TETRACYCLINE COMPLEXES WITH SUSTAINED ACTIVITY
20210401996 · 2021-12-30
Inventors
- Andreas KIESOW (Halle, DE)
- Mirko BUCHHOLZ (Halle, DE)
- Sandra SAREMBE (Halle, DE)
- Karsten MÄDER (Leipzig, DE)
- Martin KIRCHBERG (Halle, DE)
- Sigrun EICK (Bolligen, CH)
Cpc classification
A61K47/34
HUMAN NECESSITIES
C07C2603/46
CHEMISTRY; METALLURGY
C07C237/24
CHEMISTRY; METALLURGY
A61K47/593
HUMAN NECESSITIES
A61K47/69
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
A61K47/59
HUMAN NECESSITIES
Abstract
The present invention generally relates to controlled release of tetracycline antibiotics. Specifically, it relates to a complex comprising a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate; a pharmaceutical preparation comprising the complex, methods for manufacturing the complex and the pharmaceutical preparation, and a complex or a pharmaceutical preparation for use in a method for treatment of the human or animal body, in particular for therapy and/or prophylaxis of a bacterial infection; and/or wherein antibiotic activity is maintained over a prolonged period of time; and/or for the therapy and/or prophylaxis of an acute, chronic or recurrent periodontal disease.
Claims
1. A complex formed of a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA.sub.2), wherein: the molar ratio TC:MA.sub.2 is in the range of 1:0.8-3.0; the divalent metal cation M is an earth alkaline metal cation; and A is a carboxylate anion derived from a C.sub.8-C.sub.24 carboxylic acid, wherein the tetracycline compound (TC) is selected from minocycline, doxycycline, chlortetracycline, rolitetracycline, tigecycline, demeclocycline, lymecycline, meclocycline, methacycline, omadacycline, sarecycline, eravacycline, clomocycline, 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline and 9-(N,N-dimethylglycylamido)-minocycline.
2. The complex according to claim 1, wherein: the ratio TC:MA.sub.2 is 1:2; or M is selected from Mg.sup.2+ and Ca.sup.2+; or A is a carboxylate anion derived from a carboxylic acid selected from arachidic acid (C.sub.20), stearic acid (C.sub.18), palmitic acid (C.sub.16), myristic acid (C.sub.14), lauric acid (C.sub.12), capric acid (C.sub.10), caprylic acid (C.sub.8), hydroxystearic acid, oleic acid, linoleic acid, linolenic acid and ricinoleic acid.
3. The complex according to claim 1, wherein: the ratio TC:MA.sub.2 is 1:2; and M is selected from Mg.sup.2+ and Ca.sup.2+; and A is a carboxylate anion derived from a carboxylic acid selected from arachidic acid (C.sub.20), stearic acid (C.sub.18), palmitic acid (C.sub.16), myristic acid (C.sub.14), lauric acid (C.sub.12), capric acid (C.sub.10), caprylic acid (C.sub.8), hydroxystearic acid, oleic acid, linoleic acid, linolenic acid and ricinoleic acid.
4. The complex according to claim 1 having the formula [(TC).2(MgA.sub.2)], wherein the tetracycline compound (TC) is minocycline or a pharmaceutically acceptable salt, hydrate or solvate thereof.
5. A pharmaceutical preparation comprising the complex according to claim 1 and optionally one or more pharmaceutically acceptable excipient(s).
6. The pharmaceutical preparation according to claim 5, which is; i) arranged for topical administration; or ii) is selected from the group consisting of extrudate, particle, granule, powder, film, strip, compact, chip, paste, cream, gel, emulsion, suspension, liniment, ointment, balm, lotion, eye drops, spray, topical aerosol, topical solution, topical suspension, skin patch and nonwoven; or iii) or both.
7. The pharmaceutical preparation according to claim 5, i) further comprising, as an excipient, one or more biodegradable polymer(s) selected from biodegradable polyesters, mixed biodegradable polyesters, biodegradable PEGylated diblock (AB) or triblock (ABA or BAB) copolymers, and pectins; or ii) wherein the total content of the tetracycline compound (TC) is in the range of 5-20 wt. %; or iii) both.
8. The pharmaceutical preparation according to claim 7, wherein the biodegradable polymer(s): i) have a weight average molecular weight range of 4-250 kDa; ii) have a glass transition temperature of 42-48° C.; iii) have an inherent viscosity [dl/g] (0.1% in CHCl.sub.3 at 25° C.) of 0.03-1.70; or iv) are PLGA copolymers (poly(D,L-lactide-co-glycolides)) having a L/G ratio of 5/95-95/5; or v) combinations thereof.
9. The pharmaceutical preparation according to claim 7, wherein the biodegradable polymer(s) are PLGA copolymers; i) having a L/G ratio of 25/75-75/25; ii) having a weight average molecular weight range (kDa) of 5-90; iii) having an inherent viscosity [dl/g] (0.1% in CHCl.sub.3 at 25° C.) of 0.14-0.82; or iv) being terminated with ester, alkyl ester or PEG groups; v) or combinations thereof.
10. The pharmaceutical preparation according to claim 7, wherein the biodegradable polymer(s) are poly(D,L-lactide-co-glycolides) with a PLA/PGA ratio of 50:50, having a weight average molecular weight range of 7,000-38,000 and/or an inherent viscosity [dl/g] (0.1% in CHCl.sub.3 at 25° C.) of 0.16-0.44, or both, or poly(D,L-lactide-co-glycolides) with a PLA/PGA ratio of 50:50 and a PEG end group (PLGA-PEG), having a weight average molecular weight range of 30-85 kDa or an inherent viscosity [dl/g] (0.1% in CHCl.sub.3 at 25° C.) of 0.45-0.80, or both.
11. The pharmaceutical preparation according to claim 5, which is in the form of a strand-shaped extrudate.
12. The pharmaceutical preparation according to claim 11 having: i) an essentially circular or an essentially elliptical cross section; or ii) a maximum cross-sectional diameter of 0.1-10 mm, 0.2-5 mm, 0.3-1 mm, 0.4-0.8 mm, 0.3-0.6 mm, 0.2-5 mm, 0.3-1 mm, 0.6-0.9 mm, 0.4-0.8 mm, 0.3-0.6 mm, 0.3 mm, 0.4 mm, 0.5 mm or 0.6 mm; or iii) combinations thereof.
13. A method for manufacturing the complex according to claim 1, the method comprising the following steps: (a) mixing a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA.sub.2) in a molar ratio of 1:0.8-3.0 with an organic solvent to obtain a dispersion, wherein the divalent metal cation M is an earth alkaline metal cation and A is a carboxylate anion derived from a C.sub.8-C.sub.24 carboxylic acid; (b) heating said dispersion to form the complex; and (c) removing the solvent to obtain the complex.
14. A method for manufacturing the pharmaceutical preparation according to claim 11, the method comprising the following steps: (d) comminuting the complex according to claim 1 and, if present, one or more pharmaceutically acceptable excipients to obtain an extrusion precursor; (e) extruding said extrusion precursor at a temperature above room temperature; and (f) cooling the product of step (e) to obtain the pharmaceutical preparation in the form of a strand-shaped extrudate.
15-19. (canceled)
20. A method for treatment of a human or animal in need thereof comprising administering a therapeutically effective amount of the complex according to claim 1 to the human or animal.
21. The method according to claim 20, wherein the method is for therapy or prophylaxis, or both of (i) a bacterial infection; or (ii) a bacterial infection caused by one or more bacteria selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Streptococcus gordonii, Fusobacterium nucleatum, Actinomyces naeslundii and Parvimonas micra.
22. The method according to claim 21, wherein antibiotic activity is maintained over a period of at least 21 days.
23. The method according to claim 20, wherein the method is for therapy or prophylaxis, or both of an acute, chronic or recurrent periodontal disease.
24. The method according to claim 23, wherein the periodontal disease is selected from dental plaque-induced gingival diseases, periodontitis, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotizing periodontal diseases, abscesses of the periodontium, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections.
25. The pharmaceutical preparation according to claim 7, wherein the biodegradable polyesters are selected from poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polyhydroxybutyric acid (PHB) and polycaprolactone (PCL); the mixed biodegradable polyesters are selected from PLA-PCL and PLGA-PCL; and the biodegradable PEGylated diblock (AB) or triblock (ABA or BAB) copolymers are selected from PEG-PLA, PEG-PLGA, PEG-PCL and PEG-PCL-PLGA.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
Complexes
[0048] The present invention provides a complex comprising a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA.sub.2), wherein: the molar ratio TC:MA.sub.2 is in the range of 1:0.8-3.0; the divalent metal cation M is an earth alkaline metal cation; and A is a carboxylate anion derived from a C.sub.8-C.sub.24 carboxylic acid.
[0049] A major problem with the controlled release of tetracyclines represents their chemical instability in water. For example, minocycline shows epimerization reactions with loss of activity. Also doxycycline degrades in an aqueous environment. Therefore, drug degradation prior release should be avoided. We established the hypothesis that the formation of a lipophilic complex might increase the stability of the drug and lead to prolonged release times.
Tetracycline Compounds
[0050] As used herein, the term “tetracycline compound” (TC) refers to any compound belonging to the family of tetracycline antibiotics, i.e. based on the 6-deoxy-6-demethyltetracycline minimum pharmacophore structure:
##STR00003##
[0051] Preferably, the tetracycline compound (TC) is selected from minocycline, doxycycline, tetracycline, chlortetracycline, oxytetracycline, rolitetracycline, tigecycline, demeclocycline, lymecycline, meclocycline, methacycline, omadacycline, sarecycline and eravacycline. Preferred tetracycline compounds are also selected from the group consisting of 7-chlortetracycline, 5-hydroxytetracycline, tetracycline, 6-demethyl-7-chlortetracycline, 2-N-pyrrolidinomethyltetracycline, 2-N-lysinomethyltetracycline, N-methylol-7-chlortetracycline, 6-methylene-5-hydroxytetracycline (methacycline), 6-deoxy-5-hydroxytetracycline (doxycycline), 7-dimethylamino-6-demethyl-6-deoxytetracycline (minocycline), 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline, 9-(N,N-dimethylglycylamido)-minocycline, 9-(t-butylglycylamido)-minocycline (tigecycline), as further described in Table 2 of Chopra et al., Microbiol Mol Biol Rev. 2001, June 65(2):232-60). Further preferred tetracycline compounds include clomocycline (N-methylol-7-chlortetracycline), 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline and 9-(N, N-dimethylglycylamido)-minocycline.
[0052] It should be noted that the term “tetracycline compound” or “TC” as used herein denotes the general class of compounds, namely any compound belonging to the family of tetracycline antibiotics, whereas the term “tetracycline” only denotes the specific, preferred compound (4S,4aS,5aS,12aS)-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide:
##STR00004##
[0053] Preferably, the tetracycline compound (TC) is selected from minocycline, doxycycline, tetracycline, chlortetracycline, oxytetracycline, rolitetracycline, tigecycline, demeclocycline, lymecycline, meclocycline, methacycline, omadacycline, sarecycline, eravacycline, clomocycline, 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline and 9-(N,N-dimethylglycylamido)-minocycline. These antibiotics have the advantage of being safe, clinically established and thus reliable and likely to be suitable for various therapeutic applications as described herein.
[0054] Most preferably, the tetracycline compound is minocycline (7-dimethylamino-6-demethyl-6-deoxytetracycline or (4S,4aS,5aR,12aR)-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide) or doxycycline (6-deoxy-5-hydroxytetracycline or (4S,4aR,5S,5aR,6R,12aR)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide), and yet further preferably minocycline.
##STR00005##
[0055] These antibiotics have the additional advantage that their applicability in the treatment of periodontal diseases is clinically established.
Molar Ratio
[0056] The molar ratio TC:MA.sub.2 is preferably in the range of 1:0.8-3.0. The present inventors found that the predominate species obtained when preparing the complexes according to the present invention are those wherein TC:MA.sub.2 is 1:1 or 1:2. Thus, it is intended that also mixtures wherein the molar ratio TC:MA.sub.2 is between 1:1 and 1:2 are also within the scope of the present invention. A minor excess of TC, e.g., wherein TC:MA.sub.2=1:0.8, is also within the scope of the present invention. Similarly, an excess of MA.sub.2, e.g., beyond a ratio of TC:MA.sub.2 of 1:2 or even 1:3, is also considered to be within the scope of the present invention, regardless of whether the excess metal cations are coordinated to the TC. Thus, also such mixtures containing complexes with a defined ratio of TC:MA.sub.2 together with a certain excess of one of the components are also intended to be covered by the definition of the complex according to the present invention. Preferably, TC:MA.sub.2 is 1:1 or 1:2; more preferably TC:MA.sub.2 is 1:2. Further preferable ranges for the molar ratio TC:MA.sub.2 are, for instance, 1:0.7-1:1.2, 1:0.7-1:1.1, 1:0.8-1:1.1, 1:0.9-1:1.2, 1:0.9-1:1.1, 1:1.1-1:2.9, 1:1.4-2.5, and 1:1.8-1:2.2; more preferably 1:0.9-1:1.1 and/or 1:1.9-1:2.1.
Carboxylate Anion
[0057] A is a carboxylate anion derived from a C.sub.8-C.sub.24 carboxylic acid. The lipophilic nature of the carboxylate anion contributes to improving the sustained release of the tetracycline antibiotic from the complexes and preparations according to the present invention.
[0058] The carboxylic acid can be saturated or unsaturated. Preferably, A is a carboxylate anion derived from a carboxylic acid selected from arachidic acid (C.sub.20), stearic acid (C.sub.18), palmitic acid (C.sub.16), myristic acid (C.sub.14), lauric acid (C.sub.12), capric acid (C.sub.10), caprylic acid (C.sub.8), hydroxystearic acid, oleic acid, linoleic acid, linolenic acid and ricinoleic acid. The hydroxystearic acid can be preferably 10-hydroxystearic acid or 12-hydroxystearic acid. Most preferably, the carboxylate anion is stearate (C.sub.18).
Pharmaceutically Acceptable Salts, Hydrates and Solvates
[0059] As used herein, the term “pharmaceutically acceptable” embraces both human and veterinary use. For example, the term “pharmaceutically acceptable” embraces a veterinarily acceptable compound or a compound acceptable in human medicine and health care. Salts, hydrates and solvates of the tetracycline compounds are those wherein the counter-ion or associated solvent is pharmaceutically acceptable. However, salts, hydrates and solvates having non-pharmaceutically acceptable counter-ions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds and their pharmaceutically acceptable salts, hydrates and solvates. Suitable salts include those formed with either organic and inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulfuric, nitric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, triphenylacetic, sulfamic, sulfanilic, succinic, oxalic, fumaric, maleic, malic, mandelic, glutamic, aspartic, oxaloacetic, methanesulfonic, ethanesulfonic, arylsulfonic (for example p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic or naphthalenedisulfonic), salicylic, glutaric, gluconic, tricarballylic, cinnamic, substituted cinnamic (for example, phenyl, methyl, methoxy or halo substituted cinnamic, including 4-methyl and 4-methoxycinnamic acid), ascorbic, oleic, naphthoic, hydroxynaphthoic (for example 1- or 3-hydroxy-2-naphthoic), naphthaleneacrylic, benzoic, 4-methoxybenzoic, 2- or 4-hydroxybenzoic, 4-chlorobenzoic, 4-phenylbenzoic, benzeneacrylic (for example 1,4-benzenediacrylic), isethionic acids, perchloric, propionic, glycolic, hydroxyethanesulfonic, pamoic, cyclohexanesulfamic, salicylic, saccharinic and trifluoroacetic acid. Particularly preferred acid addition salts are hydrochloride, hyclate (hydrochloride hemiethanolate hemihydrate, .HCl.½EtOH.½H2O) and acid addition salts formed from maleic or oxalic acid. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases such as dicyclohexylamine and N-methyl-D-glucamine. All pharmaceutically acceptable acid addition salt forms of the compounds of the present invention are intended to be embraced by the scope of the present invention.
[0060] The complex is formed of a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a divalent metal carboxylate (MA.sub.2), In a preferred embodiment, the ratio TC:MA.sub.2 is 1:2; and/or M is selected from Mg.sup.2+ and Ca.sup.2+; and/or A is a carboxylate anion derived from a carboxylic acid selected from arachidic acid (C.sub.20), stearic acid (C.sub.18), palmitic acid (C.sub.16), myristic acid (C.sub.14), lauric acid (C.sub.12), capric acid (C.sub.10), caprylic acid (C.sub.8), hydroxystearic acid, oleic acid, linoleic acid, linolenic acid and ricinoleic acid. Preferably, the complex consists of said tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof, and the divalent metal carboxylate (MA.sub.2),
[0061] In a further preferred embodiment, the complex has the formula [(TC).2(MgA.sub.2)], wherein the tetracycline compound (TC) is minocycline or a pharmaceutically acceptable salt thereof. Preferably, TC is minocycline free base or minocycline hydrochloride, and A is stearate.
Pharmaceutical Preparations
[0062] The present invention provides a pharmaceutical preparation comprising the complex as described above and optionally one or more pharmaceutically acceptable excipient(s).
[0063] As used herein, the term “pharmaceutical preparation” is intended to encompass a product comprising the claimed compounds in the therapeutically effective amounts, as well as any product that results, directly or indirectly, from combinations of the claimed compounds.
[0064] In the pharmaceutical preparation according to the present invention, the total content of the tetracycline compound (TC) is preferably in the range of 5-30 wt. %, 30-50 wt. %, more preferably 8-28 wt. %, 32-48 wt. %, further preferably 10-20 wt. %, yet further preferably 11-15 wt. %, still further preferably 11.5±5 wt. %, 27.9 wt. %±5 wt. %, 11.5±2 wt. %, 27.9 wt. %±2 wt. %, 11.7-14.75 wt. %, 15.25-19.75 wt. %, and most preferably 11.5 wt. % or 27.9 wt. %, Furthermore, the overall content of the tetracycline complex (preferably, the complex having the formula [(TC).2(MgA.sub.2)]) in the pharmaceutical preparation is in the range of 5-95 wt. %, more preferably 10-80 wt. %, further preferably 15-75 wt. %, yet further preferably 20-60 wt. %, still further preferably 30-50 wt. %, and most preferably 41.2 wt. %.
Excipients
[0065] As used herein, the term “excipient” refers to a carrier, a binder, a disintegrator and/or a further suitable additive for a galenic formulation, such as extrudate, cream, gel, emulsion, suspension, liniment, ointment, powder, paste, balm, lotion, eye drops, spray, topical aerosol, topical solution, topical suspension, skin patch and nonwoven. Carriers, which can be added to the mixture, include necessary and inert pharmaceutical excipients, including, but not limited to, suitable release retardants, suspending agents, lubricants, flavorants, sweeteners, preservatives, coatings, granulating agents, dyes, and coloring agents.
[0066] A particularly preferable class of excipients are biodegradable polymers, which will be discussed in greater detail below.
[0067] Further preferable types of excipients are emulsifiers, such as glycerol monostearate, which may be present preferably in an amount of 5 to 15 wt. %, more preferably 8 to 12 wt. %, most preferably 10 wt. %. By adding such excipients, the release behavior of the preparations, in particular extrudates, can be modulated (e.g., accelerated).
[0068] Further preferable types of excipients are plasticizers, such as polyethylene glycol (PEG), which may be present preferably in an amount of 5 to 15 wt. %, more preferably 8 to 12 wt. %, most preferably 10 wt. %. A preferable type of plasticizer is PEG 1500. By adding such excipients, the mechanical behavior of the preparations, in particular of extrudates, can be modulated (e.g., brittleness and/or a tendency towards hardening over time can be reduced).
Routes of Administration and Types of Preparations
[0069] Preferably, the pharmaceutical preparation according to the present invention is arranged for topical administration. Topical administration means application to body surfaces such as the skin or mucous membranes to treat ailments via a large range of classes including creams, foams, gels, lotions, and ointments. Many topical medications are epicutaneous, meaning that they are applied directly to the skin. Topical medications may also be inhalational, such as asthma medications, or applied to the surface of tissues other than the skin, such as eye drops applied to the conjunctiva, or ear drops placed in the ear, or medications applied to the surface of a tooth, gum or periodontal pocket.
[0070] Further preferably, the pharmaceutical preparation is selected from the group consisting of extrudate, particle, granule, powder, film, strip, compact, chip, paste, cream, gel, emulsion, suspension, liniment, ointment, balm, lotion, eye drops, spray, topical aerosol, topical solution, topical suspension, skin patch and nonwoven. Yet further preferably, the pharmaceutical preparation is arranged for application into a periodontal pocket.
Extrudates
[0071] We aimed to develop hot-melt extrudates that emulate the ideals that an ideal drug delivery system for the treatment of periodontitis should combine a simple production, an easy application, the absence of solvents and a controlled release over a prolonged period with a high bioactivity. Hot melt extrusion is a favorable technique because it is a continuous and well established process in the pharmaceutical industry. The dimensions and shapes can be adapted by the extrusion parameters. In addition, the flexibility and swelling behavior can be modified by appropriate selections of the excipients. The extrudates can be cut to the appropriate length. The insertion of the extrudates is an easy and fast process. It requires—in contrast to the application of microparticles and highly viscous gels—no special device.
[0072] As used herein, the term “extrudate” refers to any material that has been extruded through a die. Specifically, extrudates refer to dosage forms obtained by shaping a precursor by extrusion into rods which can be either cut into pieces or used as such, e.g. as a sustained release dosage form for topical or a different route of administration. Preferably, the pharmaceutical preparation is in the form of a strand-shaped extrudate, e.g., as shown in
Biodegradable Polymers
[0073] The pharmaceutical preparation according to the present invention preferably comprises, as a possible excipient, one or more biodegradable polymer(s). These may be selected from biodegradable polyesters, such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polyhydroxybutyric acid (PHB) or polycaprolactone (PCL); mixed biodegradable polyesters, such as PLA-PCL and PLGA-PCL; biodegradable PEGylated diblock (AB) or triblock (ABA or BAB) copolymers, such as PEG-PLA, PEG-PLGA, PEG-PCL and PEG-PCL-PLGA; and pectins,
[0074] Poly(glycolic acid) (PGA), Poly(lactic acid) (PLA) and their copolymers are biodegradable polyesters suitable for use the present invention. They degrade in the body by simple hydrolysis of the ester backbone to non-harmful and non-toxic compounds. The degradation products are either excreted by the kidneys or eliminated as carbon dioxide and water through well-known biochemical pathways. Since PLA/PGA polymers are considered safe, non-toxic and biocompatible by regulatory agencies in virtually all developed countries, additional applications of these materials can be brought to market sooner and are more cost effective than those utilizing novel polymers with unproven biocompatibility. Examples of PLA/PGA polymers suitable for use in the present invention are for instance the RESOMER polymers manufactured by Evonik Röhm Pharma GmbH shown in Table 1 below (RESOMER® Biodegradable Polymers for Medical Device Applications Research. RESOMER® Materials by Evonik Röhm GmbH, https://www.sigmaaldrich.com/technical-documents/articles/materials-science/polymer-science/resomerhtml, retrieved 25 Oct. 2018).
Poly(glycolic acid) (PGA)
[0075] PGA is a highly crystalline material with a high melting point (225-230° C.) and variable solubility in organic solvents that is generally low and depends on polymer molecular weight. It is still susceptible to hydrolysis due to the ester bond in the polymer backbone. In spite of its low solubility, this polymer has been fabricated into a variety of forms and structures. Techniques used to develop PGA-based structures include extrusion, injection and compression molding as well as particulate leaching and solvent casting. Fibers of PGA have a high strength and modulus and are particularly rigid.
Poly(lactic acid) (PLA)
[0076] Unlike glycolide, lactide is a chiral molecule and exists in two distinct optically active forms—L-lactide and D-lactide. When each of these monomers is polymerized, the resulting polymer is semi-crystalline. Polymerization of a racemic mixture of L- and D-lactides forms poly-D,L-lactide (PDLLA), which is amorphous and has a glass transition temperature of 55-60° C. The degree of crystallinity can be tuned by altering the ratio of D to L enantiomers within the polymer. Selection of the PLA stereochemistry can have a major effect on the polymer properties, processability and biodegradability. Poly (L-lactide) or PLLA is often the polymer of choice for cast/extruded biomedical devices because it breaks down into L(+)-lactic acid units, which is the naturally occurring stereoisomer and is therefore excreted with minimal toxicity.
Poly (Lactide-co-Glycolide) Copolymers (PLGA)
[0077] Both L- and DL-lactides have been used for co-polymerization. The ratio of glycolide to lactide at different compositions allows control of the degree of crystallinity of the polymers. When the crystalline PGA is co-polymerized with PLA, the degree of crystallinity is reduced and as a result this leads to increases in rates of hydration and hydrolysis. It can therefore be concluded that the degradation time of the copolymer is related to the ratio of monomers used in synthesis. In general, the higher the content of glycolide, the quicker the rate of degradation. However, an exception to this rule is the 50:50 ratio of PGA: PLA, which exhibits the fastest degradation.
[0078] As end groups, three types of end-groups functional groups are preferred: (i) free carboxylic acid group, (ii) ester terminated group, (iii) alkyl ester group. Polymers capped with ester terminated and alkyl ester groups typically show longer degradation lifetimes than the free carboxylic analogs.
TABLE-US-00001 TABLE 1 RESOMER ® Biodegradable Polymers Molecular Weight Viscosity Product No. RESOMER Type Product Name Range [dl/g] 719854 RESOMER L 206 S Poly(L-lactide) — 0.8-1.2 719951 RESOMER R 202 S Poly(D,L-lactide) 10,000-18,000 0.16-0.24 719978 RESOMER R 202 H Poly(D,L-lactide) 10,000-18,000 0.16-0.24 719935 RESOMER R 203 S Poly(D,L-lactide) 18,000-28,000 0.25-0.35 719943 RESOMER R 203 H Poly(D,L-lactide) 18,000-28,000 0.25-0.35 719889 RESOMER RG 502 Poly(D,L-lactide-co-glycolide) 50:50 7,000-17,000 0.16-0.24 719897 RESOMER RG 502 H Poly(D,L-lactide-co-glycolide) 50:50 7,000-17,000 0.16-0.24 739952 RESOMER RG 503 Poly(D,L-lactide-co-glycolide) 50:50 24,000-38,000 0.32-0.44 719870 RESOMER RG 503 H Poly(D,L-lactide-co-glycolide) 50:50 24,000-38,000 0.32-0.44 739944 RESOMER RG 504 Poly(D,L-lactide-co-glycolide) 50:50 38,000-54,000 0.45-0.60 719900 RESOMER RG 504 H Poly(D,L-lactide-co-glycolide) 50:50 38,000-54,000 0.45-0.60 739960 RESOMER RG 505 Poly(D,L-lactide-co-glycolide) 50:50 54,000-69,000 0.61-0.74 719862 RESOMER RG 653 H Poly(D,L-lactide-co-glycolide) 50:50 24,000-38,000 0.32-0.44 719919 RESOMER RG 752 H Poly(D,L-lactide-co-glycolide) 50:50 4,000-15,000 0.14-0.22 719927 RESOMER RG 756 S Poly(D,L-lactide-co-glycolide) 50:50 76,000-116,000 0.71-1.0 739979 RESOMER RG 858 S Poly(D,L-lactide-co-glycolide) 50:50 190,000-240,000 1.3-1.7 719846 RESOMER X Polydioxanone — 1.5-2.2 (0.1% in HFIP, 30° C.) Tg Tm Product No. Application [° C.] [° C.] End Group 719854 Medical Device 60-65 180-185 ester terminated 719951 Controlled Release 38-42 amorphous ester terminated 719978 Controlled Release 44-48 amorphous free carboxylic 719935 Controlled Release 46-50 amorphous ester terminated 719943 Controlled Release 48-52 amorphous free carboxylic 719889 Controlled Release 42-46 amorphous alkyl ester 719897 Controlled Release 42-46 amorphous free carboxylic 739952 Controlled Release 44-48 amorphous ester 719870 Controlled Release 44-48 amorphous free carboxylic 739944 Controlled Release 46-50 amorphous ester 719900 Controlled Release 46-50 amorphous free carboxylic 739960 Controlled Release 48-52 amorphous ester 719862 Controlled Release 46-50 amorphous free carboxylic 719919 Controlled Release 42-46 amorphous free carboxylic 719927 Controlled Release 49-55 amorphous ester terminated 739979 Controlled Release — amorphous alkyl ether 719846 Medical Device (−10)-(−5) 110-115 — *Inherent Viscosity [dl/g], (0.1% in CHCl.sub.3, 25° C., except where noted)
[0079] Further suitable biodegradable polymers for use in the present invention are available under the name EXPANSORB® from Merck KGaA (PLA & PLGA Polymer Portfolio for Peptide & Small Molecule Formulation http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-DE-Site/de_DE/-EUR/ShowDocument-Pronet?id=201604.101, retrieved 25 Oct. 2018):
TABLE-US-00002 TABLE 2 EXPANSORB ® Biodegradable Polymers I.V. Poly (D,L- range MW lactic (dL/g, (indicative acid) Ratio End CHCl.sub.3, range, Product Order No. (PLA) L/DL group 25° C.) kDa) EXPANSORB ® DL 100-1A 5.43108 PDLLA 0/100 COOH 0.05-0.20 6-20 EXPANSORB ® DL 100-2A 5.43109 PDLLA 0/100 COOH 0.15-0.30 10-25 EXPANSORB ® DL 100-5A 5.43110 PDLLA 0/100 COOH 0.40-0.60 45-80 EXPANSORB ® DL 100-7A 5.43111 PDLLA 0/100 COOH 0.60-0.80 70-120 EXPANSORB ® DLL 10-15A 5.43112 P(L/DL)LA 90/10 COOH 1.30-1.70 200-250 EXPANSORB ® DL 100-2E 5.43113 PDLLA 0/100 ESTER 0.15-0.30 10-25 Poly(D,L- I.V. lactic-co- range MW glycolic (dL/g, (indicative acid) Ratio End CHCl.sub.3, range, Product Order No. (PLGA) L/DL group 25° C.) kDa) EXPANSORB ® DLG 45-2A 5.43114 PLGA 45/55 COOH 0.15-0.30 15-30 EXPANSORB ® DLG 50-2A 5.43115 PLGA 50/50 COOH 0.15-0.25 5-20 EXPANSORB ® DLG 50-3A 5.43116 PLGA 50/50 COOH 0.25-0.40 15-40 EXPANSORB ® DLG 50-5A 5.43117 PLGA 50/50 COOH 0.40-0.55 42-65 EXPANSORB ® DLG 50-6A 5.43118 PLGA 50/50 COOH 0.55-0.65 60-85 EXPANSORB ® DLG 50-8A 5.43119 PLGA 50/50 COOH 0.65-0.90 80-130 EXPANSORB ® DLG 55-5A 5.43120 PLGA 55/45 COOH 0.40-0.50 42-65 EXPANSORB ® DLG 65-3A 5.43121 PLGA 65/35 COOH 0.25-0.35 15-40 EXPANSORB ® DLG 65-6A 5.43122 PLGA 65/35 COOH 0.50-0.65 45-85 EXPANSORB ® DLG 75-2A 5.43124 PLGA 75/25 COOH 0.08-0.21 5-20 EXPANSORB ® DLG 75-5A 5.43125 PLGA 75/25 COOH 0.38-0.64 37-84 EXPANSORB ® DLG 75-7A 5.43126 PLGA 75/25 COOH 0.60-0.70 65-85 EXPANSORB ® DLG 75-8A 5.43127 PLGA 75/25 COOH 0.70-0.90 76-130 EXPANSORB ® DLG 75-10A 5.43123 PLGA 75/25 COOH 0.80-1.10 110-166 EXPANSORB ® DLG 85-2A 5.43129 PLGA 85/15 COOH 0.15-0.25 10-15 EXPANSORB ® DLG 85-7A 5.43128 PLGA 85/15 COOH 0.55-0.75 65-95 EXPANSORB ® DLG 90-5A 5.43130 PLGA 90/10 COOH 0.35-0.55 30-70 EXPANSORB ® DLG 90-7A 5.43131 PLGA 90/10 COOH 0.60-0.75 50-95 EXPANSORB ® DLG 95-2A 5.43132 PLGA 95/5 COOH 0.15-0.25 5-20 EXPANSORB ® DLG 95-4A 5.43133 PLGA 95/5 COOH 0.25-0.50 35-60 EXPANSORB ® DLG 50-2E 5.43134 PLGA 50/50 ESTER 0.15-0.25 5-20 EXPANSORB ® DLG 50-6E 5.43135 PLGA 50/50 ESTER 0.55-0.65 60-85 EXPANSORB ® DLG 50-7E 5.43136 PLGA 50/50 ESTER 0.60-0.70 70-100 EXPANSORB ® DLG 65-6E 5.43137 PLGA 65/35 ESTER 0.50-0.65 45-85 EXPANSORB ® DLG 75-2E 5.43138 PLGA 75/25 ESTER 0.15-0.30 10-25 EXPANSORB ® DLG 75-4E 5.43139 PLGA 75/25 ESTER 0.30-0.50 25-60 EXPANSORB ® DLG 75-7E 5.43140 PLGA 75/25 ESTER 0.66-0.80 80-115 EXPANSORB ® DLG 75-9E 5.43141 PLGA 75/25 ESTER 0.75-1.00 100-160 EXPANSORB ® DLG 85-7E 5.43142 PLGA 85/15 ESTER 0.55-0.75 65-95 EXPANSORB ® DLG 85-12E 5.45583 PLGA 85/15 ESTER 1.00-1.30 185-210 EXPANSORB ® DLG 50-6P 5.43143 PLGA-PEG 50/50 PEG 0.45-0.65 30-60 EXPANSORB ® DLG 50-7P 5.43144 PLGA-PEG 50/50 PEG 0.65-0.80 60-85 EXPANSORB ® DLG 75-4A 5.45223 PLGA 75/25 COOH 0.30-0.50 25-60
[0080] The biodegradable polymers used in the present invention preferably have a weight average molecular weight range (kDa) of 4-250, 4-240, 4-15, 5-210, 5-90, 5-20, 7-17, 10-25, 10-18, 10-15, 15-40, 15-30, 18-28, 24-38, 25-60, 30-70, 30-60, 35-60, 37-84, 42-65, 45-85, 50-95, 60-85, 65-95, 65-85, 70-100, 76-130, 76-116, 80-130, 80-115, 100-160, 110-166, 185-210, 190-240, more preferably 7-85, 8-16, 25-37, 0,45-0,80, 0,46-0,64, 0,66-0,79, most preferably 7-17, 24-38, 45-65 and/or 65-80. The biodegradable polymers used in the present invention preferably have a glass transition temperature of 42-48° C., more preferably 43-47° C., even more preferably 42-46 or 44-48° C. The biodegradable polymers used in the present invention preferably have an inherent (intrinsic) viscosity [dl/g] (0.1% in CHCl.sub.3 at 25° C.) of 0.03-1.70, 0.03-1.75, 0.05-1.60, 0.05-0.20, 0.06-1.60, 0.08-0.21, 0.13-1.32, 0.16-1.28, 0.12-0.46, 0.14-0.22, 0.14-1.70, 0.15-0.21, 0.15-0.25, 0.15-0.30, 0.15-0.43, 0.16-0.24, 0.18-0.22, 0.25-0.35, 0.25-0.35, 0.25-0.40, 0.25-0.50, 0.30-0.46, 0.30-0.50, 0.32-0.44, 0.35-0.42, 0.35-0.55, 0.38-0.64, 0.40-0.50, 0.40-0.55, 0.40-0.60, 0.42-0.67, 0.42-0.82, 0.45-0.60, 0.45-0.65, 0.47-0.63, 0.47-0.78, 0.50-0.65, 0.55-0.65, 0.55-0.75, 0.61-0.74, 0.60-0.70, 0.60-0.75, 0.60-0.80, 0.63-0.82, 0.65-0.80, 0.67-0.78, 0.66-0.80, 0.65-0.90, 0.70-0.90, 0.71-1.00, 0.75-1.00, 0.80-1.10, 0.80-1.20, 1.00-1.30, 1.30-1.70. Polymers terminated (capped) with ester, alkyl ester and/or PEG groups are preferred.
[0081] The biodegradable polymers used in the present invention are preferably PLGA copolymers (poly(D,L-lactide-co-glycolides)) having a L/G ratio of 5/95-95/5, 10/90-90/10, 15/85-85/15, 25/75-75/25, 35/65-65/35, 45/55-55/45, 45/55-50/50, 50/50-55/45, 55/45-65/35, 65/35-75/25, 75/25-85/15, 85/15-90/10, 90/10-95/5, more preferably 25/75, 45/55, 50/50, 55/45, 65/35, 75/25, 75/25, 85/15, 90/10, 95/5, 95/5, most preferably 50/50.
[0082] Preferred biodegradable polymers are PLGA copolymers having a L/G ratio of 25/75-75/25, preferably 35/65-65/35, more preferably 45/55-55/45, most preferably 50/50; and having a weight average molecular weight range (kDa) of 5-90, preferably 7-85, more preferably 7-17, 24-38, 30-60, 60-85, further preferably 8-18, 25-37, 31-59, 61-84; and having an inherent (intrinsic) viscosity [dl/g] (0.1% in CHCl.sub.3 at 25° C.) of 0.14-0.82, preferably 0.16-0.80, more preferably 0.16-0.24, 0.32-0.44, 0.45-0.65, 0.65-0.85, further preferably 0.17-0.23, 0.323-0.43, 0.46-0.64, 0.66-0.84; and being terminated with ester, alkyl ester and/or PEG groups; optionally having a glass transition temperature of 42-48° C., preferably 42-46 or 44-48° C., futher preferably 43-45 or 45-47° C.
[0083] Preferred biodegradable polymers are poly(D,L-lactide-co-glycolides) with a PLA/PGA ratio of 50:50; preferably having a weight average molecular weight range of 7,000-38,000, more preferably 7,000-17,000 or 24,000-38,000; and/or an intrinsic viscosity of 0.16-0.44, more preferably 0.16-0.24 or 0.32-0.44; and/or a glass transition temperature of 42-48° C., more preferable 42-46 or 44-48° C. Further preferably, the biodegradable polymer is selected from Resomer® RG 502 (“Resomer 502”), Resomer® RG 503 (“Resomer 503”) or a mixture thereof.
[0084] Preferred biodegradable polymers are also poly(D,L-lactide-co-glycolides) with a PLA/PGA ratio of 50:50 and a PEG end group (PLGA-PEG); preferably having a weight average molecular weight range of 30-85, more preferably 30-60 kDa; and/or preferably having an intrinsic viscosity of 0.45-0.80, more preferably 0.45-0.65. Further preferably, the biodegradable polymer is EXPANSORB® DLG 50-6P.
Methods for Manufacturing
[0085] The present invention further provides a method for manufacturing the complex as described above, the method comprising the following steps: (a) mixing a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA.sub.2) in a molar ratio of 1:0.8-3.0 with an organic solvent to obtain a dispersion, wherein the divalent metal cation M is an earth alkaline metal cation and A is a carboxylate anion derived from a C.sub.8-C.sub.24 carboxylic acid; (b) heating said dispersion to form the complex; (c) removing the solvent to obtain the complex. Preferable tetracycline compounds, molar ratios, carboxylate anions, pharmaceutically acceptable salts, hydrates and solvates are as described in the section “Complexes” above. In a preferred embodiment, the complex according to the present invention is obtained/obtainable by a method for manufacturing as described herein.
[0086] In the mixing step (a), a preferable solvent is, e.g., an alcohol, such as methanol, ethanol or mixtures thereof in the presence or in the absence of water. Preferably, a mass/volume ratio of TC to solvent of 1:100 to 1:300, preferably 1:200 may be used.
[0087] Heating the dispersion (b) accelerates the formation of the complex, preferably at a temperature of 50° C. or more, more preferably 60° C. or most preferably 70° C. or more and up to the boiling point of the solvent. Depending on the solvent chosen, the temperature and the mass/volume ratio of TC to solvent, complete dissolution may occur during step (b), such that a solution of the complex is obtained.
[0088] In step (c), the solvent can be removed by a suitable method known in the art. Preferably, the solvent may be evaporated at room temperature or elevated temperature; and/or in a shaker, an oven or on/in a film; and/or under atmospheric or preferably reduced pressure.
[0089] The pharmaceutical preparations according to the present invention, unless otherwise specified, may be prepared according to a suitable method known in the art. However, when the pharmaceutical preparations are in the form of strand-shaped extrudates, the latter are preferably prepared according to a method for comprising the following steps: (d) comminuting said complex and, if present, one or more pharmaceutically acceptable excipients to obtain an extrusion precursor; (e) extruding said extrusion precursor at a temperature above room temperature; (f) cooling the product of step (e) to obtain the pharmaceutical preparation in the form of a strand-shaped extrudate. preferred excipients, routes of administration and types of preparations, extrudates, and biodegradable polymers are as described in the section “Pharmaceutical Preparations” above. In a preferred embodiment, the pharmaceutical preparation according to the present invention is obtained/obtainable by a method for manufacturing as described herein.
[0090] In the comminution step (d), the complex is mixed, comminuted and homogenized together with one or more pharmaceutically acceptable excipient(s) (as described above, and if present) to obtain an extrusion precursor. Preferably, one or more biodegradable polymer(s), emulsifier(s) and/or plasticizer(s) as described above may be added as excipients. Further preferably, the content of the tetracycline compound (TC) added is in the range of 5-30 wt. %, more preferably 8-28 wt. %, further preferably 10-20 wt. %, yet further preferably 11-15 wt. %, and most preferably 11.5 wt. % or 27.9 wt. % relative to the total amount of components mixed. Comminution is preferably performed by milling or grinding, preferably in a mill or a grinder, more preferably at a low temperature, e.g., in a cryomill at −196° C. When a cryomill is used, performing 6, preferably 5 milling cycles at a frequency of 30 Hz for 150 s with intermediate cooling (30 s) at a frequency of 5 Hz between the cycles is particularly preferred to obtain an extrusion precursor.
[0091] The extrusion step (e) is performed by extruding the extrusion precursor through a die. Preferably, a twin-scree extruder may be used. The extrusion is performed at a suitable temperature above room temperature which is sufficient to ensure plastic deformability of the extrusion precursor material. Depending on the composition of the precursor, this is preferably a temperature about equal to or above the softening point (in the case of a pure complex without polymer ingredients); or to or above the glass transition temperature ((in the case where polymer ingredient, such as biodegradable polymers are present). Typically, the extrusion temperature may be preferably in the range of 48 to 56° C., more preferably from 49 to 55° C., even more preferably selected from 49, 52, 53 and 55° C.
[0092] The cross-sectional shape of the extrudate is mainly determined by the die at the exit of the extruder, which may be preferably essentially circular or an essentially elliptical cross section and/or a maximum cross-sectional diameter in the range of 0.1-10 mm, more preferably 0.2-5 mm, further preferably 0.3-1 mm, yet further preferably 0.4-0.8 mm, most preferably 0.6 mm. Greenstein and Tonetti (Greenstein, G. et al. J Periodontol 2000, 76, 1237-1247) demonstrated in their work the difficulties of the dental sulcus as targeted application site. The inflamed dental pocket is at least about 5 mm deep, 3 mm wide and 1 mm thick. The sulcus volume of 15 μl is renewed approximately 40 times per hour. This limited volume enables high local concentrations of the API. Hence, a diameter less than 1 mm and/or a length of less than 5 mm, preferably less than 3 mm, is preferred for a comfortable application. Preferable diameters of the extrudates are, e.g., 125-975 μm, 425-575 μm, 625-775 μm. Preferably, the extrusion step yields homogeneous extrudates with a diameter from 600 to 900 μm, e.g., 620-880 μm. The use of different diameters adapted to the pocket size and the simultaneous application of different diameters (e.g., 600 μm surrounded by 300 μm) seems highly promising. The diameter and shape of the extrudates can be adapted. For instance, the die of the extruder can be exchanged as desired by a plate with different diameters or geometric forms.
[0093] Upon cooling (f) of the extruded product of step (e), a pharmaceutical preparation in the form of a strand-shaped extrudate is obtained, as shown, e.g., in
Therapeutic Applications
[0094] The present invention further provides a complex and/or a pharmaceutical preparation as defined above for use in a method for treatment of the human or animal body. The present disclosure also provides a method for treatment of the human or animal body wherein the method comprises administration of a therapeutically effective amount of a complex and/or a pharmaceutical preparation as defined above to a subject in need thereof.
[0095] The present invention further provides a complex and/or a pharmaceutical preparation as defined above for use in a method for therapy and/or prophylaxis of a bacterial infection, preferably caused by one or more bacteria selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Streptococcus gordonii, Fusobacterium nucleatum, Actinomyces naeslundii and Parvimonas micra; and/or wherein antibiotic activity is maintained over a period of at least 21 days, preferably at least 25, more preferably at least 28, further more preferably at least 35 days, most preferably 42 days; and/or for use in a method for therapy and/or prophylaxis of an acute, chronic or recurrent periodontal disease. The disclosure further provides a method for therapy and/or prophylaxis of a bacterial infection, preferably caused by one or more bacteria selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Streptococcus gordonii, Fusobacterium nucleatum, Actinomyces naeslundii and Parvimonas micra; and/or wherein antibiotic activity is maintained over a period of at least 21 days preferably at least 25, more preferably at least 28, further more preferably at least 35 days, most preferably 42 days; and/or a method for therapy and/or prophylaxis of an acute, chronic or recurrent periodontal disease, each comprising administration of a therapeutically effective amount of a complex and/or a pharmaceutical preparation as described above to a subject in need thereof, respectively.
[0096] It is currently considered that P. gingivalis, P. intermedia, T. forsythia, F. nucleatum and P. micros are pathogens particularly associated with periodontitis. Additionally, A. naeslundii and S. gordonii, belonging to the so-called “early colonizers”, are considered to be essential for the formation of a biofilm (e.g., an oral periopathogenic biofilm) and are also present in the cases of periodontal diseases.
[0097] The major categories of periodontal diseases and conditions are classified in the groups of dental plaque-induced gingival diseases, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotizing periodontal diseases, abscesses of the periodontium, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis, and endodontic infections. In the present invention, the acute, chronic or recurrent periodontal disease is preferably selected from the group consisting of dental plaque-induced gingival diseases, periodontitis, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotizing periodontal diseases, abscesses of the periodontium, periodontitis associated with endodontic lesions, pen-implant mucositis, peri-implantitis and endodontic infections. The present disclosure also provides a method for therapy or prophylaxis of an acute, chronic or recurrent periodontal disease which is preferably selected from the group consisting of dental plaque-induced gingival diseases, periodontitis, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotizing periodontal diseases, abscesses of the periodontium, periodontitis associated with endodontic lesions, pen-implant mucositis, peri-implantitis and endodontic infections, wherein the method comprises administration of a therapeutically effective amount of a complex and/or a pharmaceutical preparation as described above to a subject in need thereof. Further acute, chronic or recurrent periodontal diseases are described, e.g., in Armitage, A. Ann Periodontol 1999, 4, 1-6.
[0098] The term “subject” as used herein refers to an animal, preferably a mammal, most preferably a human, who is or has been the object of treatment, therapy, prophylaxis, observation or experiment.
[0099] The term “therapeutically effective amount” as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
EXAMPLES
Formation and Properties of Complexes
Example 1: Formation of a 1:2 Complex of minocycline (Free Base) and magnesium stearate
[0100] Minocycline (free base) was weight in a round-bottom flask together with magnesium stearate in a molar ratio of 1:2. Specifically, minocycline (10 mg, M=457.48 g/mol, 218.8 μmol) was combined with magnesium stearate (25.848 mg, M=591.24 g/mol, 43.717 μmol). Both components were briefly mixed in a hand-shaker. The mixture was suspended in undenatured ethanol (96% v/v, 2 mL) to afford a light yellow, non-translucent suspension. The closed flask was placed in a water bath under gentle swirling. Beginning at about 50° C., formation and dissolution of the complex started. At about 60-70° C., a clear, orange solution was formed which remained clear upon cooling to room temperature. The solvent was removed in a tempered shaker.
Example 2: Formation of a 1:1 Complex of minocycline (Free Base) and magnesium stearate
[0101] A complex having a molar ratio of 1:1 was prepared in an analogous way as described in Example 1 by using an appropriately adapted amount of minocycline (free base).
Example 3: Formation of a 1:2 Complex of minocycline hydrochloride and magnesium stearate
[0102] A complex of minocycline hydrochloride and magnesium stearate having a molar ratio of 1:2 was prepared in a similar manner as described in Example 1 above, wherein an appropriate amount of minocycline hydrochloride was used such that the molar ratio of minocycline and magnesium stearate was 1:2.
Example 4: Formation of a 1:1 Complex of minocycline hydrochloride and magnesium stearate
[0103] A complex of minocycline hydrochloride and magnesium stearate having a molar ratio of 1:1 was prepared in a similar manner as described in Example 1 above, wherein an appropriate amount of minocycline hydrochloride was used such that the molar ratio of minocycline and magnesium stearate was 1:1.
Example 5: Formation of a 1:2 Complex of doxycycline (Free Base) and magnesium stearate
[0104] A complex of doxycycline (free base) and magnesium stearate having a molar ratio of 1:2 was prepared in a similar manner as described under Example 1 above, wherein doxycycline was used instead of minocycline.
Example 6: Formation of a 1:1 Complex of doxycycline (Free Base) and magnesium stearate
[0105] A complex of doxycycline (free base) and magnesium stearate having a molar ratio of 1:1 was prepared in a similar manner as described under Example 1 above, wherein however doxycycline free base was used instead of minocycline an appropriate amount of doxycycline was such that the molar ratio was 1:1.
[0106] The products from the above examples have different properties as compared to those of the respective tetracycline compounds (minocycline, minocycline hydrochloride and doxycycline), which is indicative for the formation of distinct, new complexes.
[0107] The solubility of the complexes as compared to the respective tetracycline compound (e.g., pure minocycline) is reduced. Minocycline is soluble in most polar solvents, such as water, dimethyl sulfoxide, ethanol, methanol and N-methyl pyrrolidone. The complexes, in contrast, are sparingly soluble in water but are well-soluble in chloroform. The stearate counter ion is considered to contribute to the modified solubility properties. Variations of the counter ion may be considered in order to tune the solubility.
[0108] The products having a molar ratio of the tetracycline compound to magnesium stearate of 1:1 crystallize upon drying, whereas those having a molar ratio of 1:2 solidify to afford a glassy solid (
Example 7: Stabilization in Aqueous Environment
[0109] Minocycline (10 mg;
Example 8: Spectroscopic Properties
a) UV/Vis Absorption Spectra
[0110] Furthermore, changes in the absorption spectra were observed (
b) IR Absorption Spectra
[0111] FTIR-ATR spectra were measured in potassium bromide pellets using a Bruker IFS 28 instrument equipped with a Sensir ATR unit in the area of 4000 to 400 cm.sup.−1. The IR spectra as well provide indications for the complexation (
Formation and Properties of Pharmaceutical Preparations (Extrudates)
Example 9: Extrusion of a 1:2 Complex of minocycline (Free Base) and magnesium stearate
a) Complex Preparation
[0112] For extrusion, typically a larger amount of the complexes is required (e.g., an amount corresponding to 100 mg minocycline or more). The components were weighed and treated as described above under Example 1. Subsequently, the complex solution was spread onto multiple Petri dishes coated with Teflon film, and was covered with perforated aluminum foil. The Petri dishes were dried in a vacuum dying oven at room temperature over at least 8 hours (or overnight). A yellow powder was obtained, which was then removed from the Teflon film by means of a card sheet.
b) Extrusion Precursor Preparation
[0113] Then, comminution and homogenization was performed by means of a Retsch CryoMill. The complex powder (1.00 g, corresponding to 289 mg of pure minocycline) was weighed in the cryomill jar together with two milling balls of 1 cm size in the cryomill jar. The jar was fitted into the cryomill and the following parameters were adjusted: upon automatic precooling phase, 6 milling cycles at a frequency of 30 Hz for 150 s each were performed; between the individual milling cycles, intermediate cooling (30 s) was performed at a frequency of 5 Hz. Upon thawing up to room temperature, the extrusion precursor was obtained.
c) Extrusion
[0114] For extrusion, the three heating zones of a twin-screw extruder were pre-heated at H1=49° C., H2=52° C. and H3=55° C. The control unit of the twin-screw extruder was set to 800 rpm, which corresponds to an actual rotation frequency of estimated 150 turns of the screws per minute. The extrusion precursor was carefully added to the extruder by hand and in small portions. Upon sufficient filling and after the required amount of time, the material reached the softening point and was capable of being deformed plastically. The complex exited the extruder through a 0.6 mm wide die plate as a continuous strand and was captured on a Teflon film coated surface, where it was cooled down to room temperature to afford the final extrudate.
Example 10: Extrusion of a 1:2 Complex of minocycline (Free Base) and magnesium stearate Together with Resomer 502
a) Complex Preparation
[0115] The components for the complex formation were weighed and treated as described above under Example 1. Subsequently, the complex solution was spread onto multiple Petri dishes coated with Teflon film, and was covered with perforated aluminum foil. The Petri dishes were dried in a vacuum dying oven at room temperature over at least 8 hours (or overnight). A yellow powder was obtained, which was then removed from the Teflon film by means of a card sheet.
b) Extrusion Precursor Preparation
[0116] Then, comminution and homogenization was performed by means of a Retsch CryoMill. The complex powder (412.3 mg, corresponding to 115 mg of pure minocycline) was weighed in the cryomill chamber together with 587.7 mg of the desired biodegradable polymer (Resomer 502) and two milling balls of 1 cm size in the cryomill jar. The jar was fitted in the cryomill and the following parameters were adjusted: upon automatic precooling phase, 6 milling cycles at a frequency of 30 Hz for 150 s each were performed; between the individual milling cycles, intermediate cooling (30 s) was performed at a frequency of 5 Hz. Upon thawing up to room temperature, the extrusion precursor was obtained (1 g, containing 11.5 wt. % pure minocycline).
c) Extrusion
[0117] For extrusion, the three heating zones of a twin-screw extruder were pre-heated at H1=49° C., H2=49° C. and H3=53° C. The control unit of the twin-screw extruder was set to 800 rpm, which corresponds to an actual rotation frequency of estimated 150 turns of the screws per minute. The extrusion precursor was carefully added by hand in small portions to the extruded. Upon sufficient filling and after the required amount of time, the material reached the glass transition temperature and was capable of being deformed plastically. The mixture exited the extruder through a 0.6 mm wide die plate as a continuous strand and was captured on a Teflon film coated surface, where it was cooled down to room temperature to afford the final extrudate. The obtained extrudate is shown in
Example 11: Extrusion of a 1:2 Complex of minocycline (Free Base) and magnesium stearate Together with Resomer 503
[0118] An extrudate containing the 1:2 complex of minocycline (free base) and magnesium stearate from Example 1 together with Resomer 503 was prepared in analogous manner as described in Example 10, wherein Resomer 503 was used instead of Resomer 502. The obtained extrudate is shown in
Example 12: Extrusion of a 1:2 Complex of minocycline (Free Base) and magnesium stearate Together with Resomer 502/503 (1:1) and 10% glycerol monostearate (GMS)
[0119] An extrudate was prepared in analogous manner as described in Example 10, wherein additionally glycerol monostearate (10 wt. % based on the total weight of the mixture) were used, and a mixture of Resomer 502 and Resomer 503 (1:1 w/w) in total amount adding up to 100 wt. % was used instead of Resomer 502.
Example 13: Extrusion of a 1:2 Complex of minocycline (Free Base) and magnesium stearate Together with PEG-PLGA (Expansorb DLG 50-6P) and 10% PEG 1500.
[0120] The complex powder prepared as described above under Examples 1 and 9 (412.3 mg, corresponding to 115 mg of pure minocycline and 297.3 mg magnesium stearate) was weighed in a Retsch CryoMill chamber together with the desired biodegradable polymer (Expansorb DLG 50-6P, 487.7 mg), plasticizer (PEG 1500, 100 mg) and two milling balls of 1 cm size. Then, comminution and homogenization was performed. The container was fitted in the cryomill and the following parameters were adjusted: upon automatic precooling phase, 5 milling cycles at a frequency of 30 Hz for 150 s each were performed; between the individual milling cycles, intermediate cooling (30 s) was performed at a frequency of 5 Hz. Upon thawing up to room temperature, the extrusion precursor was obtained (1 g, containing 11.5 wt. % pure minocycline).
Extrusion was Performed in an Analogous Manner as Described in Example 10.
Example 14: Release Properties of the Extrudates
[0121]
[0122] As can be seen from the curves for Examples 10-12, the addition of PLGA polymers leads to further release delay. In this case, about 10-13% of remaining minocycline were still present at the end of the experiment. This may be partially attributed to the acidic microenvironment in PLGA polymers and the slightly better stability in acidic medium, as demonstrated in
[0123] Furthermore, the addition of an excipient such as glycerol mono stearate (Example 12) can accelerate the release if desired.
[0124] Finally, the PEG-PLGA-based extrudate of Example 13 containing Expansorb DLG50-6P and 10% of PEG 1500 as a plasticizer also shows advantageous sustained release properties. Additionally, this extrudate has an optimized flexibility and mechanical formability.
Example 15: Further Properties of the Extrudates
a) Mechanical Properties
[0125]
b) Mucoadhesive Properties
[0126] The retention ability of extrudate preparations was tested in a retention analysis experimental setup as shown in
Release Kinetics and Antimicrobial Activity
Example 16: Release Kinetics and Antimicrobial Activity (Minocycline Preparations)
[0127] In order to assess the release kinetics and biological activity of the active substances, an approach was chosen taking into consideration the conditions in the oral cavity, namely the flow rate (turnover) and composition of the sulcus in the gingival crevicular fluid (as described in Goodson, J M (2003), Periodontol 2000 31:43-54; Tew, J G et al. (1985), Infect Immun 49:487-93).
a) Release Kinetics
[0128] Specifically, samples of the following substances were tested, each containing an amount of active substance equivalent to 1 mg minocycline (except the vehicle extrudate):
[0129] minocycline hydrochloride powder (purchased from SIGMA, Product No. M9511-25MG; in
[0130] minocycline/PLGA microspheres (Arestin® from Orapharma Inc. Europe, PZN 1295115; 1 capsule=1 mg minocycline; in
[0131] Example 11 (in
[0132] Example 10 (in
[0133] control consisting of biodegradable polymer without tetracycline compound (in
[0134] Samples were placed in dark tubes and an appropriate amount of buffered saline containing serum albumin (phosphate buffered saline with 15 mg/mL BSA) was added. At given time intervals, the samples were centrifuged, a predetermined amount of the buffer was removed and stored (eluate), and a fresh portion of buffer was then added. Eluates were collected over the course of seven weeks, as shown in Table 4, and tested with respect to their antimicrobial activity using the methods described below (minimal inhibitory concentration determination and biofilm formation inhibition).
TABLE-US-00003 TABLE 4 Pipetting chart for eluate samples drawn for release kinetics evaluation: Time Removal (μL) Addition (μL) 0 23.5 30 min 22 22 60 min* 22 44 2 h* 44 44 3 h 44 44 4 h* 44 88 6 h* 44 792 24 h* .sup.# 792 1′056 2 d* .sup.# 1′056 1′056 3 d 1′056 1′056 4 d* 1′056 1′056 × 3 = 3′168 7 d* .sup.# 3′168 3′500 10.5 d 3′500 3′500 14 d* .sup.# 3′500 3′304 17.5 d 3′304 3′304 21 d* 3′304 3′108 24.5 d 3′108 3′108 28 d* .sup.# 3′108 2′912 31.5 d 2′912 2′912 35 d* 2′912 2′716 38.5 d 2′716 2′716 42 d* 2′716 2′520 45.5 d 2′520 2′520 49 d* 2′520 2′324 52.5 d 2′324 2′324 56 d* 2′324 *denotes eluates removed at these times were used in the MIC determination; .sup.# denotes eluates removed at these times were used in the biofilm formation inhibition.
TABLE-US-00004 TABLE 5 Effective concentration of minocycline versus Streptococcus gordonii ATCC 10558 and Porphyromonas gingivalis ATCC 33277 up to 42 d, calculated based on the MIC of the eluates (dilutions) Minocycline Arestin Extrudate 502 Extrudate 503 S. gord. P. ging. S. gord. P. ging. S. gord. P. ging. S. gord. P. ging. 1 h >16000 >32000 >16000 >32000 1000 4000 4000 >32000 2 h 8000 2000 8000 4000 2000 1000 4000 2000 4 h 2000 1000 2000 1000 1000 1000 2000 2000 24 h 250 500 1000 1000 500 1000 500 1000 2 d 62.5 62.5 125 250 125 250 250 500 7 d 4 62.5 15.6 62.5 15.6 62.5 125 250 14 d <2 4 8 8 15.6 31.3 31.3 62.5 21 d <2 <1 <2 <1 15.6 15.6 31.3 62.5 28 d <2 <1 <2 <1 15.6 15.6 15.6 15.6 35 d <2 <1 <2 <1 15.6 15.6 8 8 42 d <2 <1 <2 <1 8 2 8 2
b) Minimal Inhibitory Concentration (MIC) Determination
[0135] The dilutions were determined at which growth of two indicator strains (Porphyromonas gingivalis ATCC 33277, Streptococcus gordonii ATCC 10558) was still inhibited (minimal inhibitory concentration, MIC). For this purpose, samples the eluates were mixed with nutrition medium and the respective bacterial strain and incubated over 18 h at 37° C. under aerobic conditions. The approach and the media used were in accordance with the recommendations of The European Committee on Antimicrobial Susceptibility Testing—EUCAST. Each determination was performed double; growth control was performed each time in one well, respectively.
[0136]
[0137] In Table 5, based on the MIC for minocycline against the two strains (P. gingivalis ATCC 33277: 0.25 μg/mL, S. gordonii ATCC 10558: 0.5 μg/mL), the effective concentration of the active substance was calculated. These data also highlight the activity of the eluates over a prolonged period of time.
c) Biofilm Formation Inhibition
[0138] The surface of a 96-well plate was coated with 10 μL of eluate. Upon addition of a 6-species mixture of periodontitis-associated bacteria, a biofilm was formed over 6 h (as described in, e.g., Jurczyk K., Nietzsche S., Ender C., Sculean A., Eick S. In-vitro activity of sodium-hypochlorite gel on bacteria associated with periodontitis. Clin Oral Investig. 2016 November; 20(8):2165-2173). Then, the number of still vital bacteria in the biofilm (colony forming units=cfu) was determined.
[0139]
Example 17: Release Kinetics and Antimicrobial Activity (Doxycycline Preparations)
[0140] In order to assess the release and kinetics and the biological activity of the doxycycline preparations, an analogous approach was chosen. Specifically, samples of the following substances were tested, each containing an amount of active substance equivalent to 1 mg doxycycline (except the vehicle extrudate):
[0141] doxycycline hyclate (1.15 mg doxycycline hyclate=1 mg doxycycline)
[0142] doxycycline gel (Ligosan®, 1 cartridge=260 mg, 14% doxycycline)
[0143] doxycycline extrudate (corresponding to 11.5% doxycycline, prepared in an analogous manner as described in Example 13)
[0144] vehicle extrudate.
[0145] The tests were performed in an analogous manner as described in Example 16 above. The release kinetics and antimicrobial activity of the preparation according to the present invention relative to the reference samples were found to be comparable with the results obtained for minocycline preparations.
Further Examples
[0146] As potential complexing agents, we selected magnesium stearate and calcium stearate because of their high theoretical potential, their low toxicity, biodegradability and their broad use in drug delivery applications. The drug complex formation was followed by UV/VIS- and IR-spectroscopy. In addition, we wanted to study the impact of glycerol-monostearate in order to modify the release rate and the mechanical properties.
[0147] The complex formation might also negatively impact the activity of the drug. Hence, bioactivity measurements were carried out. Finally, in vitro release experiments were performed to evaluate the impact on the formulation on the release kinetics.
A. Materials and Methods
[0148] Minocycline and Doxycycline were purchased from Ontario Chemicals Inc. (Ontario, Canada). Magnesium stearate and calcium stearate were kindly provided by Magnesia Germany (Müllheim, Germany). PLGA polymers and Tegin® 4100 pellets (Glycerolmonostearate abbreviated as GMS) were a gift from Evonik (Darmstadt, Germany). Nile red was acquired from Sigma Aldrich (Munich, Germany). Staphylococcus aureus ATCC 29213 was used for screening of antimicrobial activity. All used solvents were acquired from Sigma Aldrich and at least of purissimum grade.
I. Chelation of the tetracycline Derivates
[0149] The formation of a lipophilic drug complex was proposed as a potential method to increase the drug stability and to extend the release time. For this purpose, different molar compositions of the antibiotics and magnesium- or respectively calcium stearate were investigated. For the complex formation tetracyclines and the stearate salts were weighed into flasks, in a molar ratio of 1:1, 1:2 and 2:1. For instance, 10 mg of minocycline were paired with 25.48 mg of magnesium stearate (Molar ratio 1:2). 2 ml of ethanol were added to each flask, resulting in yellow suspensions. The flasks were heated in a water bath at 70° C. for 2 minutes.
II. Methods Used for Complex Characterization
[0150] To prove the existence of the minocycline complex, investigations of the chelates were necessary. Therefore, absorption spectra of the pure tetracycline derivatives and their complex solutions were examined with a Shimadzu UV-1800 spectrophotometer (Shimadzu, Duisburg, Germany). Beforehand, 10 μl of the freshly prepared 5 mg/ml stock solutions were diluted with 3 ml ethanol, and absorption spectra were taken in the range from 250 to 450 nm in a quartz cuvette. For the recording of the infrared (IR) spectra, homogeneous compacts of the API and zinc selenide were produced. Subsequently, this compacts were surveyed with a Bruker IFS 28 equipped with a Sensir ATR unit (Bruker, Billerica, United States) operating from 4000 to 680 cm.sup.−1. Additionally, the solutions were dried in a rotary evaporator, and the remains were scraped from the flask. These remains were surveyed under a microscope (Carl Zeiss Microscopy, Jena, Germany). These remnants were also exposed to different solvents, to reveal changes in solubility.
III. Disc Diffusion Test
[0151] After deciding for minocycline as favored active component, an agar disc diffusion test was performed, to examine its antimicrobial activity after complexation. The procedure followed the recommendations set by EUCAST. Test substance was the minocycline complex containing 10 mg/ml minocycline and controls were minocycline dissolved in ethanol (10mg/ml), and pure ethanol. Solutions were diluted 3:7 with ethanol and immediately thereafter each 10 μl were pipetted to unloaded antibiotic test discs with a diameter of 6 mm (BD, Allschwil, Switzerland), thus each discs (except for ethanol control) contained 30 μg of minocycline. Overnight culture of S. aureus ATCC 29213 was suspended to McFarland 0.5, each 100 μl was spread on Mueller-Hinton-agar plate (Oxoid, Basingstoke, GB). Following the evaporation of the solvent, the test discs were placed on the plates. After incubation at 35° C. for 18 h, the diameters of the inhibition zones were measured.
IV. Extrusion Precursor Production with Cryomilling and Hot-Melt Extrusion
[0152] In advance of the cryomilling, an ethanolic minocycline complex solution was spread on Teflon foil covered petri dishes in a thin layer of droplets. The petri dishes were stored in a vacuum drying oven for at least 12 hours at 25° C. The dried minocycline complex powder was then collected from the Teflon foil.
[0153] Batch sizes of around 1 g are necessary to achieve a sufficient filling of the extrusion chamber. Hence, for cryomilling, 412.3 mg of minocycline complex powder (containing 115 mg minocycline) and 587.7 mg of the desired PLGA polymer were inserted into the grinding chamber together with two 10 mm steel spheres. The extrudates containing 10% GMS, therefore enclosed only 487.7 mg of PLGA polymer. After proper adjustment in the Retsch CryoMill (Retsch, Haan, Germany), the mixture was submitted to an automatic pre-cooling phase, followed by 5 milling cycles at 30 Hz for 150 s. After each milling cycle a 30 s long cooling phase reassured suitable process temperatures at 5 Hz.
[0154] Subsequently, the mixture was ready for extrusion. Therefore, a twin-screw extruder ZE 5 Eco (Three-Tec, Seon, Switzerland) with an integrated SK 500 E frequency converter was utilized. A die with a 600 μm diameter and screws with L/D-ratio of 21.25 were used. The extrusion temperature depended on the polymer and the intended diameter. Starting at 49° C. in the first heating zone, the temperature was progressively elevated up to 53° C. in the third heating zone for Resomer 502, 503 and their mixture. For the extrudates containing 10% GMS, 55° C. were applied in the last heating zone. The minocycline-PLGA powder was slowly and manually filled into the extruders opening at 800 rpm according to the frequency converter. The emerging continuous polymer string was captured on a Teflon foil covered surface. The final product was cut into pieces of desired length with a scalpel.
V. Determination of Drug Content
[0155] The minocycline content of the extrudates was quantified via a HPLC method after extraction of the drug from the extrudate. For the extraction a method inspired by A. Holmkvist et al. 2016 (24) was used. After weighing, the extrudate was transferred in a 15 ml centrifuge tube. 2 ml of acetonitrile containing 0.1% trifluoric acid (TFA) were added to dissolve the polymer. After 1 minute of shaking with an IKR-VIBRO-FIX-shaker 4 ml of methanol were added to solve the minocycline and precipitate the polymer. Afterwards the tube was centrifuged at 1000 rpm for 5 minutes and sterile filtered through a 0.45 μm Teflon filter. HPLC was performed on a Waters 600 E system with an XTerra RP18 5 μm 3.9×150 column (Waters). 20 μl sample volume were injected at a flowrate of 1.0 ml/min. A gradient program was chosen for the mobile phase. Starting with 25 mM KH.sub.2PO.sub.4+0.06% H.sub.3PO.sub.4 (A), which shifts over 10 minutes to 60% acetonitrile (B). After 5 minutes the mobile phase was gradually changed back to A within 1 minute. The retention time was 9.5 minutes and the drug was detected with a UV/VIS detector at 355 nm. Linear calibration curves (r.sup.2>0.999) were obtained in the range of 2×50 μg/ml. A recovery rate of 96% was achieved.
[0156] For the stability investigations an Agilent 1200 Series system with a XTerra RP18 5 μm 3.9×150 column (Waters) was utilized. 10 μl sample volume were injected at a flowrate of 1.0 ml/min. For this method was also a gradient program applied. The mobile phase started with a composition of water/methanol (95/5+0.1% TFA) (A) and was shifted over 7 minutes to pure methanol (0.1% TFA) (B). After 3 minutes the mobile phase returned to solvent A within 1 minute. The retention time was 4.8 minutes. For the evaluation and extraction of the chromatograms, the Chem32 software was used.
VI. Release Studies and Stability Investigation
[0157] The extrudates were cut into pieces of 4 mm+/−100 μm length. For each polymer type, 5 extrudates (length 4 mm) were weighed and exposed to 1 ml of phosphate buffered saline (PBS) pH 7.0 in a 2 ml glass vial. The vials were incubated in a light protected water bath at 37° C. For 3 weeks every 2 days samples were taken. From day 22 on, the sample taking interval was stretched to 4 days. Sample taking included a complete substitution of buffer. The samples were stored in a fridge at 2-8° C. until their drug content was evaluated by the above explained HPLC-method. The release profile was examined for 42 days. After this time period, the residue of the extrudates were also investigated by this HPLC-method to quantify the remaining minocycline content.
[0158] For the stability investigation, 10 mg samples for minocycline and 20 mg for the complex were weighed into 4 ml glass vials. One of each vial was incubated in 4 ml of PBS pH 7.0 and another vial was exposed to 4 ml of an acidic solution at pH 2.3, which was adjusted with hydrochloric acid. Until the 9.sup.th day, samples were taken every day, while the removed volume was not replaced. From there on the intervals were stretched to 3 to 4 days.
B. Results
I. Complex Formation
Molar Ratios
[0159] Molar ratios of 2:1, 1:1 and 1:2 (Tetracycline derivate: fatty acid salt) have been tested as potential chelate complexes. The combination of minocycline and magnesium stearate yielded for all ratios a yellow solution. Contrary, the pairing of minocycline with calcium stearate resulted in a suspension for the 1:2 ratio. Therefore, this ratio had been excluded from further investigations. Doxycycline was similar to minocycline with regard to the magnesium stearate complexes. Clear solutions emerged with a less intensive yellow coloring. The pairing of doxycycline with calcium stearate revealed slightly cloudy solutions for the ratios of 1:1 and 1:2.
II. Complex Characterization
1. UV/Vis-Spectroscopy
[0160] A prominent indication for a successful chelation are changes in the absorption spectra. Minocycline has an absorption maximum at 255 and 344 nm in ethanol (
[0161] In combination with calcium stearate, minocycline's absorption spectrum experiences comparable changes. The 1:1 ratio leads to a hypsochromic shift for the shortwave maximum and bathochromic shift for the longwave maximum, while the 1:2 ratio has already been excluded. Analogous, the 2:1 ratio resembles an overlapping spectrum of pure API and the complex. Both also have in common, that after addition of small amounts of hydrochloric acid (HCl) the absorption maxima of minocycline and the complexes shift to 264 nm and approximately 355 nm (
[0162] For doxycycline the results are as expected quite close to minocycline, due to the structural similarity. It is noteworthy however, that both of doxycycline absorption maxima are shifted bathochromically, independent from the chelating ion (
[0163] In conclusion, this experiment lead to the exclusion of all 2:1 ratios, and the 1:1 ratio of doxycycline and calcium stearate, due to the insufficient complex formation.
2. IR-Spectroscopy
[0164] The IR-spectra also give evidence for the chelation (
[0165] For doxycycline however, the IR spectrum exposes a broader peak at approximately 3300 cm.sup.−1 (a), which cannot be distinguished between O—H and N—H vibration. Nevertheless, this peak is also widened and smoothed in the presence of divalent cations (
3. Complex Drying and Microscopy
[0166] For further processing, it was necessary to transfer the complex into a powder form. Therefore, the complex solutions were slowly dried and their remnants closer examined. The dried minocycline and doxycycline-magnesium stearate complexes both appeared as glasslike, amorphous structures for the molar ratio of 1:2.
[0167] Changes in solubility were also observed. The minocycline complex exhibited a reduced water solubility compared to the parent drug. In return, the solubility in organic solvents was improved, especially in chloroform. These changes also imply the formation of a chelate complex.
[0168] At this point, minocycline was selected for further experiments, even though the doxycycline magnesium stearate complex seemed evenly suitable. The reason therefore was the broader spectrum of activity and the superior capability of tissue penetration (Cunha, B A. et al. Eur J Clin Microbiol Infect Dis. 2018, 37(1), 15-20). So, the following paragraphs focus on minocycline magnesium stearate complex in the ratio of 1:2.
III. Disc Diffusion Test
[0169] It is well known, that polyvalent cations can have a negative influence on the antibiotic potential of tetracycline antibiotics (Naz, S. et al. Arzneimittefforschung 1996, 46(7), 701-704). On account of that fact, a disc diffusion test was carried out, to investigate minocycline's antibiotic activity after complexation. The diameter of the inhibition zone is decisive for the antibiotic activity (
IV. Hot-Melt Extrusion
[0170] In advance of the extrusion process, cryomilling ensures a homogeneous extrusion precursor while keeping the thermal stress at a minimum.
[0171] With Resomer 502 RG and 503 RG extrudates in a size range from 600 to 800 μm were achieved (
[0172] Nevertheless, these extrudates were still within the desired size range. Additionally, the extrusion of the pure minocycline complex powder was carried out. It was possible to obtain extrudates from this approach, but they were brittle and exhibited fragile mechanic resilience. So, these extrudates only served as reference for the in vitro release experiments.
V. In Vitro Release
[0173] The in vitro release profile (
[0174] The addition of a PLGA polymer can modify the release rate to a desired level. As expected, Resomer 502 offers a faster release compared to Resomer 503, due to its lower molecular weight. This difference is especially examinable in the first two weeks, until their release rates start to align. Remarkable is also the higher standard variation for Resomer 502. For the 1:1 composition of Resomer 502 and 503, a release between the curves of the single polymers was expected. These expectations were met until day 7, when the polymer blend fell behind. After 42 days the polymer blend nearly reaches the release level of Resomer 503. So, in contrast the impact of GMS seems even greater. Initially GMS was used as softener for the extrudates, but the effect was relatively small, even though the overall stability was enhanced. The greater benefit lay in the significant increase of the release rate. Within 10 days the doubled amount had been released compared to the batch without GMS. From day 10 on, their release rates also align and no greater difference prevails.
[0175] The in vitro release experiments demonstrate the controlled release over 42 days.
VI. Stability Studies
[0176] This study was carried out to clarify the destiny of the at least 40% of undetected minocycline.
[0177] The acidic environment ensures a longer lifetime of the API. A more moderate decay is observable for minocycline compared to the neutral pH conditions. The same statement is valid for the complex, while here an additional solving over time is again noticeable. From this point on, minocycline and the complex experience a similar degree of degradation. As previously demonstrated, minocycline is released from the complex at acidic conditions. Therefore, similar chromatograms and degradation rates are observable.
Discussion
[0178] Tetracyclines possess a wide range of antimicrobial acitvity, and have proven as locally antimicrobial agents in periodontal therapy. A problem for all tetracylines is their limited stability in water. Minocycline degrades faster than doxycline, and is a pronene to epimerization in aqueous media and several organic solvents, especially N-methyl pyrrolidone (NMP). These epimers provide only 5% of the initial antibiotic activity
[0179] A delay of such processes can be achieved if minocycline has been chelated in advance. Of these complexes, magnesium complexes proved to be more stable than their calcium counterparts, probably due to their higher charge density. Such influence of the charge density was noticeable during the chelation process described herein. Magnesium and calcium are both divalent cations, but due to the smaller atom diameter (Mg: 145 pm, Ca: 194 pm), magnesium with its therefore higher charge density can interact more easily with the chelation site. This explains the formation of turbid solutions in the case of doxycycline and a suspension the ratio of 1:2 for minocycline. Additionally, by choosing magnesium and calcium fatty acid salts as second compound, avoidance of water and the alteration of the APIs physical properties can be achieved.
[0180] The UV/Vis- and IR-spectroscopy investigations gave evidence about the complex formation and the suitability for the further processing. The shifts of the absorption maxima depending on the concentration of the cations, and the smoothing and widening of the OH-vibration peaks demonstrate the interactions during the chelation. There, minocycline and doxycycline exhibited equal compatibility for the complexation process. Also, both antibiotics demonstrate anti-inflammatory properties, which are beneficial for the treatment of an inflammatory disease like periodontitis. But as mentioned beforehand, minocycline was chosen for the further development, due to its more lipophilic characteristics and its superior spectrum of activity.
[0181] The durability of this activity had to be clarified, due to the might hindering interactions between API and polyvalent cation. Fortunately, the disc diffusion test revealed a high activity. A possible explanation for the slightly reduced diameter can be a decreased diffusion of the minocycline molecules through the agar matrix, due to the interaction with the magnesium stearate molecules, creating a larger and more hydrophobic molecule. Nevertheless, these results enabled the progression to the next step of development—the extrusion.
[0182] The production process itself is comparable fast and simple. The preparation of the precursor takes little effort and the absence of toxic solvents lowers the risks during manufacturing, creates a compatible product, and ensures the stability of the API. A limitation of the laboratory scale production is the manual extruder filling, resulting in a discontinuous precursor flow.
[0183] The final product as preformed insert exhibits several advantageous properties regarding the intended application. Also, there is no necessity for special equipment for the application—a simple tweezer will be sufficient. The diameter of the extrudates is not fixed, but can be adapted. The use of different diameters—adapted to the pocket size and the simultaneous application of different diameters (e.g. 600 μm surrounded by 300 μm) seems highly promising.
[0184] The achieved drug load of 11.5% is close to the reasonable limit due to the complex composition with magnesium stearate. The overall share of the complex in the extrudate is 41.2%, leaving 58.8% for the release controlling polymer. The high magnesium stearate quantity contributes to the slightly flexible traits of the extrudates. Various dosing options are imaginable through the simultaneous placement of several extrudates in one sulcus. Also, the release rate is adjustable through the choice of polymer and additional excipient.
[0185] The in vitro release period of 42 days is so far exceptional compared to the commercially available systems. As expected, Resomer 502 with its lower molecular weight exhibited a faster release rate, compared to Resomer 503. Remarkable is the higher standard variation for Resomer 502, which can also be linked to the lower molecular weight and a therefore faster autocatalysis and degradation (Fredenberg, S. et al. Int J Pharm 2011, 415, 34-52). However, the blend of both polymers behaved surprisingly unexpected. The sudden drop of its release rate is challenging to bring into context. A possible explanation can be a batch to batch variation, analytical difficulties or polymer-polymer interactions, which either create a micro-environment that promotes decay of the API, or impedes the release of minocycline.
[0186] For all samples, one observation is striking: a higher percentage of the API remains undetected. Therefore, the stability experiment was scheduled. The known fragility of minocycline in aqueous media leads to the first assumption of decay over the period of the release experiment. The elevated temperature of 37° C. promotes these processes. To gain greater insight, minocycline and the complex were investigated at pH 7.0 and pH 2.3. The neutral pH resembles the physiological pH, while the acidic pH simulates conditions that can occur inside degrading PLGA polymers. The stability investigations revealed a protective function for the complex at neutral pH values. Nevertheless, a part of the API falls to decay. The residue analysis performed at the end of the releasing experiment revealed 10 to 15% of the API to remain inside the extrudates. Considered together, these results enlighten the destiny of the missing API to a great part.
Summary and Conclusion
[0187] The objective of this study was to develop an alternative controlled release device for the local antimicrobial treatment of periodontitis. As an inflammatory and bacterial disease, periodontitis can be treated with a local antibiotic or antiseptic formulation. One class of possible antibiotics in such formulations is the group of tetracyclines. Our aim was to provide a preformed insert that offers enhanced drug stability, easy application and controlled release over several weeks. Therefore, a chelate complex consisting of a tetracycline antibiotic and a fatty acid salt was proposed. In this case, minocycline and doxycycline were paired with magnesium- and calcium stearate in different molar ratios. These chelate complexes stabilize the active pharmaceutical ingredient and enable the incorporation into a PLGA polymer matrix via hot melt extrusion. The chelate complexes were characterized via UV/Vis- and IR-spectroscopy, leading to a favoritism of the minocycline-magnesium complexes. For the reevaluation of the antibiotic activity of the complex, a disc diffusion test was carried out. The complex was mixed with different PLGA-polymers and cryomilled in advance of the extrusion. The hot melt extrusion yielded homogeneous extrudates with a diameter from 600 to 900 μm. They contain 11.5% of minocycline, are adjustable in length and offer an easy handling. In vitro release studies revealed a controlled release of the drug over 42 days.
[0188] Our approach to a novel application form for the periodontal patient offers several benefits. Minocycline's stability is enhanced by the proposed complexation before and after application. The manufacturing process is versatile, free of any toxic solvents, offers accessibility for upscaling and provides a solid preformed insert. This preformed insert features an easy application without any special equipment, adjustability, and a 42 days lasting controlled, prolonged release, which has not yet been reached by other formulations. In summary, the extrudates possess promising potential to advance as a valuable therapeutic option, in particular in the field of periodontal therapy.
Comparative Example
Comparative Example 18: Treatment with Na.SUB.2.HPO.SUB.4
[0189] Published international application WO 2012/128417 A1 describes in Example 1 the formation of an “ion complex” through the mixing of 5 wt % aqueous tetracycline hydrochloride solution with a 5 wt % aqueous solution of Na.sub.2HPO.sub.4 in a 1:1 volume ratio. Minocycline.HCl ion complexes were also claimed by WO 2012/128417 A1. Therefore, a similar reaction was carried out to compare the minocycline phosphate with our minocycline chelate com plex.
[0190] In a first attempt 375 mg of minocycline.HCl and Na.sub.2HPO.sub.4 were each weighed into a 15 ml falcon tube. Afterwards 7.125 ml of bidistilled water were added to each powder. Minocycline.HCl was not soluble in such a high concentration, resulting in a yellow suspension. Na.sub.2HPO.sub.4 was effortless soluble (
[0191] The experiment was repeated with 1 wt % minocycline HCl and 1 wt % Na.sub.2HPO.sub.4 solutions. Therefore, 75 mg of each compound were each weighed into 15 ml falcon tubes and 7,425 ml of bidistilled water were added. At this concentration, minocycline.HCl was soluble, and yielded a yellow solution (
[0192] So, for minocycline.HCl no successful precipitation of a low-soluble ion complex was observable with disodium phosphate as anionic counterpart.
[0193] The present disclosure further includes the following aspects.
[0194] [1] A complex comprising a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA.sub.2), wherein: the molar ratio TC:MA.sub.2 is in the range of 1:0.8-3.0; the divalent metal cation M is an earth alkaline metal cation; and A is a carboxylate anion derived from a C.sub.8-C.sub.24 carboxylic acid. [2] The complex according to aspect [1], wherein the tetracycline compound (TC) is selected from minocycline, doxycycline, tetracycline, chlortetracycline, oxytetracycline, rolitetracycline, tigecycline, demeclocycline, lymecycline, meclocycline, methacycline, omadacycline, sarecycline, eravacycline, clomocycline, 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline and 9-(N,N-dimethylglycylamido)-minocycline. [3] The complex according to any one of the preceding aspects, wherein: the ratio TC:MA.sub.2 is 1:2; and/or M is selected from Mg.sup.2+ and Ca.sup.2+; and/or A is a carboxylate anion derived from a carboxylic acid selected from arachidic acid (C.sub.20), stearic acid (C.sub.18), palmitic acid (C.sub.16), myristic acid (C.sub.14), lauric acid (C.sub.12), capric acid (C.sub.10), caprylic acid (C.sub.8), hydroxystearic acid, oleic acid, linoleic acid, linolenic acid and ricinoleic acid. [4] The complex according to any one of the preceding aspects having the formula [(TC).2(MgA.sub.2)], wherein the tetracycline compound (TC) is minocycline or a pharmaceutically acceptable salt, hydrate or solvate thereof. [5] A pharmaceutical preparation comprising the complex according to any one of aspects [1]-[4] and optionally one or more pharmaceutically acceptable excipient(s). [6] The pharmaceutical preparation according to aspect [5], which is arranged for topical administration and/or is selected from the group consisting of extrudate, particle, granule, powder, film, strip, compact, chip, paste, cream, gel, emulsion, suspension, liniment, ointment, balm, lotion, eye drops, spray, topical aerosol, topical solution, topical suspension, skin patch and nonwoven. [7] The pharmaceutical preparation according to aspect [5] or [6] further comprising, as an excipient, one or more biodegradable polymer(s) selected from biodegradable polyesters, such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polyhydroxybutyric acid (PHB) or polycaprolactone (PCL); mixed biodegradable polyesters, such as PLA-PCL and PLGA-PCL; biodegradable PEGylated diblock (AB) or triblock (ABA or BAB) copolymers, such as PEG-PLA, PEG-PLGA, PEG-PCL and PEG-PCL-PLGA; and pectins, and/or wherein the total content of the tetracycline compound (TC) is in the range of 5-20 wt. %.[8] The pharmaceutical preparation according to any one of aspects [5]-[7], which in the form of a strand-shaped extrudate, preferably having an essentially circular or an essentially elliptical cross section and/or a maximum cross-sectional diameter in the range of 0.1-10 mm. [9] A method for manufacturing the complex according to any one of aspects [1]-[4], the method comprising the following steps: (a) mixing a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA.sub.2) in a molar ratio of 1:0.8-3.0 with an organic solvent to obtain a dispersion, wherein the divalent metal cation M is an earth alkaline metal cation and A is a carboxylate anion derived from a C.sub.8-C.sub.24 carboxylic acid; (b) heating said dispersion to form the complex; (c) removing the solvent to obtain the complex. [10] A method for manufacturing the pharmaceutical preparation according to aspect [8], the method comprising the following steps: (d) comminuting the complex according to any one of aspects [1]-[4] and, if present, one or more pharmaceutically acceptable excipients to obtain an extrusion precursor; (e) extruding said extrusion precursor at a temperature above room temperature; (f) cooling the product of step (e) to obtain the pharmaceutical preparation in the form of a strand-shaped extrudate. [11] The complex according to any one of aspects [1]-[4] or the pharmaceutical preparation according to any one of aspects [5]-[8] for use in a method for treatment of the human or animal body. [12] The complex according to any one of aspects [1]-[4] or the pharmaceutical preparation according to any one of aspects [5]-[8] for use in a method for therapy and/or prophylaxis of (i) a bacterial infection; or (ii) a bacterial infection caused by one or more bacteria selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Streptococcus gordonii, Fusobacterium nucleatum, Actinomyces naeslundii and Parvimonas micra. [13] The complex according to any one of aspects [1]-[4] or the pharmaceutical preparation according to any one of aspects [5]-[8] for the use according to aspect [12], wherein antibiotic activity is maintained over a period of at least 21 days. [14] The complex according to any one of aspects [1]-[4] or the pharmaceutical preparation according to any one of aspects [5]-[8] for use in a method for therapy and/or prophylaxis of an acute, chronic or recurrent periodontal disease. [15] The complex according to any one of aspects [1]-[4] or the pharmaceutical preparation according to any one of aspects [5]-[8] for use in the method according to aspect [14], wherein the periodontal disease is selected from dental plaque-induced gingival diseases, periodontitis, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotizing periodontal diseases, abscesses of the periodontium, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections.