reliable LC-MS/MS method.

[0174] After application in animals, the tacrolimus content of the applied film was determined (drug content post treatment). The active dose corresponds to the tacrolimus content of the film minus the residual content after application. The biologically active dose was calculated in relation to the weight of the individual animal (active dose/body weight).

[0175] Pharmacokinetic parameters were calculated from the time course of blood concentration. The calculated AUC was normalized with the active dose and set in relation to the individual weight of the animal (AUC_all/dose).

TABLE-US-00015 TABLE 15 Application of MFK10 in three pigs: blood level of tacrolimus and pharmacokinetic parameters. Animal ID #1 #2 #1 #2 #3 Species Goettingen Minipig. naïve Sex Female Body weight/kg 15.0 14.7 1.5.6 15.6 16.6 Age/months 7.8 7.8 8.9 8.9 8.9 Date of treatment 28 Oct. 2020 28 Oct. 2020 17 Nov. 2020 17 Nov. 2020 17 Nov. 2020 Dosage form MFK10 MFK10 Prograf Prograf Prograf capsule capsule capsule Dose/mg 4.35 4.35 5 5 5 Drug content post 2.36 2.54 — — — treatment/mg (54.3%) (58.4%) (% of dose) Active dose/mg 1.99 1.81 5 5 5 time/hours Blood concentration/ng/ml 0 0 0 0 0 0 0.25 0 0 0.162 0.0775 0.177 0.5 0 0 0.675 0.96 0.903 1 0.36 0 3.39 3.57 1.78 1.5 0.556 0.0625 7.49 4.20 1.90 2 0.473 0.101 9.04 4.15 2.36 3 0.253 0.0855 8.40 3.85 3.20 4 0.158 0.069 8.54 2.75 4.03 6 0.209 0.0608 7.74 2.90 3.27 8 0.142 0 5.41 2.97 2.79 12 0.161 0 3.80 2.25 2.03 18 0.153 0 2.50 1.40 1.6 24 0.104 0 1.99 1.12 2.08 Pharmacokinetic parameters T_max/hours 1.5 2 2 1.5 4 C_max/ng/ml 0.556 0.101 9.04 4.20 4 AUC_all/h * ng/ml 4.18 0.418 105 531 54.1 Active dose/body weight/ 0.133 0.123 0.321 0.321 0.301 mg/kg AUC_aIl/dose (h * 31.5 3.39 329 166 180 ng/ml/mg/kg) Intraindividual 9.6% 2.0% 100% 100% comparison: AUC_all/dose of MFKio/Prograf

[0176] The residual macrolide levels of the films were between 54.3% and 58.4%. Tacrolimus was successfully detected in the blood of both pigs after buccal administration; the maximum concentration was reached after 1.5 and 2 hours, respectively (t_max). The film did not cause local irritation of the buccal mucosa.

[0177] These results demonstrate that a buccal resorption of micronized crystalline macrolides is effective.

[0178] There was no treatment-related mortality, clinical signs or changes in body weights. There was no irritation at the application sites of MBF loaded with micronized tacrolimus.

[0179] Mean blood concentrations-time profiles from the three animals following buccal application of MBF loaded with micronized tacrolimus, and oral administration of a 5 mg Prograf capsule. It was successfully demonstrated that micronized tacrolimus can pass the buccal mucosa following application as MBF.

Example 10: In Vivo Application (Second Study)

[0180] The first study was repeated with a film comprising molecularly dissolved macrolide (FK1) with five further animals. The animals were not the same animals as tested in the first study.

[0181] The film remained on the buccal mucosa for 60 min and was then removed. The further procedure was analogous to the first study.

TABLE-US-00016 TABLE 16 Application of FK1 in five pigs: blood level of tacrolimus and pharmacokinetic parameters. Animal ID #1 #2 #3 #4 #5 Species Goettingen Minipig. naïve Sex Female Body weight/kg 16.0 14.1 15.8 14.4 14.8 Age/months 6.7 6.7 6.7 6.7 6.7 Date of treatment 17 May 2021 17 May 2021 17 May 2021 17 May 2021 17 May 2021 Dosage form FK1 Dose/mg 4.7 4.7 4.7 4.7 4.7 Drug content post treatment/ 2.1 2.2 1.9 2.3 2.1 mg Active dose/mg 2.6 2.5 2.8 2.4 2.6 time/hours Blood concentration/ng/ml 0.25 0 0 0 0 0 0.5 0.0916 0.127 0.201 0 0.104 1 0.88 1.08 1.4 0.655 1.01 1.5 2.3 2.95 4.24 1.71 2.52 2 2.74 4.44 4.72 2.37 3.89 3 1.9 4.4 3.52 1.71 3.98 4 1.2 2.83 2.02 0.995 2.87 6 0.655 1.8 1.08 0.554 2.67 8 0.406 0.822 0.756 0.366 0.948 12 0.29 0.473 0.468 0.23 0.643 18 0.188 0.317 0.313 0.176 0.417 24 0.132 0.255 0.245 0.115 0.306 Pharmacokinetic parameters T_max/hours 2 2 2 2 3 C_max/ng/ml 2.74 4.44 4.72 2.37 3.98 AUC_all/h * ng/ml 12.9 25.1 22.4 11.0 27.8 Active dose/body weight/ 0.163 0.177 0.177 0.167 0.176 mg/kg AUC_all/dose (h *ng/ml/ 79.3 142 126 65.7 158 mg/kg)

[0182] The analysis of the drug content of the films post treatment shows that about 55% of the macrolide is biologically active and 45% remains in the film.

[0183] Tacrolimus was detected in the blood of all five pigs after buccal application (FIG. 4). The maximum concentration was reached after 2 to 3 hours (t_max). The film did not cause local irritation of the buccal mucosa.

[0184] The blood concentrations achieved were in the range of those obtained with oral administration of the commercial product Prograf. The time necessary for reaching maximum blood levels, namely 2-3 hours, is comparable to the commercial product. Accordingly, both the film comprising micronized crystalline macrolide and the film comprising molecularly dissolved macrolide allowed to effectively administer macrolide. Furthermore, unexpectedly, the films allowed to achieve blood level concentrations of macrolide which are suitable for in vivo application to a patient.

Example 11: Further Optimization of the MBF for Preventing Migration of Macrolide into the Backing Layer

[0185] The backing layer was based on ethyl cellulose dissolved in ethanol and was casted on the dried mucoadhesive layer containing Tacrolimus. Because of the excellent solubility of Tacrolimus in ethanol, a part of the active substance was extracted into the backing layer during the manufacturing.

[0186] The composition of the MBF and the manufacturing process were optimized by including an intermediate layer, particularly an adhesive interlayer, between the mucoadhesive layer and backing layer. The combined backing layer and adhesive interlayer were dried before lamination on the dried mucoadhesive layer (FIG. 5). Experiments using this revised manufacturing process and MBF composition showed that no Tacrolimus is detectable in the backing layer/adhesive interlayer, as examined using a dissolution test and HPLC. Accordingly, an adhesive interlayer prevents macrolide dissipation from the adhesive layer into the backing layer.

Example 12: In Vivo Application (Third Study)

[0187] Further variants of the MBF were developed. In all cases Tacrolimus was incorporated molecularly dissolved into the film matrix.

[0188] The composition of the layers is shown below:

TABLE-US-00017 TABLE 17 Characteristics and composition of the layers Batch no FK14 FK15 FK16 Function Appearance Rectangular Rectangular Rectangular Size 6 cm.sup.2 6 cm.sup.2 4.4 cm.sup.2 Length × width 3 cm × 2 cm 3 cm × 2 cm 2.1 cm × 2.1 cm Target Area weight Target Weight Target API content 10 mg/film 5 mg/film 5 mg /film Mucoadhesive layer [mg/film] [mg/film] [mg/film] Tacrolimus 10.0 5.0 5.0 Active Substance Hydropropyl cellulose EF (Klucel EF) 33.4 35.6 25.5 Amphiphilic film former (Matrix polymer) Hydropropyl cellulose GF (Klucel GF) 7.2 7.7 5.5 Amphiphilic film former (Matrix polymer) Ethylcellulose (Aqualon N50NF) 26.9 28.6 20.5 Lipophilic film former Gantrez MS 955 3.2 3.4 2.4 Mucoadhesive Polyvinyl acetate/povidone 2.3 2.3 1.6 Hydrophilic film (Kollidon VA64) former, solubilizer Glycerol 4.0 4.3 3.0 Softening agent Titanium dioxide 3.0 3.2 2.3 Colorant Ethanol / / / Process solvent Target Area weight (dry) 150 g/m.sup.2 150 g/m.sup.2 150 g/m.sup.2 Adhesive interlayer FK17 FK17 FK17 Kollidon SR 33.6 33.6 24.6 Lipophilic film former (Matrix polymer) Ethylcellulose (Aqualon N50NF) 2.1 2.1 t.5 Lipophilic film former Glycerol 6.3 6.3 4.6 Softening agent Ethanol / / / Process solvent Area weight (dry) 70 g/m.sup.2 70 g/m.sup.2 70 g/m.sup.2 Backing layer FK18 FK18 FK18 Ethylcellulose (Aqualon N50NF) 18.0 18.0 13.2 Lipophilic film former (Matrix polymer) Kollidon SR 9.0 9.0 6.6 Lipophilic film former (Matrix polymer) Triacetin 3.0 3.0 2.2 Softening agent Brilliant Blue 0.03 0.03 0.02 Colorant Ethanol/Aceton (80:20) / / / Process solvents Target Area weight (dry) 50 g/m2 50 g/m.sup.2 50 g/m.sup.2 1

[0189] The study was performed with five pigs which were the identical individuals as in the second study (example 10). The film remained on the buccal mucosa for 60 min and was then removed. The further procedure was analogous to the previous studies. In the session 1, two films were placed at the same time and same side of the oral cavity. In the sessions 2, 3 and 4, one film was placed to the one side of the oral cavity in the morning hours and after approx. 4 hours the second one was placed on the same side. Thus, session 1 analyzed a treatment once a day, and sessions 2, 3, and 4 analyzed a twice-a-day treatment.

TABLE-US-00018 TABLE 18 Design of third study Parameter Session 1 Session 2 Session 3 Session 4 Batch number FK14 FK14 FK15 FK16 Formulation No. Residence time 60 min 60 min 60 min 60 min Film area 6 cm.sup.2 6 cm.sup.2 6 cm.sup.2 4.4 cm.sup.2 Dose per animal 19 mg 19 mg 9.8 mg 9.6 mg Dose per film 9.5 mg 9.5 mg 4.9 mg 4.8 mg Treatment once a day twice a twice a twice a day.4 h day.4 h day.4 h between between between Application site Unilateral unilateral unilateral Unilateral Area concentration/mg/cm.sup.2 1.58 1.58 0.82 1.09 Pharmacokinetic parameters (mean of 5 animals) Active dose/% of dose per film 23% 25% 30% 36% AUC/h * ng/ml 21 35 14 16 AUC /active dose/body weight (h * ng/ml/ 123 152 103 116 mg/kg)

TABLE-US-00019 TABLE 19 Third study, Session 1: Blood concentration of Tacrolimus and pharmacokinetic parameters Animal ID #1 #2 #3 #4 #5 Species Gottingen Minipig. Non-naïve Sex Female Body weight/kg 19.3 22.3 22.9 19.4 20.7 Dosage form Dose/mg 19 19 19 19 19 Drug content post treatment/mg 12.1 16.9 9.9 17 17 Active dose/mg 6.9 2.1 9.1 2 2 time/hours Blood concentration/ng/ml 0 0 0 0 0 0 0.5 0.337 0.0878 0.363 0.174 5.06 1 1.9 0.633 2.07 1.07 2.12 1.5 5.28 1.9 5.67 2.26 2.31 2 4.75 2.87 6.51 2.61 2.37 3 2.71 2.86 6.34 1.97 1.65 4 1.65 1.75 3.86 1.2 1.12 6 0.811 0.828 2.07 0.596 0.6 8 0.561 0.552 1.42 0.412 0.426 12 0.488 0.342 1.06 0.285 0.293 18 0.345 0.223 0.737 0.222 0.194 24 0.268 0.195 0.564 0.156 0.146 Pharmacokinetic parameters T_max/hours 1.5 2 2 2 2 C_max/ng/ml 5.28 2.87 6.51 2.61 5.06 AUC_all/h * ng/ml 21.13 15.89 40.88 13.13 15.40 Active dose/body weight/mg/kg 0.358 0.094 0.397 0.103 0.097 AUC_all/active dose (h * 59 169 102.9 127 159 ng/ml/mg/kg)

TABLE-US-00020 TABLE 20 Third study, Session 2: Blood concentration of Tacrolimus and pharmacokinetic parameters Animal ID #1 #2 #3 #4 #5 Species Gottingen Minipig. Non-naïve Sex Female Body weight/kg 19.3 21.8 22.4 19.8 20.6 Dosage form Dose/mg 19 19 19 19 19 Drug content post treatment/mg 13.4 11.3 14.9 16.3 15.3 Active dose/mg 5.6 7.7 4.1 2.7 3.7 time/hours Blood concentration/ng/ml 0 0 0 0 0 0 0.5 0 0 0 0 0 1 0.106 0.204 0.181 0.138 1.5 0.298 0.717 0.578 0.508 0.209 2 0.453 1.22 1.06 0.658 0.337 3 0.423 0.793 0.953 0.561 0.458 4 0.296 (*) 0.489 (*) 0.628 0.405 0.341 6 4.23 9.69 5.3 4.5 1.34 8 2.46 4.51 3.26 2.02 1.33 12 1.56 2.77 1.47 0.856 0.564 18 0.928 1.75 0.808 0.478 0.321 24 0.677 1.29 0.607 0.332 0.204 Pharmacokinetic parameters C_max/ng/ml 4.23 9.69 5.3 4.5 1.34 AUC_all/h * ng/ml 32.65 64.03 37.47 25.19 13.40 Active dose/body weight/mg/kg 0.290 0.353 0.183 0.136 0.180 AUC_all/active dose (h * 113 181 204.7 185 75 ng/ml/mg/kg) (*) removed 45 min after placing due to the damage of the film

TABLE-US-00021 TABLE 21 Third study, Session 3: Blood concentration of Tacrolimus and pharmacokinetic parameters Animal ID #1 #2 #3 #4 #5 Species Gottingen Minipig. Non-naïve Sex Female Body weight/kg 20.3 22.6 23.6 20.8 21.6 Dosage form Dose/mg 9.8 9.8 9.8 9.8 9.8 Drug content post treatment/mg 6.1 6.4 7 7.6 7 Active dose/mg 3.7 3.4 2.8 2.2 2.8 time/hours Blood concentration/ng/ml 0 0 0 0 0 0 0.5 0 0.109 0 0 0 1 0.188 0.21 0.138 0.137 0.176 1.5 0.642 0.635 0.468 0.556 0.601 2 0.944 0.868 0.715 0.83 0.817 3 0.681 0.709 0.659 0.987 0.746 4 0.455 0.492 0.607 0.92 0.527 6 2.83 1.4 1.18 2.18 1.13 8 1.27 0.887 0.685 1.32 0.98 12 0.533 0.421 0.314 0.515 0.3841 18 0.374 0.265 0.224 0.311 0.25 24 0.241 0.21 0.161 0.206 0.176 Pharmacokinetic parameters C_max/ng/ml 2.83 1.4 1.18 2.18 1.13 AUC_all/h * ng/ml 17.59 12.36 10.22 16.72 11.69 Active dose/body weight/mg/kg 0.182 0.150 0.119 0.106 0.130 AUC_all/active dose (h * 96 82 86.1 158 90 ng/ml/mg/kg)

TABLE-US-00022 TABLE 22 Third study, Session 4: Blood concentration of Tacrolimus and pharmacokinetic parameters Animal ID #1 #2 #3 #4 #5 Species Gottingen Minipig. Non-naïve Sex Female Body weight/kg 21.8 24.3 25.2 23.4 25.1 Age/months Dosage form Dose/mg 9.6 9.6 9.6 9.6 9.6 Drug content post treatment/mg 5.1 4.6 7.4 6.7 7 Active dose/mg 4.5 5 2.2 2.9 2.6 time/hours Blood concentration/ng/ml 0 0 0 0 0 0 0.5 0 0 0 0 0 1 0.229 0.147 0.267 0.392 0.0704 1.5 0.681 0.83 0.732 0.702 0.318 2 0.966 1.43 1.28 0.895 0.578 3 0.844 1.37 1.39 0.82 0.733 4 0.616 0.819 1.08 0.61 0.609 6 1.71 3.64 1.35 1.7 0.87 8 0.994 1.89 0.925 1.01 0.613 12 0.526 1.04 0.528 0.501 0.323 18 0.336 0.581 0.36 0.302 0.203 24 0.224 0.411 0.21 0.194 0.133 Pharmacokinetic parameters C_max/ng/ml 1.71 3.64 1.39 1.7 0.87 AUC_all/h * ng/ml 14.67 27.03 15.37 14.28 909 Active dose/body weight/mg/kg 0.206 0.206 0.087 0.124 0.104 AUC_all/active dose (h * 71 131 176.1 115 88 ng/ml/mg/kg)

[0190] Interestingly, a treatment twice a day achieved higher blood concentrations than a treatment once a day, although the same dose was applied with both treatments. Particularly, as seen in the tables above, a higher AUC was achieved with a treatment twice a day than with a once daily treatment. Moreover, the achieved blood concentrations after the second application (4 hours after the first application) were much higher than after the first application although the same MBF was applied. Such pharmacokinetic properties of the mucoadhesive film are highly advantageous for fine-tuning a dosing scheme to a patient's needs. For example, the mucoadhesive film can be administered twice daily to patients requiring a higher blood concentration, while the mucoadhesive film can be administered once daily to patients with a need for lower blood concentrations. Furthermore, the time between the two applications can be adjusted to the individual need of a patient. For example, a suitable macrolide blood concentration of a patient can be fine-tuned and controlled by adjusting the time window between two subsequent applications of a mucoadhesive film.

[0191] Furthermore, interestingly, it was observed that a higher blood concentration was achieved with a mucoadhesive film having a higher area concentration. The pharmacokinetic properties of the mucoadhesive film are highly advantageous for fine-tuning a dosing scheme to a patient's needs. The inventors have observed that mucoadhesive films comprising non-nanoparticulate macrolide, particularly in the form of micronized macrolide or in a molecularly dissolved non-nanoparticulate form, allow to achieve higher blood concentrations than mucoadhesive films comprising nanoparticulate macrolide.

[0192] The features of the present invention disclosed in the specification, the claims, and/or in the accompanying figures may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.