RU2806036C2 - Tetracycline complexes with sustainable activity - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: complex formed from a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA₂), in which the TC:MA₂ molar ratio is in the range of 1:0.8-3.0, the divalent metal cation M is an alkaline earth metal cation and A is a carboxylate anion derived from a C₈-C₂₄ carboxylic acid. The tetracycline (TC) compound is selected from minocycline, doxycycline, chlortetracycline, rolitetracycline, tigecycline, demeclocycline, lymecycline, meclocycline, metacycline, omadacycline, sarecycline, eravacycline, clomocycline, 9-(N,N-dimethylglycilamido)-6-demethyl-6 -deoxytetracycline and 9-(N,N-dimethylglycylamido)minocycline. The invention also relates to a pharmaceutical preparation, methods for obtaining the specified complex and pharmaceutical preparation, the use of the specified complex and a method for treatment using the specified complex or pharmaceutical preparation. The tetracycline compound complexes of the present invention degrade more slowly in aqueous media than the corresponding tetracycline compounds and have lower solubility in water and provide a more delayed and sustained release than the corresponding tetracycline compounds.

EFFECT: proposed complexes can be used for the treatment and/or prevention of bacterial infection or acute, chronic or recurrent periodontal disease.

20 cl, 23 dwg, 4 tbl, 18 ex

Description

Field of technology to which the invention relates

The present invention generally relates to the controlled release of tetracycline antibiotics. In particular, it refers to a complex containing a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate; a pharmaceutical preparation containing a complex, methods for producing the complex and a pharmaceutical preparation, and a complex or pharmaceutical preparation for use in a method of treating a human or animal body, in particular for the therapy and/or prevention of a bacterial infection; and/or in which the antibiotic activity is maintained for an extended period of time; and/or for the treatment and/or prevention of acute, chronic or recurrent periodontal disease.

State of the art

Tetracycline compounds are a class of antibiotics that inhibit protein synthesis by preventing aminoacyl-tRNA from attaching to the ribosomal acceptor (A) site. Tetracyclines are broad-spectrum agents that are active against a wide range of gram-positive and gram-negative bacteria, atypical organisms such as chlamydia, mycoplasma and rickettsia, and protozoan parasites. The beneficial antimicrobial properties of these agents and the lack of serious side effects have led to their widespread use in the treatment of human and animal infections, such as periodontal disease (Chopra et al.,Microbiol Mol Biol Rev. 2001, June 65(2):232-60). Tetracycline molecules contain a linear fused tetracyclic core (rings designated A, B, C, and D) to which various functional groups are attached. The simplest tetracycline that exhibits detectable antibacterial activity is 6-deoxy-6-demethyltetracycline, and therefore this structure can be considered a minimal pharmacophore (Mitscher, L.A.1978.The chemistry of the tetracycline antibiotics. Marcel Dekker, Inc., New York, N.Y):

Tetracycline antibiotics that are widely used in clinical practice include doxycycline, minocycline, and tetracycline. An overview of the most commonly used tetracycline antibiotics is presented below:

Among them, especially doxycycline and minocycline have been used to treat periodontal diseases. However, there are problems with the therapeutic use of these active substances due to their relatively short retention time and their instability in the aqueous environment.

Periodontal diseases are widespread. Approximately 30% of the population worldwide is affected. They have a significant impact on individuals and society, and treatment is costly. The cost of dental care ranks fourth among all diseases and consumes 5 to 10% of all health care resources (Batchelor, P. British Dental Journal 2014, 217, 405-409). Representative population studies show that periodontal disease is widespread and has been increasing in prevalence since 1997 (Micheelis , W. et al . Vierte Deutsche Mundgesundheitsstudie (DMS IV), Deutscher Ärzte-Verlag, Köln , 2006 ). Among the German adult population, 52.7% were found to suffer from moderate periodontitis and 20.5% from severe periodontitis. Health insurance costs in Germany for direct treatment of periodontitis amounted to approximately €1.1 billion ( Statistisches Bundesamt , 2008 ), excluding costs for secondary diseases.

Periodontitis is a general term describing an inflammatory condition of the periodontium that is caused by multibacterial induction and is closely associated with various systemic diseases such as cardiovascular disease, rheumatoid arthritis, chronic obstructive pulmonary disease and Alzheimer's disease.

According to the recommendations of the German Society of Dental, Oral and Craniomandibular Sciences, currently used therapy for periodontitis is usually carried out by manual supragingival and subgingival debridement (removal of bacterial plaque) together with the use of antiseptics (daily disinfection with mouthwashes), which destroys all biofilm of the cavity mouth and provides an opportunity for recolonization by potential pathogens (Sanz, I. et al. J Evid Based Dent Practice 2012 , 12(3), 76-86). In addition, adjuvant systemic therapy with broad-spectrum antibiotics is used for advanced forms of the disease, such as persistent or recurrent localized deep sites (Jepsen, K. et al. Periodontol 2000 2016 , 71(1), 812-112). The latter also leads to non-selective biofilm destruction and must be administered in high doses and over a long period of time to achieve sufficient therapeutic levels at the specific site of action, i.e. the periodontal pocket. Standard adjuvant therapy for periodontitis includes, for example, systemic doxycycline (PO) 1 × 200 mg/stamp for 1 day and 2 × 100 mg/stamp for an additional 18 days (Wissenschaftliche Stellungnahme: Adjuvante Antibiotika in der Parodontitistherapie, Deutsche Gesellschaft für Zahn- Mund- und Kieferheilkunde, DZZ 2003 ). As a result, resistance to oral pathogens develops. Additionally, the patient's gut microbiome is disrupted, resulting in loss of metabolic support, immune modulation, and allowing recolonization by potential pathogens. Targeted and targeted therapies will mean significant improvements in the treatment of periodontitis and related conditions. A systematic review showed a significant effect of tetracycline formulations on reducing periodontal pocket depth (Matesanz-Pérez, P. et al. J Clin Periodontol 2013 , 40(3), 227-241).

The presence of periodontopathogenic bacteria varies among patients with periodontitis. However, the detection of certain bacterial species in subgingival plaques has been closely associated with the etiology of periodontal disease (Socransky et al.,Journal of Clinical Periodontology,1998, 25, 134-144). The first step to the development of periodontitis is the colonization of yellow complex, green complex, and purple complex bacteria in healthy periodontal areas. KindsActinomyces are closely related to members of the “yellow complex” (Streptococcus sanguis,Streptococcus mitis,Streptococcus gordonii AndStreptococcus intermedius), "green complex" (Eikenella corrodens,Capnocytophaga gingivalis,Capnocytophaga sputigena) and the “purple complex” (Veillonella parvula,Actinomyces odontolyticus). These complexes are accompanied by colonization by members of the so-called “orange complex” bacteria, including members of the subspeciesFusobacterium nucleatum/periodonticum, Prevotella intermedia,Prevotella nigrescensAndParvimonas micra (formerly known asPeptostreptococcus micros orMicromonas micros). Colonization of healthy periodontal sites by members of the "orange complex" has been found to correlate with the occurrence of gingivitis. Orange complex bacteria also promote colonization by red complex bacteria, which in turn are associated with deep pockets and chronic periodontitis. "Red complex", consisting of a closely related groupTannerella forsythia,Porphyromonas gingivalisAndTreponema denticola, largely relates to clinical indicators of periodontal disease and in particular to pocket depth and bleeding on probing. Additionally, Lamont et al. (Microbiology 2002,148, 1627-1636) and Daep et al. (Infect. Immun. 2006, 74, 5756-5762) demonstrated thatS. gordonii plays a role in colonizationPorphyromonas gingivalis.

The use of antibiotics or antiseptics in adhesive, sustained-release formulations for topical administration is technically challenging. The mucous membrane of the mouth is covered with a liquid film and is partially populated with bacteria. In addition, it is subject to mechanical stress due to talking, swallowing and chewing. Although these stress sources are not directly present in the periodontal pocket, the presence of gingival fluid (the flow rate of which greatly increases in the case of periodontitis) leads to increased leaching of active substances from the pocket.

In view of this difficult initial situation, none of the existing products, either topical antibiotic therapy or antiseptic therapy, has been able to adequately address the existing problems. For example, Actisit® is a tetracycline-containing fiber for placement in the periodontal pocket, requiring complex application by a qualified dentist into the periodontal pocket and further intervention for removal after 10 days. This product is no longer available on the German market. Elisol®, a gel containing metronidazole, does not meet expectations for release of the active ingredient for more than two days due to insufficient gel adhesion. This product is also no longer available on the German market. Atridox®, a controlled-release doxycycline product consisting of a two-syringe mixing system, has also been withdrawn from the German market.

Ligosan®, which is an application system containing doxycycline, and PerioChip®, which is a biodegradable chip containing chlorhexidine, are the only topical products still available on the market. Ligosan® is an application system containing 14% doxycycline in a biodegradable gel matrix for application into the periodontal pocket using a special localization system that provides continuous release for 12 days and results in improved periodontal pockets. However, handling and controllability of the gel is difficult and applying the appropriate amount in all parts of the pocket is difficult, especially when it comes to the directly invisible interdental spaces in the posterior teeth. The gel must be administered using a special application system. In addition, although it has been shown to be effective against bacteria in deep periodontal pockets, it is not suitable as a complete therapy and does not replace classical treatments (Eickholz, P et al.; J Clin Periodontol 2002 ; 108-117; Ratka-Krüger , P. et al., J Periodontol . 2005 /1,76: 66-74). In addition, due to the high cost and low motivation for dentists, the general use of this therapy appears to be difficult (see https://www.parodontitis.com/ligosan-slow-releaseR-zur-behandlung-von-zahnfleischtaschen.html ). Periochip® contains 2.5 mg chlorhexidine in a biodegradable gelatin chip that can be inserted into periodontal pockets using tweezers. The chip remains in the pocket for 7-10 days and over time the drug is continuously released. However, it has been found not to be effective against bacterial composition as a topical antibiotic. In addition, health insurance does not pay the costs (see https://www.parodontitis.com/lokale-behandlung-der-zahnfleischtaschen-mit-periochip-ec40-chxhtml.html). Thus, although some improvements in periodontitis parameters (pocket depth and inflammation parameters) have been achieved with these systems, they require complex application and, in addition, are not reimbursed by public health insurance companies in Germany, so they cause huge costs that have to be paid patients.

Systemic low-dose doxycycline (Periostat®) has also been used as long-term therapy. However, the expected pharmacological effect does not always correlate with a successful clinical outcome. For example, the therapy appears to have reduced effectiveness in smokers. In addition, a high level of patient compliance is required; and research findings regarding the presence of clinical improvement are controversial. Thus, no general recommendations can be made regarding this therapy (G. Greenstein, Efficacy of submicrobial-dose doxycycline in the treatment of periodontal diseases: a critical evaluation,The international journal of Periodontics & Restorative Dentistry, 2004, 24(6):528-543; I. Needleman et al., A randomized-controlled trial of low-dose doxycycline for periodontitis in smokers,Journal of Clinical Periodontology, 2007, 34(4):325-333).

Finally, Arestin® is a sustained-release subgingival product consisting of microspheres containing the antibiotic minocycline hydrochloride embedded in a bioresorbable polymer, poly(glycolide-co-DL-lactide) or PGLA, for professional subgingival administration into periodontal pockets. Each single dose capsule delivers minocycline hydrochloride equivalent to 1 mg of minocycline free base. The continuous release of the drug is described to be 14 days. However, the product is no longer available on the German market (see https://www.parodontitis.com/vom-markt-genommen-atridoxR-actisiteR-arestinR-und-elyzolR.html). The consistency of the material (microspheres) requires application using special carpules and makes handling and placement in the periodontal pocket difficult and not very reliable.

As presented herein, there are several effective therapeutic options, but all offer room for improvement. Thus, there is a great need to develop new, improved forms of administration of tetracycline antibiotics with sustained activity, particularly for the treatment of periodontitis and related conditions. An ideal drug delivery system for the treatment of periodontitis should combine simple manufacture, easy administration, no solvents, and controlled release over a long period with high biological activity.

Problems solved by the invention

In view of the above, the present invention is directed to providing an administration form for tetracycline compounds having improved stability; and/or prolonged activity; and/or simpler, easier and/or more reliable use and handling. In particular, the present invention is directed to the creation of topical forms that allow the dentist to administer the antibiotic in a simple, reliable and as painless manner as possible. In addition, it aims for a significantly prolonged and uniform release so that the required level of activity is maintained over a long period of time (i.e., beyond 14 days). Finally, it would be desirable for new administration forms to be preferably safe, reliable and cheap enough to be reimbursed by public health insurance.

Additional objects of the present invention are to provide a method for making such administration forms; providing a method of treating a human or animal body and/or a compound or pharmaceutical for use in such a method; providing a method of treating or preventing a bacterial infection and/or a compound or pharmaceutical for use in such a method; providing a method of treating or preventing acute, chronic or recurrent periodontal disease and/or a compound or pharmaceutical compound for use in such a method.

The essence of the invention

The present invention provides a complex containing a tetracycline compound (TC) or a pharmaceutically acceptable salt thereof and a divalent metal carboxylate (MA ₂ ), in which: the molar ratio of TC:MA ₂ is in the range of 1:0.8-3.0; The divalent metal cation M is an alkaline earth metal cation and A is a carboxylate anion derived from a C ₈ -C ₂₄ carboxylic acid.

The present invention further provides a pharmaceutical preparation containing the complex in accordance with any one of aspects 1-4 and optionally one or more pharmaceutically acceptable excipients, preferably in the form of a filamentary extrudate.

The present invention further provides a method for preparing the said complex, comprising the following steps: (a) mixing a tetracycline compound (TC) or a pharmaceutically acceptable salt thereof and a divalent metal carboxylate (MA ₂ ) in a molar ratio of 1:0.8-3.0 with an organic solvent to obtain a dispersion in which the divalent metal cation M is an alkaline earth metal cation and A is a carboxylate anion derived from a C ₈ -C ₂₄ carboxylic acid; (b) heating said dispersion to form a complex; (c) removing the solvent to obtain a complex. The present invention further provides a method of manufacturing a pharmaceutical preparation in the form of a filamentary extrudate, comprising the following steps: (d) grinding the specified 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 a pharmaceutical preparation in the form of a threaded extrudate.

The present invention further provides a complex as defined above and/or a pharmaceutical preparation as defined above for use in a method of treating a human or animal body; and/or for use in a method of treating and/or preventing 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 in which the antibiotic activity is maintained for a period of at least 21 days; and/or for use in a method of therapy and/or prevention of acute, chronic or recurrent periodontal disease.

EFFECTS ACHIEVED BY THE INVENTION

The present inventors have discovered that the complexes of the tetracycline compounds of the present invention degrade more slowly in aqueous media than the corresponding tetracycline compounds. In addition, these complexes have been found to have lower water solubility and provide a more delayed and sustained release compared to corresponding tetracycline compounds or even sustained release systems such as Arestin®. Although chelate complexes of tetracycline compounds with magnesium or calcium ions in a ratio of 1:0.5 have been previously described, the present complexes differ in their stoichiometry. Complexation in accordance with the present invention results in altered chemical properties of the compounds. In particular, solubility, photometric characteristics and reactivity have a beneficial effect. The active substance remains biologically active for an extended period of time and is released in a delayed manner due to lower solubility in water, increased resistance to degradation in aqueous solution and the lipophilic properties of the carboxylate anion. In addition, complex formation appears to prevent the conversion of tetracycline compounds into less active epimers and thus helps maintain the biological activity of the active substances over a longer period of time. These effects may persist when the complexes of the present invention are administered either alone or in combination with one or more pharmaceutical excipients in a pharmaceutical preparation, eg, a topical preparation.

Moreover, when the complex is provided in a pharmaceutical preparation such as an extrudate (either as a pure substance or together with an excipient such as a biodegradable polymer), even longer release and activity can be achieved. Such extrudates can be easily formed and processed (eg, for placement in a periodontal pocket), further resulting in further delayed drug release. Finally, they demonstrate good adhesion to the oral mucosa and thus excellent application reliability.

The manufacturing method described herein results in the formation of new complexes of tetracycline compounds with unexpectedly pronounced sustained drug release, which is therapeutically desirable. In addition, the complexes obtained in accordance with the manufacturing method of the present invention have increased solubility in organic solvents and decreased solubility in water. Methods for the preparation of complexes and preparations in accordance with the present invention are simple, universal, do not require the use of toxic organic solvents and can be based solely on the use of well-known, reliable and safe pharmaceutical ingredients. In addition, the complexes of the present invention are easily moldable and can be extruded either alone or together with one or more excipients to form an extrudate having a suitable desired geometry, for example, suitable for easier placement in a periodontal pocket.

The complexes and formulations have been shown to have superior release characteristics and sustained antibacterial activity, particularly against oral pathogens associated with the onset and progression of periodontitis. This may lead to increased effectiveness of non-surgical periodontal therapy and therefore a reduction in the amount of surgical treatment required, as well as significant cost savings. In addition, the patient burden caused by treatment with systemically administered antibiotics can be reduced by substituting an effective topical therapeutic formulation instead.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1 shows glass vials with dried minocycline/magnesium stearate complexes in various molar ratios. From left to right: minocycline hydrochloride/magnesium stearate in molar ratios 1:1, 1:2; minocycline free base/magnesium stearate in ratios of 1:1 and 1:2 (according to Examples 4, 3, 2 and 1, respectively).

In FIG. 2 shows micrographs of dried minocycline/magnesium stearate having a minocycline/magnesium stearate molar ratio of 1:1 (left, Example 2) and 1:2 (right, Example 1).

In FIG. 3 shows micrographs of dried doxycycline magnesium stearate having a doxycycline/magnesium stearate molar ratio of 1:1 (left, Example 6) and 1:2 (right, Example 5).

In FIG. 4 shows chromatograms of minocycline (left) and the complex according to Example 1 (right) during incubation in buffer at pH 7.1 (top) and 2.3 (bottom) at 37°C over time (day zero is always the top crooked).

In FIG. 5 shows the UV/visible absorption spectra of minocycline and minocycline complex according to Example 1 in ethanol before (left) and after (right) addition of hydrochloric acid.

In FIG. 6 shows the FTIR-ATR spectra of minocycline, magnesium stearate and the complex in accordance with Example 1 in the region of 680-4000 cm ^-1 .

In FIG. 7 shows the release curves of the extrudate of Example 9, consisting of a pure minocycline/magnesium stearate 1:2 complex, as well as with various PLGA polymers upon extrusion (Examples 10-13).

In FIG. Figure 8 shows load-displacement diagrams during deformation of extrudates on a texture analyzer equipped with a knife with a sharp blade.

In FIG. 9 is a photograph of extrudates prepared in accordance with the present invention containing Resomer 502 RG (top) and Resomer 503 RG (bottom) containing 11.5% minocycline. Both extrudates were extruded using a 600 μm extruder. The larger diameter of the 503 RG extrudate indicates the viscoelastic properties of the polymer.

In FIG. Figure 10 shows the experimental setup for assessing the adhesion force to mucosal membranes (A) and the geometry of the flow channel specifically used for measurements (B).

In FIG. 11 shows the minimum inhibitory concentrations of eluates (dilutions) compared to Streptococcus gordonii ATCC 10558 and Porphyromonas gingivalis ATCC 33277 up to 42 days.

In FIG. 12 shows the inhibitory effect of eluates obtained after 24 hours, 2 days, 7 days, 14 days and 28 days compared to biofilm formation.

In FIG. 13 shows the UV/visible spectra (230-450 nm) of minocycline and doxycycline in combination with magnesium and calcium stearate and their complexes after the addition of HCl. The ratios are molar ratios.

In FIG. 14 shows the FTIR-ATR spectra of minocycline, magnesium and calcium stearate and their complexes; 680-4000 cm ^-1 . Minocycline: a - ʋ OH; b - ʋ N-H; c - ʋCH; d - ʋ CONH ₂ ; e - ʋ C=C (aromatic); f - ʋCN.

Magnesium stearate/calcium stearate: g - ʋ CH; h - ʋ _as COO ^- ; i - δCH; j - CH ₂ swing-oscillation.

In FIG. 15 shows the FTIR-ATR spectra of doxycycline, magnesium and calcium stearate and their complexes; 680-4000 cm ^-1 . Doxycycline: a - ʋOH/ʋNH; b - ʋ S-N; c - ʋ CONH ₂ ; d - ʋ C=C (aromatic); f - ʋCN.

Magnesium stearate/calcium stearate: e - ʋ CH; f - ʋ _as COO ^- ; g - δ С-Н; h - CH ₂ swing-oscillation.

In FIG. Figure 16 shows agar plates incubated with S. aureus ATCC 29213. Left plate: ethanol-filled disks; middle cup: disks filled with an ethanol solution of minocycline complex; right cup: discs filled with ethanol solution of minocycline.

In FIG. 17 shows the release profile of several formulations of PLGA with minocycline complex in PBS pH 7.0 at 37°C.

In FIG. Figure 18 shows chromatograms of minocycline and minocycline complex at pH 7.0 and pH 2.3 at 37°C.

In FIG. 19 shows a 5 wt.% suspension of minocycline HCl (left) and a 5 wt.% solution of disodium phosphate (right).

In FIG. 20 shows a mixed solution of minocycline HCl and disodium phosphate (2.5 wt.% minocycline HCl, 2.5 wt.% disodium phosphate).

In FIG. 21 shows a 1 wt.% solution of minocycline HCl (left) and a 1 wt.% solution of disodium phosphate (right).

In FIG. 22 shows a mixed solution of minocycline HCl and disodium phosphate (0.5 wt.% minocycline HCl, 0.5 wt.% disodium phosphate).

In FIG. 23 shows a mixed solution of minocycline HCl and disodium phosphate (0.5 wt.% minocycline HCl, 0.5 wt.% disodium phosphate, but with the addition of 400 μl of 0.1 M HCl).

DETAILED DESCRIPTION OF THE INVENTION

Complexes

The present invention provides a complex containing a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA ₂ ), wherein: the TC:MA ₂ molar ratio is in the range of 1:0.8-3.0 ; The divalent metal cation M is an alkaline earth metal cation and A is a carboxylate anion derived from a C ₈ -C ₂₄ carboxylic acid.

The main problem with controlled release of tetracyclines is their chemical instability in water. For example, minocycline exhibits epimerization reactions with loss of activity. Doxycycline also degrades in an aqueous environment. Therefore, degradation of the drug before release should be avoided. The inventors hypothesized that formation of a lipophilic complex could increase drug stability and lead to increased release time.

Tetracycline compounds

As used herein, the term “tetracycline compound” (TC) refers to any compound belonging to the tetracycline family of antibiotics, that is, based on the minimal pharmacophore structure of 6-deoxy-6-demethyltetracycline:

Preferably, the tetracycline (TC) compound is selected from minocycline, doxycycline, tetracycline, chlortetracycline, oxytetracycline, rotetracycline, tigecycline, demeclocycline, lymecycline, meclocycline, methacycline, omadacycline, sarecycline and eravacycline. Preferred tetracycline compounds are also selected from the group consisting of 7-chlorotetracycline, 5-hydroxytetracycline, tetracycline, 6-demethyl-7-chlorotetracycline, 2-N-pyrrolidinomethyltetracycline, 2-N-lisinomethyltetracycline, 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-(tert-butylglycylamido)minocycline (tigecycline), as further described in Table 2 by Chopra et al.,Microbiol Mol Biol Rev. 2001, June 65(2):232-60. Additional preferred tetracycline compounds include clomocycline (N-methylol-7-chlorotetracycline), 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline and 9-(N,N-dimethylglycylamido)minocycline.

It should be noted that as used herein, the term "tetracycline compound" or "TC" refers to a general class of compounds, namely any compound belonging to the tetracycline family of antibiotics, while the term "tetracycline" refers only to a 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:

tetracycline

Preferably, the tetracycline (TC) compound is selected from minocycline, doxycycline, tetracycline, chlortetracycline, oxytetracycline, rotetracycline, tigecycline, demeclocycline, lymecycline, meclocycline, metacycline, 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 proven and thus reliable and are likely to be suitable for various therapeutic applications as described herein.

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 even more preferably minocycline .

These antibiotics have the added benefit of being clinically proven to be useful in the treatment of periodontal disease.

Molar ratio

The TC:MA ₂ molar ratio is preferably in the range of 1:0.8-3.0. The present inventors have found that the predominant species obtained in the preparation of complexes according to the present invention are those in which the TC:MA ₂ is 1:1 or 1:2. Thus, it is intended that also mixtures in which the TC:MA ₂ molar ratio is from 1:1 to 1:2 are also included within the scope of the present invention. A slight excess of TC, for example where TC:MA ₂ =1:0.8, is also within the scope of the present invention. Likewise, excess MA ₂ , for example above a TC:MA ₂ ratio 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 TC. Thus, it is also intended that such mixtures containing complexes with a certain TC:MA ₂ ratio together with a certain excess of one of the components are also covered by the definition of a complex in accordance with the present invention. Preferably the TC:MA ₂ is 1:1 or 1:2; more preferably the TC:MA ₂ is 1:2. Additional preferred ranges for the TC:MA ₂ molar ratio are, for example, 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

A is a carboxylate anion derived from a C ₈ -C ₂₄ carboxylic acid. The lipophilic nature of the carboxylate anion helps to improve the sustained release of the tetracycline antibiotic from the complexes and formulations of the present invention.

The carboxylic acid may be saturated or unsaturated. Preferably, A is a carboxylate anion derived from a carboxylic acid selected from arachidic acid ( _C20 ), stearic acid ( _C18 ), palmitic acid ( _C16 ), myristic acid ( _C14 ), lauric acid ( _C12 ) , capric acid (C ₁₀ ), caprylic acid (C ₈ ), hydroxystearic acid, oleic acid, linoleic acid, linolenic acid and ricinoleic acid. The hydroxystearic acid may preferably be 10-hydroxystearic acid or 12-hydroxystearic acid. Most preferably, the carboxylate anion is stearate (C ₁₈ ).

Pharmaceutically acceptable salts, hydrates and solvates

As used herein, the term "pharmaceutically acceptable" covers both medical and veterinary uses. For example, the term "pharmaceutically acceptable" includes a compound acceptable for veterinary use or a compound acceptable for medical and public health purposes. Salts, hydrates and solvates of tetracycline compounds are compounds in which the counterion or associated solvent is pharmaceutically acceptable. However, salts, hydrates and solvates containing pharmaceutically unacceptable counterions 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 both 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, almond , glutamic, aspartic, oxaloacetic, methanesulfonic, ethanesulfonic, arylsulfonic (for example, p-toluenesulfonic, benzenesulfonic, naphthalene sulfonic or naphthalene disulfonic), salicylic, glutaric, gluconic, tricarballyl, cinnamic, substituted cinnamic (for example, phenyl-, methyl-, meth hydroxy or halogenated cinnamic acid, including 4-methyl- and 4-methoxycinnamic acid), ascorbic acid, oleic acid, naphthoic acid, hydroxynaphthoic acid (for example, 1- or 3-hydroxy-2-naphthoic acid), naphthalene acrylic acid, benzoic acid, 4-methoxybenzoic acid, 2-or 4- hydroxybenzoic, 4-chlorobenzoic, 4-phenylbenzoic, benzolacrylic (eg 1,4-benzenediacrylic), isethionic acids, perchloric, propionic, glycolic, hydroxyethanesulfonic, pamic, cyclohexanesulfamic, salicylic, saccharic and trifluoroacetic acid. The most preferred acid addition salts are the hydrochloride, hyclate (hemiethanolate hydrochloride 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 sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, 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 included within the scope of the present invention.

The complex is formed from a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a divalent metal carboxylate (MA ₂ ). In a preferred embodiment, the TC:MA ₂ ratio is 1:2; and/or M is selected from Mg ²⁺ and Ca ²⁺ ; and/or A is a carboxylate anion derived from a carboxylic acid selected from arachidic acid (C ₂₀ ), stearic acid (C ₁₈ ), palmitic acid (C ₁₆ ), myristic acid (C ₁₄ ), lauric acid (C ₁₂ ), capric acid (C ₁₀ ), caprylic acid (C 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 a divalent metal carboxylate (MA ₂ ).

In a further preferred embodiment, the complex has the formula [(TC)⋅2(MgA 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.

Pharmaceuticals

The present invention provides a pharmaceutical preparation containing a complex as described above, and optionally one or more pharmaceutically acceptable excipient(s).

As used herein, the term "pharmaceutical" is intended to cover a product containing the claimed compounds in therapeutically effective amounts, as well as any product that is the direct or indirect result of combinations of the claimed compounds.

In the pharmaceutical preparation according to the present invention, the total content of tetracycline compound (TC) is preferably in the range of 5-30 wt%, 30-50 wt%, more preferably 8-28 wt%, 32-48 wt%, more more preferably 10-20 wt.%, even more preferably 11-15 wt.%, even more preferably 11.5±5 wt.%, 27.9±5 wt.%, 11.5±2 wt.%, 27 .9±2 wt.%, 11.7-14.75 wt.%, 15.25-19.75 wt.% and most preferably 11.5 wt.% or 27.9 wt.%. In addition, the total content of the tetracycline complex (preferably the complex having the formula [(TC)⋅2(MgA ₂ )]) in the pharmaceutical preparation is in the range of 5-95 wt.%, more preferably 10-80 wt.%, even more preferably 15-75 wt%, even more preferably 20-60 wt%, even more preferably 30-50 wt% and most preferably 41.2 wt%.

Excipients

As used herein, the term "excipient" refers to a carrier, binder, disintegrant and/or other suitable additive for a herbal formulation, such as an extrudate, cream, gel, emulsion, suspension, liniment, ointment, powder, paste, balm, lotion, eye drops, spray, topical aerosol, topical solution, topical suspension, skin patch and non-woven fabric. Carriers that may be added to the mixture include necessary and inert pharmaceutical excipients, including, but not limited to, suitable release retardants, suspending agents, lubricants, flavoring agents, sweeteners, preservatives, coatings, granulating agents, dyes and coloring agents.

The most preferred class of excipients are biodegradable polymers, which will be discussed in more detail below.

Additional preferred 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 pattern of drugs, in particular extrudates, can be controlled (eg, accelerated).

Additional preferred 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%. The preferred type of plasticizer is PEG 1500. By adding such excipients, the mechanical behavior of the preparations, in particular extrudates, can be controlled (eg, the brittleness and/or tendency to harden over time can be reduced).

Routes of administration and types of drugs

Preferably, the pharmaceutical preparation according to the present invention is intended for topical administration. Topical administration means application to body surfaces such as the skin or mucosal membranes to treat diseases through a wide range of classes including creams, foams, gels, lotions and ointments. Many topical medications are cutaneous, meaning they are applied directly to the skin. Topical medications may also be inhaled, such as asthma medications, or applied to the surface of tissues other than the skin, such as eye drops placed on the conjunctiva or ear drops placed in the ear, or medications applied to the surface tooth, gum or periodontal pocket.

Even more preferably, the pharmaceutical preparation is selected from the group consisting of extrudate, particle, granule, powder, film, strip, compressed powder, chip, paste, cream, gel, emulsion, suspension, liniment, ointment, balm, lotion, eye drops, spray, topical aerosol, topical solution, topical suspension, skin patch and non-woven fabric. Even more preferably, the pharmaceutical preparation allows for use in a periodontal pocket.

Extrudates

The inventors sought to develop hot-melt extrudates that replicate the optimal conditions in which an ideal drug delivery system for the treatment of periodontitis should combine simple manufacturing, convenient administration, solvent-free, and controlled release over a long period of time with high biological activity. Hot melt extrusion is a suitable technology because it is a continuous and well-established process in the pharmaceutical industry. Sizes and shapes can be changed by extrusion parameters. In addition, flexibility and swelling behavior can be modified by appropriate selection of excipients. Extrudates can be cut to the appropriate length. Installation of extrudates is a simple and quick process. This does not require any special device, unlike the application of microparticles and highly viscous gels.

As used herein, the term “extrudate” refers to any material that has been extruded through a die. In particular, extrudates refer to dosage forms obtained by extruding a precursor into rods that can either be cut into pieces or used as such, for example, as a sustained release dosage form for topical or other routes of administration. Preferably, the pharmaceutical preparation is in the form of a filamentary extrudate, for example as shown in FIG. 9. The filamentary extrudate preferably has a substantially circular or substantially elliptical cross-section and/or a maximum cross-sectional diameter in the range of 0.1-10 mm, 0.2-5 mm, 0.3-1 mm, 0.4-0 .8 mm, 0.3-0.6 mm, more preferably 0.2-5 mm, even more preferably 0.3-1 mm, even more preferably 0.4-0.8 mm, most preferably 0.3- 0.6 mm, 0.3, 0.4, 0.5 or 0.6 mm. The appropriate diameter can be easily, painlessly and securely placed into a periodontal pocket of a given size.

Biodegradable polymers

The pharmaceutical preparation according to the present invention preferably contains one or more biodegradable polymer(s) as a possible excipient. They can 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 ); biodegradable polyester blends 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.

Poly(glycolic acid) (PGA), poly(lactic acid) (PLA) and their copolymers are biodegradable polyesters suitable for use in the present invention. They degrade in the body by simple hydrolysis of the ester chain into harmless and non-toxic compounds. Decomposition products are either excreted by the kidneys or excreted in the form of carbon dioxide and water through well-known biochemical pathways. Because PLA/PGA polymers are considered safe, non-toxic, and biocompatible by regulatory authorities in virtually all developed countries, additional applications of these materials can be brought to market sooner and are more cost-effective than those using new polymers with unproven biocompatibility. Examples of PLA/PGA polymers suitable for use in the present invention are, for example, 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/resomer.html, found 10/25/2018).

Poly(glycolic acid) (PGA)

PGA is a highly crystalline material with a high melting point (225-230°C) and variable solubility in organic solvents, which is usually low and depends on the molecular weight of the polymer. It is still susceptible to hydrolysis due to the ester bond in the polymer backbone. Despite its low solubility, this polymer has been manufactured into various shapes and structures. Techniques used to develop PGA-based structures include extrusion, injection molding and compression molding, as well as solid leaching and solution molding. PGA fibers have high strength and elastic modulus and are particularly tough.

Poly(lactic acid) (PLA)

Unlike glycolide, lactide is a chiral molecule and exists in two different optically active forms - L-lactide and D-lactide. When each of these monomers is polymerized, the resulting polymer is semicrystalline. Polymerization of a racemic mixture of L- and D-lactides results in the formation of poly-D,L-lactide (PDLLA), which is amorphous and has a glass transition temperature of 55-60°C. The degree of crystallinity can be adjusted by changing the ratio of D- and L-enantiomers in the polymer. The choice of PLA stereochemistry can have a major impact on the properties, processability, and biodegradability of the polymer. Poly(L-lactide) or PLLA is often the polymer of choice for cast/extruded biomedical devices because it decomposes into L(+)-lactic acid units, which are naturally occurring stereoisomers and are therefore excreted with minimal toxicity.

Poly(lactide-co-glycolide) copolymers (PLGA)

Both L- and DL-lactides have been used for copolymerization. The ratio of glycolide and lactide in various compositions makes it possible to control the degree of crystallinity of polymers. When crystalline PGA is copolymerized with PLA, the degree of crystallinity decreases, resulting in increased rates of hydration and hydrolysis. Thus, we can conclude that the degradation time of the copolymer is related to the ratio of monomers used in the synthesis. As a rule, the higher the glycolide content, the faster the rate of destruction. However, the exception to this rule is the 50:50 PGA:PLA ratio, which exhibits the fastest degradation.

As end groups, three types of functional end groups are preferred: (i) free carboxylic acid group, (ii) ester end group, (iii) alkyl ester group. Ester- and alkylether-capped polymers generally exhibit longer degradation times than their free carboxyl analogues.

Table 1 : Biodegradable polymers RESOMER®

Product no. Type RESOMER The product's name Molecular weight range Viscosity [dl/g] Application Tg [°C] Tm [°C] End group 719854 RESOMER L 206 S Poly(L-lactide) -- 0.8-1.2 Medical product 60-65 180-185 ether terminal 719951 RESOMER R 202 S Poly(D, L-lactide) 10000-18000 0.16-0.24 Controlled release 38-42 amorphous ether terminal 719978 RESOMER R 202 H Poly(D, L-lactide) 10000-18000 0.16-0.24 Controlled release 44-48 amorphous free carboxylic acid 719935 RESOMER R 203 S Poly(D, L-lactide) 18000-28000 0.25-0.35 Controlled release 46-50 amorphous ether terminal 719943 RESOMER R 203 H Poly(D, L-lactide) 18000-28000 0.25-0.35 Controlled release 48-52 amorphous free carboxylic acid 719889 RESOMER RG 502 Poly(D, L-lactide-co-glycolide) 50:50 7000-17000 0.16-0.24 Controlled release 42-46 amorphous alkyl ether 719897 RESOMER RG 502 H Poly(D, L-lactide-co-glycolide) 50:50 7000-17000 0.16-0.24 Controlled release 42-46 amorphous free carboxylic acid 739952 RESOMER RG 503 Poly(D, L-lactide-co-glycolide) 50:50 24000-38000 0.32-0.44 Controlled Release 44-48 amorphous ethereal 719870 RESOMER RG 503 H Poly(D, L-lactide-co-glycolide) 50:50 24000-38000 0.32-0.44 Controlled release 44-48 amorphous free carboxylic acid 739944 RESOMER RG 504 Poly(D, L-lactide-co-glycolide) 50:50 38000-54000 0.45-0.60 Controlled Release 46-50 amorphous ethereal 719900 RESOMER RG 504 H Poly(D, L-lactide-co-glycolide) 50:50 38000-54000 0.45-0.60 Controlled Release 46-50 amorphous free carboxylic acid 739960 RESOMER RG 505 Poly(D, L-lactide-co-glycolide) 50:50 54000-69000 0.61-0.74 Controlled Release 48-52 amorphous ethereal 719862 RESOMER RG 653 H Poly(D, L-lactide-co-glycolide) 65:35 24000-38000 0.32-0.44 Controlled Release 46-50 amorphous free carboxylic acid 719919 RESOMER RG 752 H Poly(D, L-lactide-co-glycolide) 75:25 4000-15000 0.14-0.22 Controlled Release 42-46 amorphous free carboxylic acid 719927 RESOMER RG 756 S Poly(D, L-lactide-co-glycolide) 75:25 76000-116000 0.71-1.0 Controlled Release 49-55 amorphous ether terminal 739979 RESOMER RG 858 S Poly(D, L-lactide-co-glycolide) 85:15 190000-240000 1.3-1.7 Controlled Release - amorphous alkyl ether 719846 RESOMER X Polydioxanone -- 1.5-2.2 (0.1% in GFIP, 30ºC) medical device (-10)-(-5) 110-115 -- ⃰Specific viscosity [dl/g], (0.1% in CHCl ₃ , 25°C, except where noted)

Additional 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, found 10/25/2018):

table 2 : Biodegradable polymers EXPANSORB®

Product Serial number Poly(D,L-lactic acid) (PLA) L/DL ratio End group V.V. range (dl/g, CHCl ₃ , 25°C) MW (approximate range, 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 ETHER 0.15-0.30 10-25 Product Serial number Poly(D,L-lactic-co-glycolic acid) (PLGA) L/G ratio End group V.V. range (dl/g, CHCl ₃ , 25°C) MW (approximate range, 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 ETHER 0.15-0.25 5-20 EXPANSORB® DLG 50-6E 5.43135 PLGA 50/50 ETHER 0.55-0.65 60-85 EXPANSORB® DLG 50-7E 5.43136 PLGA 50/50 ETHER 0.60-0.70 70-100 EXPANSORB® DLG 65-6E 5.43137 PLGA 65/35 ETHER 0.50-0.65 45-85 EXPANSORB® DLG 75-2E 5.43138 PLGA 75/25 ETHER 0.15-0.30 10-25 EXPANSORB® DLG 75-4E 5.43139 PLGA 75/25 ETHER 0.30-0.50 25-60 EXPANSORB® DLG 75-7E 5.43140 PLGA 75/25 ETHER 0.66-0.80 80-115 EXPANSORB® DLG 75-9E 5.43141 PLGA 75/25 ETHER 0.75-1.00 100-160 EXPANSORB® DLG 85-7E 5.43142 PLGA 85/15 ETHER 0.55-0.75 65-95 EXPANSORB® DLG 85-12E 5.45583 PLGA 85/15 ETHER 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

The biodegradable polymers used in the present invention preferably have a weight average molecular weight (kDa) range 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 intrinsic viscosity [dl/g] (0.1% in CHCl ₃ at 25°C) 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. Ester, alkyl ester and/or PEG-terminated polymers are preferred.

The biodegradable polymers used in the present invention are preferably PLGA (poly(D, L-lactide-co-glycolides)) copolymers having an 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.

Preferred biodegradable polymers are PLGA copolymers having an 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 (kDa) range of 5-90, preferably 7-85, more preferably 7-17, 24-38, 30-60, 60-85, even more preferably 8-18, 25-37, 31-59 , 61-84; and having an intrinsic (true) viscosity [dl/g] (0.1% in CHCl ₃ at 25°C) 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, even more preferably 0.17-0.23, 0.323-0.43, 0.46-0.64 , 0.66-0.84; and which end 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, even more preferably 43-45 or 45-47°C.

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 7000-38000, more preferably 7000-17000 or 24000-38000; and/or intrinsic viscosity 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 preferably 42-46 or 44-48°C. Even more preferably, the biodegradable polymer is selected from Resomer® RG 502 (“Resomer 502”), Resomer® RG 503 (“Resomer 503”), or mixtures thereof.

Preferred biodegradable polymers are also poly(D,L-lactide-co-glycolides) with a 50:50 PLA/PGA ratio 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. Even more preferably, the biodegradable polymer is EXPANSORB® DLG 50-6P.

Manufacturing methods

The present invention further provides a method for preparing 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 ₂ ) in a molar ratio of 1:0 .8-3.0 with an organic solvent to obtain a dispersion in which the divalent metal cation M is an alkaline earth metal cation and A is a carboxylate anion derived from a C ₈ -C ₂₄ carboxylic acid; (b) heating said dispersion to form a complex; (c) removing the solvent to obtain a complex. Preferred tetracycline compounds, molar ratios, carboxylate anions, pharmaceutically acceptable salts, hydrates and solvates are as described in the "Complexes" section above. In a preferred embodiment, the complex in accordance with the present invention is/can be obtained using the manufacturing method as described in this application.

In mixing step (a), the preferred solvent is, for example, an alcohol such as methanol, ethanol or mixtures thereof, in the presence or absence of water. Preferably, a weight/volume ratio of TC and solvent from 1:100 to 1:300, preferably 1:200, can be used.

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 chosen solvent, temperature and mass/volume ratio of TC to solvent, complete dissolution may occur during step (b), resulting in a solution of the complex.

In step (c), the solvent can be removed by a suitable method known in the art. Preferably, the solvent may be evaporated at room or elevated temperature; and/or in a shaker, oven or on/in film; and/or at atmospheric or preferably reduced pressure.

Pharmaceutical preparations in accordance with the present invention, unless otherwise indicated, can be prepared by a suitable method known in the art. However, when the pharmaceuticals are in the form of filamentary extrudates, the latter are preferably prepared in accordance with a process comprising the following steps: (d) grinding 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 a pharmaceutical preparation in the form of a threaded extrudate. Preferred excipients, routes of administration and types of formulations, extrudates and biodegradable polymers are described in the Pharmaceuticals section above. In a preferred embodiment, the pharmaceutical preparation in accordance with the present invention is/can be obtained using the manufacturing method as described in this application.

In grinding step (d), the complex is mixed, crushed and homogenized together with one or more pharmaceutically acceptable excipient(s) (as described above and if present) to produce an extrusion precursor. Preferably, one or more biodegradable polymer(s), emulsifier(s) and/or plasticizer(s) as described above may be added as excipients. Even more preferably, the content of the added tetracycline compound (TC) is in the range of 5-30 wt%, more preferably 8-28 wt%, even more preferably 10-20 wt%, even more preferably 11-15 wt%. and most preferably 11.5 wt.% or 27.9 wt.% relative to the total amount of mixed components. Comminution is preferably carried out by grinding or grinding, preferably in a mill or grinder, more preferably at low temperature, for example in a cryomill at -196°C. When using a cryomill, performing 6, preferably 5 grinding cycles at 30 Hz for 150 s with intermediate cooling (30 s) at 5 Hz between cycles is most preferred to obtain an extrusion precursor.

Extrusion step (e) is carried out by extruding the extrusion precursor through a die. Preferably, a twin screw extruder can be used. Extrusion is carried out 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 approximately equal to or higher than the softening temperature (in the case of a pure complex without polymer ingredients); or up to or above the glass transition temperature (in the case of the presence of a polymeric ingredient, such as biodegradable polymers). In general, the extrusion temperature may preferably be in the range of 48 to 56°C, more preferably 49 to 55°C, even more preferably selected from 49, 52, 53 and 55°C.

The cross-sectional shape of the extrudate is mainly determined by the die at the exit of the extruder, which may preferably be a substantially circular or substantially elliptical cross-section and/or a maximum cross-sectional diameter in the range of 0.1-10 mm, more preferably 0.2-5 mm, even more preferably 0.3-1 mm, even more 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 associated with the dental sulcus as a target site for application. The inflamed gum pocket is at least approximately 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 provides high local concentrations of the API. Therefore, a diameter of less than 1 mm and/or a length of less than 5 mm, preferably less than 3 mm, is preferred for convenient use. Preferred extrudate diameters are, for example, 125-975 microns, 425-575 microns, 625-775 microns. Preferably, the extrusion step produces homogeneous extrudates with a diameter of 600 to 900 μm, for example 620 to 880 μm. The use of different diameters adapted to the pocket size and the simultaneous use of different diameters (e.g. 600 µm surrounded by 300 µm) appears to be very promising. The diameter and shape of the extrudates can be customized. For example, the extruder die can optionally be replaced with a plate with different diameters or geometric shapes.

After cooling (f) of the extruded product of step (e), the pharmaceutical preparation is obtained in the form of a threaded extrudate, as shown, for example, in FIG. 9. The thread-like shape thus obtained can be further cut into smaller pieces or deformed as desired, for example to fit into a periodontal pocket of a certain size or shape.

Therapeutic Use

The present invention further provides a complex and/or pharmaceutical preparation, as defined above, for use in a method of treating a human or animal body. The present disclosure also provides a method of treating a human or animal body, wherein the method includes administering a therapeutically effective amount of a complex and/or a pharmaceutical as defined above to a subject in need of such treatment.

The present invention further provides a complex and/or pharmaceutical preparation as defined above for use in a method of treating and/or preventing 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 in which the antibiotic activity is maintained for a period of at least 21 days, preferably at least 25, more preferably at least 28, even more preferably at least 35 days, most preferably 42 days; and/or for use in a method of therapy and/or prevention of acute, chronic or recurrent periodontal disease. The disclosure further provides a method of treating and/or preventing 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 in which the antibiotic activity is maintained for a period of at least 21 days, preferably at least 25, more preferably at least 28, even more preferably at least 35 days, most preferably 42 days; and/or a method of treating and/or preventing acute, chronic or recurrent periodontal disease, each of which comprises administering a therapeutically effective amount of a complex and/or a pharmaceutical preparation as described above to a subject in need of such treatment, respectively.

It is currently believed that P. gingivalis, P. intermedia, T. forsythia, F. nucleatum and P. micros are the pathogens most associated with periodontitis. In addition, A. naeslundii and S. gordonii , belonging to the so-called "early colonizers", are considered essential for biofilm formation (e.g. oral periopathogenic biofilm) and are also present in the case of periodontal diseases.

The main categories of periodontal diseases and conditions are divided into the groups of plaque-induced gum disease, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotizing periodontal disease, periodontal abscesses, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections. In the present invention, acute, chronic or recurrent periodontal disease is preferably selected from the group consisting of plaque-induced gum disease, periodontitis, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotizing periodontal diseases, periodontal abscesses, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections. The present disclosure also provides a method of treating or preventing acute, chronic or recurrent periodontal disease, which is preferably selected from the group consisting of plaque-induced gum disease, periodontitis, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotic periodontal disease, abscesses periodontal disease, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections, wherein the method includes administering a therapeutically effective amount of the complex and/or a pharmaceutical preparation as described above to the subject in need of such treatment. Additional acute, chronic or recurrent periodontal diseases are described, for example, in Armitage, A. Ann Periodontol 1999 , 4 , 1-6.

As used herein, the term “subject” refers to an animal, preferably a mammal, most preferably a human, that is or has been the subject of treatment, therapy, prophylaxis, observation or experiment.

As used herein, the term “therapeutically effective amount” means that amount of an active compound or pharmaceutical agent that produces a biological or drug response in a tissue system, animal, or human that is sought by a researcher, veterinarian, physician, or other clinician, which includes alleviation of disease symptoms. 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

Minocycline (free base) was weighed into a round bottom flask along with magnesium stearate in a 1:2 molar ratio. 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 obtain a light yellow opaque suspension. The closed flask was placed in a water bath with gentle shaking. Starting at approximately 50°C, complex formation and dissolution occurred. At approximately 60-70°C a clear orange solution formed which remained clear after cooling to room temperature. The solvent was removed in a heated shaker. In FIG. 1 shows vials with dried products.

Example 2: Formation of a 1:1 complex of minocycline (free base) and magnesium stearate

A complex having a 1:1 molar ratio was prepared in the same manner as described in Example 1, using a suitably adapted amount of minocycline (free base).

Example 3: Formation of a 1:2 complex of minocycline hydrochloride and magnesium stearate

A complex of minocycline hydrochloride and magnesium stearate having a molar ratio of 1:2 was prepared in the same manner as described in Example 1 above, in which an appropriate amount of minocycline hydrochloride was used, resulting in a molar ratio of minocycline to magnesium stearate of 1:2.

Example 4: Formation of a 1:1 complex of minocycline hydrochloride and magnesium stearate

A complex of minocycline hydrochloride and magnesium stearate having a molar ratio of 1:1 was prepared in the same manner as described in Example 1 above, in which an appropriate amount of minocycline hydrochloride was used, resulting in a molar ratio of minocycline to magnesium stearate of 1:1.

Example 5: Formation of a 1:2 complex of doxycycline (free base) and magnesium stearate

A complex of doxycycline (free base) and magnesium stearate having a molar ratio of 1:2 was prepared in the same manner as described in Example 1 above, in which doxycycline was used instead of minocycline.

Example 6: Formation of a 1:1 complex of doxycycline (free base) and magnesium stearate

A complex of doxycycline (free base) and magnesium stearate having a molar ratio of 1:1 was prepared in the same manner as described in Example 1 above, in which however doxycycline free base was used instead of minocycline, the appropriate amount of doxycycline being such that the molar ratio was 1 :1.

The products from the above examples have properties different from those of the corresponding tetracycline compounds (minocycline, minocycline hydrochloride and doxycycline), indicating the formation of separate new complexes.

The solubility of the complexes is reduced compared to the corresponding tetracycline compound (for example, pure minocycline). Minocycline is soluble in most polar solvents such as water, dimethyl sulfoxide, ethanol, methanol and N -methylpyrrolidone. The complexes, on the contrary, are poorly soluble in water, but highly soluble in chloroform. The stearate counterion is thought to contribute to modified solubility properties. Counterion options can be considered to tailor solubility.

Products having a 1:1 molar ratio of tetracycline compound to magnesium stearate crystallize upon drying, while products having a 1:2 molar ratio solidify to a glassy solid (Figure 1). Samples were taken from these solids and examined under a microscope. Solids having a 1:1 molar ratio exhibited a heterogeneous composition (Figure 2, left), while samples having a 1:2 molar ratio had a uniform appearance (Figure 2, right). Similarly, a solid obtained from doxycycline and magnesium stearate in a 1:1 ratio gave heterogeneous agglomerates (Figure 3, left), while a glassy structure was formed in the case of a 1:2 molar ratio (Figure 3, right), extraction with spatula.

Example 7: Stabilization in aqueous media

Minocycline (10 mg; Figure 4, left side) and Example 1 complex (20 mg; Figure 4, right side) were placed in separate vials to obtain approximately equal amounts of minocycline. Each sample was dispersed in 4 ml of the appropriate buffer (PBS) at pH 7.1 (Fig. 4, top row) or pH 2.3 (Fig. 4, bottom row). Samples with the complex resulted in a suspension due to the lower solubility of the complex. Samples were incubated at 37°C for several days, and spectra were recorded after separation using HPLC with a UV detector at 355 nm. The dominant peak at a retention time of 4.75 min corresponds to minocycline. In the neutral pH region at 37°C, complete degradation was observed after 3 days for pure minocycline (Figure 4, top left). Under acidic conditions, the degradation process is slower, although minocycline does break down to some extent continuously. The complex, in turn, is less susceptible to degradation because it is less soluble due to its lipophilic properties and reacts less vigorously with the solvent (Figure 4, right side). It is believed that during the incubation process, additional fractions of the complex dissolve and only then undergo decomposition. Although decomposition of the active substance is then also observed, it nevertheless occurs much more slowly in the case of a complex, in particular under neutral conditions (Fig. 4, top right). Thus, complexation is believed to stabilize the composition in an aqueous environment.

Example 8: Spectroscopic properties

a) UV/visible absorption spectra

In addition, changes in the absorption spectra were observed (Figure 5). A freshly prepared ethanol solution of minocycline and the complex of Example 1 was prepared at a concentration of 5 mg/ml minocycline. From these stock solutions, 10 μl was taken and diluted with ethanol (3 ml) to give a final minocycline concentration of 16.6 μg/ml. The spectra were measured on a Shimadzu UV-1800 spectrophotometer. Minocycline shows absorption maxima at 255 and 344 nm. Upon complexation, the first maximum shifts hypsochromically to 244 nm, while the other maximum shifts bathochromically to 385 nm. When a small amount of hydrochloric acid is added, both maxima shift to 264 and 355 nm.

b) IR absorption spectra

FTIR-ATR spectra were measured in potassium bromide pellets using a Bruker IFS 28 instrument equipped with a Sensir ATR unit in the region from 4000 to 400 cm ^-1 . IR spectra also indicate complexation (Fig. 6; minocycline: a - ʋ OH; b - ʋ NH; c - ʋ CH; d - ʋ C=O/CONH ₂ ; e - ʋ C=C (aromatic); f ʋ - CN; magnesium stearate: g - ʋ CH; h - ʋ _as COO ^- ; i - δ CH; j - CH ₂ rocking-oscillation). The peaks at 3478 cm ^-1 of pure minocycline can be attributed to the vibration of the hydroxy group forming intramolecular hydrogen bonds. In contrast, the complex shows a broad peak at the same position. Due to the fact that this group is part of the coordination center for the formation of the chelate, during complexation the free vibrations of hydroxyl groups are hindered and, thus, the peak is smoothed. In addition, the C-H fluctuations of minocycline are overshadowed by the intense C-H fluctuations of magnesium stearate. The carboxylate peak at 1570 cm ^-1 remains recognizable. The peak that appeared at 1700 cm ^-1 cannot be directly attributed to the functional group. The characteristic zone area is also modified due to overlapping vibrations.

Formation and properties of pharmaceuticals (extrudates)

Example 9: Extrusion of 1:2 complex of minocycline (free base) and magnesium stearate

a) Obtaining the complex

For extrusion, a larger amount of complexes is usually required (eg, an amount corresponding to 100 mg of minocycline or more). The components were weighed and processed as described above in Example 1. Subsequently, the complex solution was distributed into several Petri dishes coated with Teflon film and covered with perforated aluminum foil. The Petri dishes were dried in a vacuum oven at room temperature for at least 8 hours (or overnight). A yellow powder was obtained, which was then removed from the Teflon film using a card.

b) Preparation of extrusion precursor

Grinding and homogenization were then carried out using a Retsch CryoMill. The complex powder (1.00 g, corresponding to 289 mg of pure minocycline) was weighed into a cryomill vessel along with two 1 cm grinding balls into a cryomill vessel. The vessel was placed in a cryomill and the following parameters were set: after the automatic pre-cooling phase, 6 grinding cycles were carried out at a frequency of 30 Hz for 150 s each; Between individual grinding cycles, intermediate cooling (30 s) was carried out at a frequency of 5 Hz. After thawing to room temperature, the extrusion precursor was obtained.

c) Extrusion

For extrusion, the three heating zones of the twin screw extruder were preheated to H1=49°C, H2=52°C and H3=55°C. The twin screw extruder control unit was set to 800 rpm, which corresponds to an actual screw speed of approximately 150 rpm. The extrusion precursor was carefully added to the extruder by hand in small portions. With sufficient filling and after the required time, the material reached its softening temperature and was able to deform plastically. The complex exited the extruder through a 0.6 mm wide spun plate as a continuous filament and landed on a Teflon film coated surface where it was cooled to room temperature to produce the final extrudate.

Example 10: Extrusion of 1:2 minocycline (free base) and magnesium stearate complex with Resomer 502

a) Obtaining the complex

The components for the formation of the complex were weighed and processed as described above in Example 1. Subsequently, the complex solution was distributed over several Petri dishes coated with Teflon film and covered with perforated aluminum foil. The Petri dishes were dried in a vacuum oven at room temperature for at least 8 hours (or overnight). A yellow powder was obtained, which was then removed from the Teflon film using a card.

b) Preparation of extrusion precursor

Grinding and homogenization were then carried out using a Retsch CryoMill. The complex powder (412.3 mg, corresponding to 115 mg of pure minocycline) was weighed into the cryomill chamber along with 587.7 mg of the desired biodegradable polymer (Resomer 502) and two 1 cm grinding balls in the cryomill vessel. The vessel was placed in a cryomill and the following parameters were set: after the automatic pre-cooling phase, 6 grinding cycles were carried out at a frequency of 30 Hz for 150 s each; Between individual grinding cycles, intermediate cooling (30 s) was carried out at a frequency of 5 Hz. After thawing to room temperature, extrusion precursor (1 g containing 11.5 wt.% pure minocycline) was obtained.

c) Extrusion

For extrusion, the three heating zones of the twin screw extruder were preheated to H1=49°C, H2=49°C and H3=53°C. The twin screw extruder control unit was set to 800 rpm, which corresponds to an actual screw speed of approximately 150 rpm. The extrusion precursor was carefully added to the extruder by hand in small portions. With sufficient filling and after the required time, the material reached the glass transition temperature and was capable of plastic deformation. The mixture exited the extruder through a 0.6 mm wide spinneret plate as a continuous filament and landed on a Teflon film coated surface where it was cooled to room temperature to produce the final extrudate. The resulting extrudate is shown in FIG. 9 (top).

Example 11: Extrusion of 1:2 minocycline (free base) and magnesium stearate complex with Resomer 503

An extrudate containing the 1:2 complex of minocycline (free base) and magnesium stearate from Example 1 along with Resomer 503 was prepared in the same manner as described in Example 10, in which Resomer 503 was used instead of Resomer 502. The resulting extrudate is shown in FIG. 9 (bottom).

Example 12: Extrusion of a 1:2 complex of minocycline (free base) and magnesium stearate along with Resomer 502/503 (1:1) and 10% glycerol monostearate (GMS)

The extrudate was prepared similarly to that described in Example 10, in which glycerol monostearate was additionally used (10 wt.% based on total mixture weight) and a mixture of Resomer 502 and Resomer 503 (1:1 wt./wt.) was used in a total amount with by adding up to 100 wt.% 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

The complex powder prepared as described above in Examples 1 and 9 (412.3 mg, corresponding to 115 mg pure minocycline and 297.3 mg magnesium stearate) was weighed into a Retsch CryoMill chamber along with the desired biodegradable polymer (Expansorb DLG 50-6P , 487.7 mg), plasticizer (PEG 1500, 100 mg) and two grinding balls 1 cm in size. Then grinding and homogenization were carried out. The container was placed in a cryomill and the following parameters were set: after the automatic pre-cooling phase, 5 grinding cycles were carried out at a frequency of 30 Hz for 150 s each; Between individual grinding cycles, intermediate cooling (30 s) was carried out at a frequency of 5 Hz. After thawing to room temperature, extrusion precursor (1 g containing 11.5 wt.% pure minocycline) was obtained.

Extrusion was carried out in the same way as described in Example 10.

Example 14: Extrudate Release Properties

In FIG. 7 clearly shows the contribution of the complexes according to the invention to the delayed release of minocycline. The melt extrudates as described in Examples 9-13 were cut into 4 mm pieces and incubated in 1 ml of buffer at pH 7.0 and 37°C. The extrudates of Examples 11-13 have a minocycline content of 11.5%; The extrudate of the pure complex of Example 9 has a minocycline content of 27.89%. Each time a sample was taken from the incubation solutions, the entire buffer was changed and the minocycline content of the old buffer was determined by HPLC. As can be seen from FIG. 7, the extrudate of the pure complex of Example 9 shows a delayed release of up to almost three weeks. At the end of the experiment, the amount of residual minocycline in the extrudate of Example 9 was analyzed and it was shown that the active substance was no longer present. We can assume that the remaining 40% decomposed over time.

As can be seen from the curves for Examples 10-12, the addition of PLGA polymers results in an additional delay in release. In this case, approximately 10-13% of the remaining minocycline was still present at the end of the experiment. This may be partly due to the acidic microenvironment in PLGA polymers and slightly better stability in acidic environments, as shown in FIG. 4.

Additionally, the addition of an excipient such as glycerol monostearate (Example 12) may speed up release if desired.

Finally, the PEG-PLGA extrudate of Example 13 containing Expansorb DLG50-6P and 10% PEG 1500 as plasticizer also exhibited favorable sustained release properties. In addition, this extrudate has optimized flexibility and mechanical formability.

Example 15: Additional properties of extrudates

a) Mechanical properties

In FIG. Figure 8 shows load-displacement diagrams during deformation of extrudates, adjusted to the geometry of a knife with a sharp blade. For these experiments, extrudates were mounted on a glass slide of a Brookfield CT3 texture analyzer equipped with a sharp bladed knife and the required weight force required for a predetermined penetration depth of 0.5 mm at a speed of 0.02 mm/sec was determined. The top diagram shows the friability properties of extrudates containing Resomer 502, 503 and their blends with and without glycerol monostearate. PEG-PLGA extrudates with different Expansorbs (bottom diagram) show an almost linear relationship between penetration depth and force required. The absence of a force change indicates irreversible deformation, which can be considered plastic. Macroscopically, the extrudates exhibit excellent flexibility. The composition with 10% PEG 1500 (Example 13) does not show any changes in mechanical properties even after long-term storage in the refrigerator. In contrast, the 5% PEG formulation partially solidifies during storage and behaves more like the corresponding extrudate without the addition of PEG.

b) Mucoadhesive properties

The retention capacity of the extrudate preparations was tested in a retention assay experimental setup as shown in FIG. 10A. Porcine intestinal mucosa obtained from Tönnies Lebensmittel GmbH & Co. KG (washed, portioned, packaged and frozen or used immediately) was used as a surface model fixed in the channel shown in FIG. 10V. Using a continuous flow of deionized water at a predetermined rinse rate (preferably 300 μL/min) and for a predetermined time, the proportion of test drug still retained by the test surface (mucosa) was determined similar to the method proposed by Batchelor et al. ( Int. J. Pharm. 2002 , May 15;238(1-2):123-32). It has been found that the preparations in accordance with the present invention exhibit adequate bioadhesion.

Release kinetics and antimicrobial activity

Example 16: Release kinetics and antimicrobial activity (minocycline preparations)

To evaluate the release kinetics and biological activity of active substances, an approach was chosen that takes into account the conditions in the oral cavity, namely the flow rate of the fluid (circulation rate) and the composition of the grooves in the periodontal fluid (as described in Goodson, JM (2003), Periodontol 2000 31:43 -54; Tew, JG et al (1985), Infect Immun 49:487-93).

a) Release kinetics

Specifically, samples of the following substances were tested, each containing an amount of active substance equivalent to 1 mg of minocycline (excluding excipient extrudate):

Minocycline hydrochloride powder (purchased from SIGMA, catalog no. M9511-25MG; in Fig. 11/12: “minocycline”)

minocycline/PLGA microspheres (Arestin® from Orapharma Inc. Europe, PZN 1295115; 1 capsule = 1 mg minocycline; in Fig. 11/12: “Arestin”)

Example 11 (in Fig. 11/12: “extrudate 503”)

Example 10 (in Fig. 11/12: “extrudate 502”)

control consisting of a biodegradable polymer without a tetracycline compound (in Fig. 11/12: “filler extrudate”).

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 specified time intervals, samples were centrifuged, a predetermined amount of buffer was removed and stored (eluate), and then a fresh portion of buffer was added. The eluates were collected over a period of seven weeks as shown in Table 4 and tested for their antimicrobial activity using the methods described below (minimum inhibitory concentration determination and biofilm inhibition).

Table 4. Pipetting scheme for eluate samples collected to evaluate release kinetics: ⃰ means that eluates removed at this time were used to determine the MIC; # indicates that eluates removed at this time were used to inhibit biofilm formation .

Time Removal (µl) Addition (µl) Time Removal (µl) Addition (µl) 0 23.5 14 d⃰ 3'500 3'304 30 min 22 22 17.5 d 3'304 3'304 60 min⃰ 22 44 21 d⃰ 3'304 3'108 2 hours⃰ 44 44 24.5 d 3'108 3'108 3 hours 44 44 28 d⃰ 3'108 2'912 4 hours⃰ 44 88 31.5 d 2'912 2'912 6 hours⃰ 44 792 35 d⃰ 2'912 2'716 24 hours⃰ 792 1'056 38.5 d 2'716 2'716 2 d⃰ 1'056 1'056 42 d⃰ 2'716 2'520 3 d 1'056 1'056 45.5 d 2'520 2'520 4 d⃰ 1'056 1’056×3=3’168 49 d⃰ 2'520 2'324 7 d⃰ 3'168 3'500 52.5 d 2'324 2'324 10.5 d 3'500 3'500 56 d⃰ 2'324

Table 5. Effective concentration of minocycline compared to Streptococcus gordonii ATCC 10558 and Porphyromonas gingivalis ATCC 33277 up to 42 days, calculated based on the MIC of eluates (dilutions)

Minicycline
S. gord. P. ging. Arestin
S. gord. P. ging. Extrudate 502
S. gord. P. ging. Extrudate 503
S. gord. P. ging. 1 hour >16000 >32000 >16000 >32000 1000 4000 4000 >32000 2 hours 8000 2000 8000 4000 2000 1000 4000 2000 4 hours 2000 1000 2000 1000 1000 1000 2000 2000 24 hours 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) Determination of minimum inhibitory concentration (MIC)

The dilutions at which the growth of two indicator strains ( Porphyromonas gingivalis ATCC 33277 , Streptococcus gordonii ATCC 10558) were still inhibited (minimum inhibitory concentration, MIC) were determined. For this purpose, eluate samples were mixed with a nutrient medium and the appropriate bacterial strain and incubated for 18 h at 37°C under aerobic conditions. The approach and media used were in accordance with the recommendations of the European Committee on Antimicrobial Susceptibility Testing - EUCAST. Each determination was performed twice; growth control was performed in one well each time, respectively.

In FIG. 11 shows at what dilution level growth inhibition can still be observed (i.e. minimum inhibitory concentrations of eluates (dilutions) compared to S. gordonii ATCC 10558 and P. gingivalis ATCC 33277 up to 42 days). Activity of pure minocycline and the commercial product (Arestin) was present until 21 days, whereas extrudate formulations 502 and 503 were active until the end of observation (42 days).

In Table 5, the effective concentration of the active substance was calculated based on the MIC for minocycline against two strains ( P. gingivalis ATCC 33277: 0.25 μg/ml, S. gordonii ATCC 10558: 0.5 μg/ml). These data also highlight the activity of eluates over long periods of time.

c) Inhibition of biofilm formation

The surface of a 96-well plate was covered with 10 μl of eluate. When a mixture of 6 species of bacteria associated with periodontitis was added, a biofilm was formed within 6 hours (as described, for example, in 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 Nov;20(8):2165-2173). The number of still viable bacteria in the biofilm (colony forming units=CFU) was then determined.

In FIG. 12 shows the inhibitory activity of eluates obtained at 24 hours, 2 days, 7 days, 14 days and 28 days against biofilm formation. Inhibition of biofilm formation may be associated with antimicrobial activity, on the one hand, and interference with the stages of biofilm formation (attachment of microorganisms to surfaces or to already attached microorganisms (coaggregation), matrix formation), on the other hand. Herein, the eluate from the vehicle also appears to exhibit some biofilm properties. After 24 hours of incubation there were no differences between the studied substances. However, later all the drugs according to the present invention show better activity than pure minocycline. After 28 days, significant inhibition by Extrudate 503 was still present (-1.9 log ₁₀ CFU).

Example 17: Release kinetics and antimicrobial activity (doxycycline preparations)

A similar approach was taken to evaluate the release, kinetics, and biological activity of doxycycline formulations. Specifically, samples of the following substances were tested, each containing an amount of active substance equivalent to 1 mg of doxycycline (excluding excipient extrudate):

doxycycline hyclate (1.15 mg doxycycline hyclate = 1 mg doxycycline)

doxycycline gel (Ligosan®, 1 cartridge=260 mg, 14% doxycycline)

Doxycycline extrudate (corresponding to 11.5% doxycycline, prepared in the same manner as described in Example 13)

filler extrudate.

The studies were carried out in a similar manner as described in Example 16 above. It was found that the release kinetics and antimicrobial activity of the drug in accordance with the present invention, compared with reference samples, are comparable to the results obtained for minocycline preparations.

ADDITIONAL EXAMPLES

The inventors selected magnesium stearate and calcium stearate as potential complexing agents due to their high theoretical potential, their low toxicity, their biodegradability, and their widespread use in drug delivery applications. The formation of the drug complex was followed by UV/visible and IR spectroscopy. In addition, the inventors wanted to study the effect of glycerol monostearate to change the release rate and mechanical properties.

Complex formation may also negatively affect the activity of the drug. Consequently, biological activity measurements were carried out. Finally, in vitro release experiments were performed to evaluate the effect of formulation on release kinetics.

A. Materials and methods

Minocycline and doxycycline were purchased from Ontario Chemicals Inc. (Ontario, Canada). Magnesium stearate and calcium stearate were kindly provided by Magnesia Germany (Mülheim, Germany). PLGA polymers and Tegin® 4100 (glycerol monostearate, abbreviated as GMS) granules were provided by Evonik (Darmstadt, Germany). Nile red was purchased from Sigma Aldrich (Munich, Germany). Staphylococcus aureus ATCC 29213 was used to screen for antimicrobial activity. All solvents used were purchased from Sigma Aldrich and were at least chemically pure.

I. Chelation of tetracycline derivatives

Formation of a lipophilic drug complex has been proposed as a potential way to improve drug stability and increase release time. For this purpose, different molar compositions of antibiotics and magnesium or calcium stearate, respectively, were studied. To form the complex, tetracyclines and stearate salts were weighed in flasks in molar ratios of 1:1, 1:2 and 2:1. For example, 10 mg of minocycline was combined with 25.48 mg of magnesium stearate (1:2 molar ratio). 2 ml of ethanol was 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 to characterize the complex

To prove the existence of the minocycline complex, chelate studies were necessary. Therefore, the absorption spectra of pure tetracycline derivatives and their complex solutions were studied on a Shimadzu UV-1800 spectrophotometer (Shimadzu, Duisburg, Germany). Previously, 10 μl of freshly prepared 5 mg/ml stock solutions were diluted with 3 ml of ethanol, and absorption spectra were recorded in the range from 250 to 450 nm in a quartz cuvette. To record infrared (IR) spectra, homogeneous pressed powders of API and zinc selenide were prepared. These compacted powders were subsequently examined using a Bruker IFS 28 equipped with a Sensir ATR (Bruker, Billerica, USA) operating in the range from 4000 to 680 cm ^-1 . In addition, the solutions were dried on a rotary evaporator and the residues were scraped off the flask. These remains were examined under a microscope (Carl Zeiss Microscopy, Jena, Germany). These residues were also exposed to different solvents to detect changes in solubility.

III. Disc diffusion method

After deciding that minocycline was the preferred active ingredient, an agar disc diffusion method was performed to examine its antimicrobial activity after complexation. The methodology followed the guidelines established by EUCAST. The test substance was a minocycline complex containing 10 mg/ml minocycline, and the controls were minocycline dissolved in ethanol (10 mg/ml) and pure ethanol. The solutions were diluted 3:7 with ethanol and immediately thereafter every 10 μl was pipetted onto unloaded 6 mm antibiotic assay disks (BD, Alschwil, Switzerland), such that each disk (except the ethanol control) contained 30 μg of minocycline. An overnight culture of S. aureus ATCC 29213 was suspended in McFarland 0.5, each 100 μl spread onto a Mueller-Hinton agar plate (Oxoid, Basingstoke, UK). After the solvent evaporated, the disks under study were placed on the dishes. After incubation at 35°C for 18 h, the diameters of the inhibition zones were measured.

IV. Preparation of extrusion precursor using cryominding and hot melt extrusion

Before cryominding, a solution of the ethanol complex of minocycline was applied to Petri dishes covered with Teflon foil with a thin layer of drops. Petri dishes were stored in a vacuum oven for at least 12 hours at 25°C. The dried minocycline complex powder was then collected from the Teflon foil.

Batch volumes of approximately 1 g are required to ensure sufficient filling of the extrusion chamber. Therefore, for cryominding, 412.3 mg of minocycline complex powder (containing 115 mg of minocycline) and 587.7 mg of the desired PLGA polymer were placed in the grinding chamber along with two 10 mm steel spheres . The extrudates containing 10% GMS therefore included only 487.7 mg of PLGA polymer. After appropriate setup in the Retsch CryoMill (Retsch, Hahn, Germany), the mixture was subjected to an automatic pre-cooling phase followed by 5 grinding cycles at 30 Hz for 150 s. After each grinding cycle, a 30-second cooling phase maintained suitable operating temperatures at 5 Hz.

Subsequently, the mixture was ready for extrusion. Therefore, a ZE 5 Eco twin-screw extruder (Three-Tec, Sion, Switzerland) with an integrated frequency converter SK 500 E was used. A die with a diameter of 600 μm and screws with an L/D ratio of 21.25 were used. The extrusion temperature depended on the polymer and the intended diameter. Starting from 49°C in the first heating zone, the temperature gradually increased to 53°C in the third heating zone for Resomer 502, 503 and their mixture. For extrudates containing 10% GMS, 55°C was used in the last heating zone. Minocycline-PLGA powder was slowly and manually loaded into the extruder orifice at 800 rpm according to the frequency converter. The emerging continuous polymer fiber was fixed on a surface covered with Teflon foil. The final product was cut with a scalpel into pieces of the desired length.

V. Determination of drug content

The minocycline content of the extrudates was quantified using the HPLC method after extraction of the drug from the extrudate. For extraction, we used the method proposed by A. Holmkvist et al. 2016 (24). After weighing, the extrudate was transferred to a 15 ml centrifuge tube. To dissolve the polymer, 2 ml of acetonitrile containing 0.1% trifluoroacetic acid (TFA) was added. After 1 minute of shaking using an IKR-VIBRO-FIX shaker, 4 mL of methanol was added to dissolve the minocycline and precipitate the polymer. The tube was then centrifuged at 1000 rpm for 5 minutes and sterilized by filtration through a 0.45 μm Teflon filter. HPLC was carried out on a Waters 600 E system with an XTerra RP18 5 µm 3.9×150 column (Waters). A sample volume of 20 μL was injected at a flow rate of 1.0 mL/min. A gradient program was selected for the mobile phase. Starting with 25 mM KH ₂ PO ₄ +0.06% H ₃ PO ₄ (A), which in 10 minutes turns into 60% acetonitrile (B). After 5 minutes, the mobile phase gradually changed back to A over 1 minute. The retention time was 9.5 minutes and the drug was detected using a UV/Vis detector at 355 nm. Linear calibration curves (r ² ˃0.999) were obtained in the range of 2-50 μg/ml. A recovery rate of 96% was achieved.

For stability studies, an Agilent 1200 Series system with an XTerra RP18 5 µm 3.9×150 column (Waters) was used. A sample volume of 10 μL was injected at a flow rate of 1.0 mL/min. A gradient program was also used for this method. The mobile phase started with water/methanol (95/5+0.1% TFA) (A) and transitioned to pure methanol (0.1% TFA) within 7 minutes (B). After 3 minutes, the mobile phase was returned to solvent A for 1 minute. The retention time was 4.8 minutes. Chem32 software was used to evaluate and extract chromatograms.

VI. Release studies and stability studies

The extrudates were cut into pieces 4 mm +/- 100 µm long. For each polymer type, 5 extrudates (4 mm long) were weighed and exposed to 1 ml phosphate buffered saline (PBS) pH 7.0 in a 2 ml glass vial. The vials were incubated in a water bath protected from light at 37°C. Samples were collected every 2 days for 3 weeks. Starting from day 22, the sampling interval was increased to 4 days. Sampling included complete buffer exchange. Samples were stored in a refrigerator at 2-8°C until their drug content was determined by the HPLC method described above. The release profile was studied over a period of 42 days. After this period of time, the remaining extrudates were also examined by this HPLC method to quantify the remaining minocycline content.

For stability studies, 10 mg samples for minocycline and 20 mg samples for the complex were weighed in 4 ml glass vials. One vial of each species was incubated in 4 ml of PBS pH 7.0 and the other vial was exposed to 4 ml of an acidic solution at pH 2.3, which was adjusted with hydrochloric acid. Until day 9, samples were taken every day and the volume removed was not replaced. From this point on, the intervals were increased to 3-4 days.

B. Results

I. Formation of the complex

Molar ratios

Molar ratios of 2:1, 1:1 and 1:2 (tetracycline derivative: fatty acid salt) were investigated as potential chelate complexes. The combination of minocycline and magnesium stearate gave a yellow solution for all ratios. In contrast, combining minocycline with calcium stearate resulted in a 1:2 suspension. Therefore, this relationship was excluded from further studies. Doxycycline was similar to minocycline with respect to magnesium stearate complexes. Transparent solutions with a less intense yellow color appeared. Combination of doxycycline with calcium stearate resulted in slightly cloudy solutions for 1:1 and 1:2 ratios.

II. Characteristics of the complex

1. UV/visible spectroscopy

A clear sign of successful chelation is changes in the absorption spectra. Minocycline has an absorption maximum at 255 and 344 nm in ethanol (Figure 13). After combination with magnesium stearate, the spectrum changed depending on the concentration of magnesium stearate. The spectra at ratios 1:1 and 1:2 have almost identical shapes and the positions of their absorption maxima are the same. For the first maximum, complexation results in a hypsochromic shift to 244 nm, while the latter exhibits a bathochromic shift to 385 nm. At the 2:1 ratio there are no clear maxima due to the overlapping spectrum of minocycline and the partially developed complex. In conclusion, the spectra indicate successful chelation in a molar ratio of 1:1 to 1:2.

In combination with calcium stearate, the absorption spectrum of minocycline undergoes comparable changes. The 1:1 ratio results in a hypsochromic shift for the short-wavelength maximum and a bathochromic shift for the long-wavelength maximum, whereas the 1:2 ratio has already been eliminated. Likewise, the 2:1 ratio resembles the overlapping spectrum of the pure API and the complex. Both also have in common that after the addition of small amounts of hydrochloric acid (HCl), the absorption maxima of minocycline and complexes shift to 264 nm and approximately 355 nm (Figure 13). This indicates decomposition of the complex, which is suitable for subsequent detection during HPLC analysis.

For doxycycline, the results are, as expected, quite similar to minocycline due to structural similarity. However, it should be noted that both absorption maxima of doxycycline are shifted bathochromically, regardless of the chelating ion (Fig. 13). Magnesium stearate shows slight differences in the position of absorption maxima depending on the amount of fatty acid salt. Interestingly, the spectra in the 2:1 and 1:1 ratios for calcium stearate are almost identical. Therefore, chelation appears to be incomplete until a 1:2 ratio is achieved. The addition of HCl also leads to the decomposition of complexes with spectra and maxima that are close to those of minocycline.

In conclusion, it should be noted that this experiment results in the exclusion of all 2:1 ratios and 1:1 ratios of doxycycline and calcium stearate due to insufficient complex formation.

2. IR spectroscopy

IR spectra also indicate chelation (Figure 14). The peaks at 3478 cm ^-1 (a) in the spectrum of pure minocycline can be attributed to the hydroxide vibration involved in intramolecular H-bonding. Instead, the complex provides a broad peak. This indicates that free vibration of the binding sites is hindered by the presence of magnesium or calcium ions. The CH (c) fluctuations of minocycline suppress the more prominent CH (g) peaks of magnesium and calcium stearate. The carboxylate peak at 1570 cm ^-1 (h) remains clearly visible. At 1700 cm ^-1 a new peak appears, which cannot be clearly assigned to a specific functional group. The signature area has also changed due to the overlapping vibrations, but does not provide any additional evidence.

However, for doxycycline, the IR spectrum exhibits a broader peak at approximately 3300 cm ^-1 (a), which cannot be distinguished between the OH and NH vibrations. However, this peak also broadens and flattens in the presence of divalent cations (Figure 15).

3. Drying of the complex and microscopy

For further processing it was necessary to transfer the complex into powder form. Therefore, solutions of the complex were slowly dried, and their residues were carefully examined. Dried minocycline and doxycycline-magnesium stearate complexes appeared as glassy amorphous structures at a molar ratio of 1:2. In FIG. Figure 2 shows this homogeneous structure, as well as the heterogeneous appearance of the 1:1 ratio in the case of minocycline. Unfortunately, calcium stearate complexes did not produce such uniform results for any of the antibiotics. Even though the ratios of the remaining complexes may be suitable for other forms of processing, the 1:2 molar ratio showed more promising properties for the intended application.

Changes in solubility were also observed. The minocycline complex showed reduced solubility in water compared to the parent drug. In turn, solubility in organic solvents was improved, especially in chloroform. These changes also suggest the formation of a chelate complex.

At this point, minocycline was chosen for additional experiments, although a complex of doxycycline and magnesium stearate seemed quite suitable. Therefore, the reason was a broader spectrum of action and superior tissue penetration (Cunha, BA. et al. Eur J Clin Microbiol Infect Dis. 2018 , 37(1), 15-20). Thus, the following paragraphs are devoted to the complex of minocycline and magnesium stearate in a 1:2 ratio.

III. Disc diffusion method

It is well known that polyvalent cations can have a negative effect on the antibiotic potential of tetracycline antibiotics (Naz, S. et al. Arzneimittelforschung 1996 , 46(7), 701-704). Therefore, the disk diffusion method was carried out to study the antibiotic activity of minocycline after complexation. The diameter of the inhibition zone is critical for antibiotic activity (Figure 16: agar plates incubated with S. aureus ATCC 29213. Left plate: disks loaded with ethanol; middle plate: disks loaded with ethanol solution of minocycline complex; right plate: disks loaded with ethanol solution of minocycline). Control disks loaded with ethanol failed to prevent the growth of the test microorganism. Pure minocycline had a diameter of 26 mm, and the complex reached 24 mm. Although the complex's zone of inhibition is slightly smaller, the complex remains active against microbiological threats.

IV. Hot melt extrusion

Before the extrusion process, the cryomill provides a homogeneous extrusion precursor while maintaining minimal thermal stress.

Resomer 502 RG and 503 RG produced extrudates in the size range from 600 to 800 μm (Figure 9). Notably, Resomer 503 has viscoelastic properties, which results in expansion immediately after extrusion. In contrast, Resomer 502 produces a continuous, reproducible 600 µm extrudate. A larger diameter results in a different amount of drug available if extrudates of the same length are used. In FIG. 9 shows the differences in size. Later, Resomer 502 and 503 compositions were tested. These mixtures did not affect the product diameter. The addition of GMS also did not change the extrudate diameter, but did reduce the brittleness.

However, these extrudates were still within the desired size range. In addition, pure minocycline complex powder was extruded. Using this approach, extrudates could be obtained, but they were brittle and exhibited fragile mechanical stability. Thus, these extrudates served only as a reference for in vitro release experiments.

V. In vitro release

The in vitro release profile (Figure 17) shows for all formulations a higher initial release rate that gradually slows down. Pure minocycline complex extrudates showed the fastest release but also no burst.

The addition of PLGA polymer can change the release rate to the desired level. As expected, Resomer 502 offers faster release compared to Resomer 503 due to its lower molecular weight. This difference is especially noticeable in the first two weeks until their release rates begin to level off. Also notable is the higher standard variation for Resomer 502. For the 1:1 formulation of Resomer 502 and 503, release was expected between the individual polymer curves. These expectations were met until the 7th day, when the polymer mixture was inferior. After 42 days, the polymer mixture almost reaches the release level of Resomer 503. Thus, on the contrary, the effect of GMS is even greater. GMS was initially used as a softener for extrudates, but the effect was relatively small, although overall stability was increased. The more significant benefit is the significantly increased release rate. Within 10 days, double the amount was released compared to the batch without GMS. Starting from the 10th day, their release rates also level out and there is no big difference.

In vitro release experiments demonstrate controlled release over 42 days.

VI. Stability studies

This study was conducted to determine the fate of at least 40% of undetected minocycline. In FIG. 18 shows the ongoing breakdown of minocycline and the complex depending on the pH of the environment. Minocycline exhibits marked degradation at neutral pH. Within 3 days, the minocycline peak at 4.8 minutes disappears, while destruction products become noticeable. Significant degradation is also evident at the macroscopic level due to a color change from yellow to completely black. However, the complex is able to protect minocycline to a certain extent. After 3 weeks, the minocycline peak is still visible, while, of course, the degradation product peaks are gradually increasing. Reduced solubility in water causes a slight tendency to decompose. However, as the incubation period progresses, more parts of the complex dissolve and become detectable. This can also be seen in the chromatogram on day 2, where there is an increase in the minocycline peak. The onset of a chemical equilibrium between the dissolved minocycline, which is undergoing decomposition, and the not yet dissolved minocycline complex is possible.

An acidic environment ensures that the API lasts longer. More moderate degradation was observed for minocycline compared to neutral pH conditions. The same statement is true for the complex, although in this case again an additional solution is noticeable over time. From this point on, minocycline and the complex experience the same degree of destruction. As previously shown, minocycline is released from the complex under acidic conditions. Consequently, similar chromatograms and degradation rates are observed.

Discussion

Tetracyclines have a wide spectrum of antimicrobial activity and have proven themselves as local antimicrobial agents in periodontal therapy. The problem with all tetracyclines is their limited stability in water. Minocycline degrades more rapidly than doxycline and is prone to epimerization in aqueous media and some organic solvents, particularly N-methylpyrrolidone (NMP). These epimers provide only 5% of the initial activity of the antibiotic.

A delay in such processes can be achieved if minocycline has been chelated in advance. Of these complexes, magnesium complexes were found to be more stable than their calcium counterparts, likely due to their higher charge density. This charge density effect was noticeable during the chelation process described herein. Magnesium and calcium are both divalent cations, but due to the smaller atomic diameter (Mg: 145 pm, Ca: 194 pm) thus magnesium with its higher charge density can interact more easily with the chelation site. This explains the formation of cloudy solutions in the case of doxycycline and a 1:2 suspension ratio for minocycline. In addition, by selecting magnesium and calcium fatty acid salts as the second compound, water ingress and alteration of the physical properties of the API can be avoided.

UV/Vis and IR spectroscopy studies confirmed complex formation and suitability for further processing. Shifts in absorption maxima as a function of cation concentration and flattening and broadening of OH vibration peaks demonstrate interactions during chelation. There, minocycline and doxycycline showed equal compatibility for the complexation process. In addition, both antibiotics have anti-inflammatory properties, which are advantageous in the treatment of inflammatory disease such as periodontitis. But, as mentioned earlier, minocycline was chosen for further development due to its more lipophilic characteristics and superior spectrum of action.

The duration of this effect was subject to clarification due to possible interference with the interaction between the API and the polyvalent cation. Fortunately, the disc diffusion method showed high activity. A possible explanation for the slightly reduced diameter could be reduced diffusion of minocycline molecules through the agar matrix due to interaction with magnesium stearate molecules, creating larger and more hydrophobic molecules. However, these results contributed to the advancement of the next stage of development - extrusion.

The production process itself is relatively quick and simple. Preparation of the precursor requires little effort, and the absence of toxic solvents reduces risks during manufacture, creates a compatible product, and ensures API stability. A limitation of laboratory scale production is manual filling of the extruder, which results in intermittent precursor flow.

The final preformed insert product exhibits several advantageous properties with respect to the intended application. There is also no need for special equipment for application - simple tweezers will be sufficient. The diameter of the extrudates is not fixed, but can be set. 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) appears to be very promising.

The achieved drug load of 11.5% is close to a reasonable limit due to the complex composition with magnesium stearate. The total proportion of complex in the extrudate is 41.2%, leaving 58.8% for the release-controlling polymer. Significant amounts of magnesium stearate contribute to the minor flexibility properties of the extrudates. Various dosing options are possible due to the simultaneous placement of several extrudates in one groove. In addition, the release rate is controlled by the choice of polymer and additional excipient.

The in vitro release period of 42 days is still exceptional compared to commercially available systems. As expected, Resomer 502 with its lower molecular weight showed a higher release rate compared to Resomer 503. Notable is the higher standard variant for Resomer 502, which may also be due to the lower molecular weight and therefore faster autocatalysis and destruction (Fredenberg, S. et al. Int J Pharm 2011 , 415, 34-52). However, the mixture of both polymers performed surprisingly unexpectedly. The sudden drop in its release rate is difficult to put into context. Possible explanations could be batch-to-batch variation, analytical difficulties, or polymer-polymer interactions that either create a microenvironment that promotes API degradation or interfere with the release of minocycline.

Across all samples, one observation is striking: a higher percentage of APIs go undetected. Therefore, a stability experiment was planned. The known fragility of minocycline in an aqueous environment leads to the first assumption of degradation during the period of the release experiment. These processes are facilitated by an elevated temperature of 37°C. For better understanding, minocycline and the complex were tested at pH 7.0 and pH 2.3. Neutral pH resembles physiological pH, while acidic pH simulates conditions that may occur within degradable PLGA polymers. Stability studies revealed the protective function of the complex at neutral pH values. However, some of the API disintegrates. Residue analysis performed at the end of the release experiment indicated that 10 to 15% of the API remained within the extrudates. Taken together, these results provide significant insight into the purpose of the missing API.

Conclusion and conclusion

The purpose of this study was to develop an alternative controlled-release device for topical antimicrobial treatment of periodontitis. As an inflammatory and bacterial disease, periodontitis can be treated with a topical antibiotic or antiseptic formulation. One class of possible antibiotics in such formulations is the tetracycline group. The inventors' goal was to provide a preformed insert that provides increased 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 combined with magnesium and calcium stearate in different molar ratios. These chelate complexes stabilize the active pharmaceutical ingredient and allow incorporation into a PLGA polymer matrix via hot melt extrusion. The chelate complexes were characterized by UV/Vis and IR spectroscopy, leading to a preference for minocycline-magnesium complexes. To re-evaluate the antibiotic activity of the complex, the disk diffusion method was performed. Before extrusion, the complex was mixed with various PLGA polymers and cryo-milled. Hot melt extrusion provided homogeneous extrudates with diameters ranging from 600 to 900 μm. They contain 11.5% minocycline, are adjustable in length and are easy to use. In vitro release studies revealed controlled drug release over 42 days.

The inventors' approach to the new form of administration for the periodontitis patient offers several advantages. The stability of minocycline is enhanced by the proposed chelation before and after use. The manufacturing process is versatile, contains no toxic solvents, is scalable and provides a solid preformed insert. This preformed insert features ease of use without any special equipment, adjustability, and a controlled extended release of 42 days, which has not yet been achieved in other formulations. Thus, extrudates have promising potential to be promoted as valuable therapeutic agents, particularly in the field of periodontal therapy.

COMPARATIVE EXAMPLE

Comparative Example 18: Na Treatment ₂ HPO ₄

Published international application WO 2012/128417 A1 describes in Example 1 the formation of an “ionic complex” by mixing a 5 wt.% aqueous solution of tetracycline hydrochloride with a 5 wt.% aqueous solution of Na ₂ HPO ₄ in a volume ratio of 1:1. Minocycline⋅ion HCl complexes have also been reported in WO 2012/128417 A1. Therefore, a similar reaction was performed to compare minocycline phosphate with the inventors' minocycline chelate complex.

In the first attempt, 375 mg of minocycline⋅HCl and Na ₂ HPO ₄ were weighed into a 15 ml Falcon tube. After this, 7.125 ml of double-distilled water was added to each powder. Minocycline⋅HCl was not soluble at this high concentration, resulting in a yellow suspension. Na ₂ HPO ₄ dissolved easily (Fig. 19). These two solutions were mixed, resulting in an orange solution (Figure 20). Unlike WO 2012/128417 A1, no precipitation was observed; solubility even increased. The solution was centrifuged at 4000 rpm for 4 minutes, but no sediment was observed.

The experiment was repeated with solutions of 1 wt.% minocycline HCl and 1 wt.% Na ₂ HPO ₄ . Therefore, 75 mg of each compound was weighed into 15 ml Falcon tubes and 7.425 ml of double-distilled water was added. At this concentration, minocycline⋅HCl was soluble and gave a yellow solution (Figure 21). Mixing both components also results in a yellow solution (Figure 22). No precipitation was observed, and centrifugation at 4000 rpm for 4 minutes did not change the state of the solutions. The final solution had a pH value of 7.1, indicating the presence of minocycline as a cation. However, to ensure the cationic state of minocyclines before mixing both solutions, the experiment was also repeated by replacing 400 μL of 7.425 mL double-distilled water with 0.1 M HCl. The change in pH also did not lead to precipitation (Fig. 23).

Thus, for minocycline⋅HCl, successful precipitation of the complex of poorly soluble ions with disodium phosphate as an anionic analog was not observed.

The present disclosure further includes the following aspects.

[1] A complex containing a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA ₂ ), in which: the TC:MA ₂ molar ratio is in the range of 1:0.8-3.0 ; The divalent metal cation M is an alkaline earth metal cation and A is a carboxylate anion derived from a C ₈ -C ₂₄ carboxylic acid. [2] The complex according to aspect [1], wherein the tetracycline (TC) compound is selected from minocycline, doxycycline, tetracycline, chlortetracycline, oxytetracycline, rotetracycline, tigecycline, demeclocycline, lymecycline, meclocycline, metacycline, omadacycline, sarecycline, eravacycline, clomocycline, 9 -(N,N-dimethylglycilamido)-6-demethyl-6-deoxytetracycline and 9-(N,N-dimethylglycilamido)minocycline. [3] The complex according to any one of the preceding aspects, wherein: the TC:MA ₂ ratio is 1:2; and/or M is selected from Mg ²⁺ and Ca ²⁺ ; and/or A is a carboxylate anion derived from a carboxylic acid selected from arachidic acid (C ₂₀ ), stearic acid (C ₁₈ ), palmitic acid (C ₁₆ ), myristic acid (C ₁₄ ), lauric acid (C ₁₂ ), capric acid (C ₁₀ ), caprylic acid (C 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 ₂ )], wherein the tetracycline compound (TC) is minocycline or a pharmaceutically acceptable salt, hydrate or solvate thereof. [5] A pharmaceutical preparation comprising a 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 intended for topical administration and/or is selected from the group consisting of extrudate, particle, granule, powder, film, strip, compressed powder, chip, paste, cream, gel, emulsion, suspension, liniment, ointment, balm, lotion, eye drops, spray, topical aerosol, topical solution, topical suspension, skin patch and non-woven material. [7] The pharmaceutical preparation according to aspect [5] or [6], further containing 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); biodegradable polyester blends 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 in which the total content of tetracycline compound (TC) is in the range of 5-20 wt.%. [8] A pharmaceutical preparation according to any one of aspects [5]-[7], which is in the form of a filamentary extrudate, preferably having a substantially circular or substantially elliptical cross-section and/or a maximum cross-sectional diameter in the range of 0.1-10 mm . [9] A method for making a complex according to any one of aspects [1]-[4], 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 ₂ ) in a molar ratio of 1:0.8-3.0 with an organic solvent to obtain a dispersion in which the divalent metal cation M is an alkaline earth metal cation and A is a carboxylate anion derived from a C ₈ -C ₂₄ carboxylic acid; (b) heating said dispersion to form a complex; (c) removing the solvent to obtain a complex. [10] The method of manufacturing a pharmaceutical preparation according to aspect [8], comprising the following steps: (d) grinding 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 a pharmaceutical preparation in the form of a threaded extrudate. [11] A complex according to any one of aspects [1]-[4] or a pharmaceutical preparation according to any one of aspects [5]-[8] for use in a method of treating a human or animal body. [12] A complex according to any one of aspects [1]-[4] or a pharmaceutical preparation according to any one of aspects [5]-[8] for use in a method of therapy and/or prevention of (i) bacterial infection or (ii) 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] A complex according to any one of aspects [1]-[4] or a pharmaceutical preparation according to any one of aspects [5]-[8] for use according to aspect [12], wherein the antibiotic activity is maintained for a period of at least at least 21 days. [14] A complex according to any one of aspects [1]-[4] or a pharmaceutical preparation according to any one of aspects [5]-[8] for use in a method of therapy and/or prevention of 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], in which periodontal disease is selected from gum diseases, caused by plaque, periodontitis, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotic periodontal diseases, periodontal abscesses, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections.

Claims (36)

1. A complex formed from a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA ₂ ) for the treatment and/or prevention of bacterial infection or acute, chronic or recurrent periodontal disease, in which: the molar ratio of TC:MA ₂ is in the range of 1:0.8-3.0; The divalent metal cation M is an alkaline earth metal cation and A is a carboxylate anion derived from a C ₈ -C ₂₄ carboxylic acid, wherein the tetracycline (TC) compound is selected from minocycline, doxycycline, chlortetracycline, rolitetracycline, tigecycline, demeclocycline, lymecycline, meclocycline, metacycline, omadacycline, sarecycline, eravacycline, clomocycline, 9-(N,N-dimethylglycilamido)-6-demethyl-6 -deoxytetracycline and 9-(N,N-dimethylglycylamido)minocycline. 2. The complex according to claim 1, in which: the TC:MA ₂ ratio is 1:2; or M is selected from Mg ²⁺ and Ca ²⁺ ; or A is a carboxylate anion derived from a carboxylic acid selected from arachidic acid (C ₂₀ ), stearic acid (C ₁₈ ), palmitic acid (C ₁₆ ), myristic acid (C ₁₄ ), lauric acid (C ₁₂ ), capric acid (C ₁₀ ), caprylic acid (C ₈ ), hydroxystearic acid, oleic acid, linoleic acid, linolenic acid and ricinoleic acid. 3. Complex according to any one of the previous paragraphs, in which: the TC:MA ₂ ratio is 1:2; And M is selected from Mg ²⁺ and Ca ²⁺ ; And A is a carboxylate anion derived from a carboxylic acid selected from arachidic acid (C ₂₀ ), stearic acid (C ₁₈ ), palmitic acid (C ₁₆ ), myristic acid (C ₁₄ ), lauric acid (C ₁₂ ), capric acid (C ₁₀ ), caprylic acid (C ₈ ), hydroxystearic acid, oleic acid, linoleic acid, linolenic acid and ricinoleic acid. 4. The complex according to any one of the preceding claims, having the formula [(TC)⋅2(MgA ₂ )], in which the tetracycline compound (TC) is minocycline or a pharmaceutically acceptable salt, hydrate or solvate thereof. 5. A pharmaceutical preparation for the treatment and/or prevention of a bacterial infection or acute, chronic or recurrent periodontal disease, containing the complex according to any one of claims 1 to 4 and optionally one or more pharmaceutically acceptable excipient(s). 6. The pharmaceutical preparation according to claim 5, which is intended for local administration and/or selected from the group consisting of extrudate, particle, granule, powder, film, strip, pressed powder, chip, paste, cream, gel, emulsion, suspension, liniment, ointment, balm, lotion, eye drops, spray, topical aerosol, topical solution, topical suspension, skin patch and non-woven material. 7. The pharmaceutical preparation according to claim 5 or 6, further containing 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); biodegradable polyester blends 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 in which the total content of tetracycline compound (TC) is in the range of 5-20 wt.%. 8. The pharmaceutical preparation according to claim 7, in which the biodegradable polymers have a weight average molecular weight range of 4-250 kDa and/or have a glass transition temperature of 42-48°C, have an intrinsic viscosity [dl/g] (0.1% in CHCl ₃ at 25°C) 0.03-1.70. 9. The pharmaceutical preparation according to claim 7 or 8, wherein the biodegradable polymers are PLGA copolymers having a weight average molecular weight range (kDa) of 5-90, having an intrinsic viscosity [dl/g] (0.1% in CHCl ₃ at 25 °C) 0.14-0.82, and/or which end with ester, alkyl ester and/or PEG groups. 10. Pharmaceutical preparation according to any one of claims 7 to 9, wherein the biodegradable polymers are poly(D, L-lactide-co-glycolides) having a weight average molecular weight range of 7000-38000 and/or intrinsic viscosity [dl/g ] (0.1% in CHCl ₃ at 25°C) 0.16-0.44. 11. Pharmaceutical preparation according to any one of claims 5 to 10, which is in the form of a thread-like extrudate. 12. A pharmaceutical preparation according to claim 11, having a substantially circular or substantially elliptical cross-section and/or 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 -0.6mm, 0.3mm, 0.4mm, 0.5mm or 0.6mm. 13. A method for obtaining a complex according to any one of claims 1-4, including the following stages: (a) mixing a tetracycline compound (TC) or a pharmaceutically acceptable salt, hydrate or solvate thereof and a divalent metal carboxylate (MA ₂ ) in a molar ratio of 1:0.8-3.0 with an organic solvent to obtain a dispersion in which the divalent metal cation M is an alkaline earth metal cation and A is a carboxylate anion derived from a C ₈ -C ₂₄ carboxylic acid; (b) heating said dispersion to form a complex; (c) removing the solvent to obtain a complex. 14. A method for producing a pharmaceutical preparation according to claim 11, including the following steps: (d) grinding the complex according to any one of claims 1 to 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 a pharmaceutical preparation in the form of a threaded extrudate. 15. Use of a complex according to any one of claims 1-4 or a pharmaceutical preparation according to any one of claims 5-12 in a method of therapy and/or prevention of (i) bacterial infection; (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 ; and/or (iii) acute, chronic or recurrent periodontal disease. 16. Use according to claim 15, wherein the antibiotic activity is maintained for a period of at least 21 days. 17. Use according to claim 15, in which periodontal disease is selected from gum diseases caused by dental plaque, periodontitis, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotic periodontal diseases, periodontal abscesses, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections. 18. A method of treating a human or animal body, including administering a therapeutically effective amount of the complex according to claim 1 or a pharmaceutical preparation according to claim 5 to an object in need of such treatment, where the method is intended for the treatment and/or prevention of (i) bacterial infection; (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 ; and/or acute, chronic or recurrent periodontal disease. 19. The method of claim 18, wherein the antibiotic activity is maintained for a period of at least 21 days. 20. The method according to claim 18, in which periodontal disease is selected from gum diseases caused by dental plaque, periodontitis, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotic periodontal diseases, periodontal abscesses, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections.