WO2007075269A2

WO2007075269A2 - Method for inhibiting tooth displacement with non-antibacterial tetracyline formulations

Info

Publication number: WO2007075269A2
Application number: PCT/US2006/046335
Authority: WO
Inventors: Johannes Wilhelmus Von Den Hoff; Miriam Margot Bildt
Original assignee: Collagenex Pharmaceuticals, Inc.
Priority date: 2005-12-27
Filing date: 2006-12-04
Publication date: 2007-07-05
Also published as: WO2007075269A3

Abstract

The invention relates to a method for inhibiting tooth displacement in a mammal in need thereof comprising administering to the mammal an effective amount of a non-antibacterial tetracycline formulation. The invention also relates to a method for reducing osteoclasts present on alveolar bone surfaces in a mammal in need thereof comprising administering to the mammal an effective amount of a non- antibacterial tetracycline formulation.

Description

METHOD FOR INHIBITING TOOTH DISPLACEMENT WITH NON- ANTIBACTERIAL TETRACYCLINE FORMULATIONS

BACKGROUND OF THE INVENTION

Orthodontic treatment involves the use of corrective appliances, e.g. braces. Corrective appliances can be used to straighten teeth, correct bite irregularities, close gaps and bring the teeth and lips into proper alignment. In young children, orthodontic treatment may also be used to guide proper jaw growth and permanent tooth eruption.

Most orthodontic treatments occur in two phases: an active phase and a retention phase. The active phase involves the use of braces or other appliances to move teeth into proper alignment and coordination.

Maintaining a good result following the active phase is one of the most difficult aspects of the orthodontic treatment process. Normal maturational changes, together with post-treatment tooth alterations, conspire against long term stability. Post-treatment retention is necessary.

The retention phase involves the use of a retainer to hold the teeth in their new position for the long term. There are two types of retainers that are used to prevent displacement of post-treatment tooth position, fixed and removable. Fixed (or permanent) retainers use bonding to fix a wire to the lingual surface of the teeth. Removable retainers are used for four hours per day plus overnight, and include elastomeric or rubber positioners, acrylic retainers, and Essix thermoplastic copolyester retainers.

Some disadvantages of fixed retainers are possible hygiene difficulties and localized relapse where there is a partial debond of the retainer. Some disadvantages of removable retainers include cost and delay in fabrication of the appliance, speech difficulties, poor esthetics, and a lack of patient compliance. Therefore, the prior art treatments for preventing changes in post-treatment tooth position are limited and not without disadvantages. There is a need for novel, alternate, and superior treatments for preventing changes in the post-active phase of the orthodontic treatment process.

The compound tetracycline is a member of a class of antibiotic compounds that is referred to as the tetracyclines, tetracycline compounds, tetracycline derivatives and the like. The compound tetracycline exhibits the following general structure:

Structure A

The numbering system of the tetracycline ring nucleus is as follows:

Structure B

Tetracycline, as well as the terramycin and aureomycin derivatives, exist in nature, and are well known antibiotics. Natural tetracyclines may be modified without losing their antibiotic properties, although certain elements must be retained. The modifications that may and may not be made to the basic tetracycline structure have been reviewed by Mitscher in The Chemistry of Tetracyclines, Chapter 6, Marcel Dekker, Publishers, New York (1978). According to Mitscher, the substituents at positions 5-9 of the tetracycline ring system may be modified without the complete loss of antibiotic properties.

Changes to the basic ring system or replacement of the substituents at positions 4 and 10-12, however, generally lead to synthetic tetracyclines with substantially less or effectively no antimicrobial activity. Some examples of chemically modified non-antibacterial tetracyclines (hereinafter COLs) are 4- dedimethylaminotetracyline, 4-dedimethylaminosancycline (6-demethyl-6-deoxy-4- dedimethylaminotetracycline), 4-dedimethylaminominocycline (7-dimethylamino-6- demethyl-6-deoxy-4-dedimethylaminotetracycline), and 4- dedimethylaminodoxycycline (5-hydroxy-6-deoxy-4-dedimethyaminotetracycline).

In addition to their antimicrobial properties, tetracyclines have been described as having a number of other uses. For example, tetracyclines are also known to inhibit the activity of collagen destructive enzymes produced by mammalian (including human) cells and tissues by non-antibiotic mechanisms. Such enzymes include the matrix metalloproteinases (MMPs), including collagenases (MMP-I, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9), and others (e.g. MMP-12, MMP-14). See Golub et al., J. Periodont. Res. 20:12-23 (1985); Golub et al. Crit. Revs. Oral Biol. Med. 2:297-322 (1991); U.S. Patent Nos. 4,666,897; 4,704,383; 4,935,411; 4,935,412. Also, tetracyclines have been known to inhibit wasting and protein degradation in mammalian skeletal muscle, U.S. Pat. No. 5,045,538, to inhibit inducible NO synthase, U.S. Patent Nos. 6,043,231 and 5,523,297, and phospholipase A₂, U.S. Patent Nos. 5,789,395 and 5,919,775, and to enhance IL-10 production in mammalian cells. These properties cause the tetracyclines to be useful in treating a number of diseases.

The object of this invention is to provide a method for inhibiting tooth displacement in a mammal in need thereof.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a method for inhibiting tooth displacement in a mammal in need thereof. The method comprises administering to the mammal an effective amount of a non-antibacterial tetracycline formulation.

In another embodiment, the invention relates to a method for reducing osteoclasts present on alveolar bone surfaces in a mammal in need thereof. The method comprises administering to the mammal an effective amount of a non- antibacterial tetracycline formulation. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. The effect of COL-3 on tooth movement.

Orthodontic tooth movement was inhibited in a dose-dependent manner. The teeth of rats that received 30 mg/kg of COL-3 moved significantly slower than those of the 0 or 6 mg/kg group.

Figure 2. Histological sections of rat molars stained with haematoxylin-eosin and ED l.

A. Overview of an experimental molar. Dentin (D), pulp (P)₅ alveolar bone (B) and the periodontal ligament (L) are indicated. ED-I positive cells are stained brown. The frame indicates a field of interest, viewed in more detail in figure 2B.

B. Experimental and control roots of rats that received 0 or 30 mg/kg COL-3. The small arrows indicate osteoclasts, the large arrows indicate the direction of force in the experimentally moved teeth.

C. Detailed picture of an ED-I stained osteoclast.

Figure 3. Osteoclast numbers in the experimental and control roots.

Osteoclasts were counted at the mesial and distal sides of the roots.

A. At the distal sides of the control teeth, far more osteoclasts were present than at the mesial sides.

B. The number of osteoclasts was significantly lower at the mesial side of the experimental teeth in the 30 mg/kg group, than in the 0 and 6 mg/kg group.

In all three, groups the number of osteoclasts at the distal sides was significantly lower than at the mesial sides.

DETAILED DESCRIPTION

The invention relates to a method for inhibiting tooth displacement by administering a non-antibacterial tetracycline formulation. Tooth displacement is the shifting in position of one or more teeth. Tooth displacement can occur for many different reasons. For example, orthodontic treatment, as discussed above, shifts the position of teeth to correct irregularities, etc. Post-active phase orthodontic treatment tooth displacement is the unwanted shifting of teeth back to the position they were in prior to orthodontic treatment.

Tooth displacement can also occur for reasons other than orthodontics. For example, crowding of lower incisors is often considered a normal part of the aging process. It has been suggested that without orthodontic treatment, approximately two thirds of adolescents with good teeth alignment and normal occlusion will develop incisor irregularity by early adulthood.

As will be discussed below, the inventors have discovered that non- antibacterial tetracycline formulations effectively inhibit tooth displacement. Post- active phase orthodontic treatment tooth displacement involves the same remodeling processes as in orthodontic tooth movement. Therefore, since non-antibacterial tetracyclines formulations are able to inhibit the rate of orthodontic tooth movement (Fig. 1), they are also able to inhibit post-active phase movement.

Orthodontic tooth movement requires extensive remodeling of the periodontal ligament and the alveolar bone. Osteoclasts resorb bone, allowing teeth to migrate in the direction of applied force, e.g. braces.

It has also been discovered that non-antibacterial tetracycline formulations effectively reduce osteoclasts present on alveolar bone surfaces (Fig 3). In another embodiment, therefore, the invention relates to a method for reducing osteoclasts present on alveolar bone surfaces in a mammal in need thereof by administering a non-antibacterial tetracycline formulation.

Non-antibacterial Tetracycline Formulation

In this specification, a non-antibacterial tetracycline formulation comprises a sub-antibacterial dose of an antibacterial tetracycline compound, a non-antibacterial tetracycline compound, or a pharmaceutically acceptable salt thereof. Any antibacterial tetracycline compound may be used in the method of the present invention. Some examples of antibacterial tetracycline compounds include doxycycline, minocycline, tetracycline, oxytetracyclϊne, chlortetracycline, demeclocycline, lymecycline. Doxycycline is preferably administered as its hyclate salt or as a hydrate, preferably monohydrate.

Non-antibacterial tetracycline compounds are structurally related to the antibacterial tetracyclines, but have had their antibacterial activity substantially or completely eliminated by chemical modification. For example, non-antibacterial tetracycline compounds have at least about two times, preferably at least about ten times, even more preferably at least about twenty five times, less antibacterial activity than that of doxycycline. In other words, non-antibacterial tetracycline compounds are incapable of achieving significant antibacterial activity comparable to that of doxycyline at comparable concentrations.

Any non-antibacterial tetracycline compound may be used in the method of the present invention. Some specific examples of non-antibacterial tetracycline compounds (COLs) include 4-de(dimethylamino)tetracycline (COL-I), tetracyclinonitrile (COL-2), 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (COL-3), 7-chloro-4-de(dimethylamino)-tetracycline (COL-4), tetracycline pyrazole (COL-5), 4-hydroxy-4-de(dimethylamino)-tetracycline (COL-6), 4- de(dimethylamino-12α-deoxytetracycline (COL-7), 6-deoxy-5α-hydroxy-4- de(dimethylamino)tetracycline (COL-8), 4-de(dimethylamino)-12α- deoxyanhydrotetracycline (COL-9), and 4-de(dimethylamino)minocycline (COL-IO).

Some additional examples of tetracycline compounds include those compounds disclosed in U.S. Patent No. 6,638,922 issued on October 28, 2003, and assigned to CollaGenex Pharmaceuticals, Inc. The specific and generic tetracycline compounds disclosed in U.S.' Patent No. 6,638,922 are herein incorporated by reference.

Preferably, the tetracycline compounds have low phototoxicity, or are administered in an amount that results in a plasma level at which the phototoxicity is acceptable. The preferred amount of the tetracycline compound produces no more phototoxicity than is produced by the administration of a 40 mg total daily dose of doxycycline.

Examples of tetracycline compounds with low phototoxicity include, but are not limited to, tetracycline compounds having general formulae:

Structure K

wherein: R7, R8, and R.9 taken together in each case, have the following meanings:

R7 R8 R9 hydrogen hydrogen amino (COL-

308) hydrogen hydrogen palmitamide (COL-

311) hydrogen hydrogen dimethylamino (COL-

306) and

Structure L

Structure M Structure N

Structure O wherein: R7, R8, and R9 taken together in each case, have the following meanings:

R7 R8 R9 hydrogen hydrogen acetamido (COL-

801) hydrogen hydrogen dimethylaminoacetamido (COL-

802) hydrogen hydrogen palmitamide (COL-

803) hydrogen hydrogen nitro (COL-

804) hydrogen hydrogen amino (COL-

805) and

Structure P wherein: R8, and R9 taken together are, respectively, hydrogen and nitro (COL- 1002).

The term "pharmaceutically acceptable salt" refers to a nontoxic, well- tolerated salt prepared from a tetracycline compound, such as those described above, and an acid or base. The acids and bases may be inorganic or organic.

Some examples of inorganic acids include hydrochloric, hydrobromic, nitric, hydroiodic, sulfuric, and phosphoric acids. Some examples of organic acids include carboxylic and sulfonic acids. The radical of the organic acids maybe aliphatic or aromatic. Some examples of organic acids include formic, acetic, phenylacetic, propionic, succinic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, panthenoic, benzenesulfonic, stearic, sulfanilic, alginic, tartaric, citric, gluconic, gulonic, arylsulfonic, and galacturonic acids. Appropriate bases may be selected, for example, from sodium carbonate, sodium acetate, potassium citrate, N₅N- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethyl enediamine, meglumine (N-methylglucamine), and procaine.

Throughout this specification, parameters are defined by maximum and minimum amounts. Each minimum amount can be combined with each maximum amount to define a range.

Dose

In one embodiment, a non-antibacterial tetracycline formulation comprising an antibacterial tetracycline compound is administered in a sub-antibacterial amount. A sub-antibacterial amount of an antibacterial tetracycline compound is any amount that results in a tetracycline plasma concentration: (i) which is effective for inhibiting post-treatment tooth displacement, but (ii) which has no, or substantially no, antibacterial activity.

A concentration of an antibacterial tetracycline compound having substantially no antibacterial activity is any concentration that does not significantly prevent the growth of bacteria. That is, a microbiologist would not consider the growth of bacteria to be inhibited from a clinical point of view. One way in which to quantify the antibacterial activities of tetracycline compounds is by a measure called minimum inhibitory concentration (MIC), as is known by a skilled artisan.

An MIC is the minimum tetracycline concentration that inhibits the growth of a particular strain of bacteria in vitro. MIC values are determined using standard procedures. Standard procedures are, for example, based on a dilution method (broth or agar), or an equivalent, using standard concentrations of inoculum and tetracycline powder. See, for example, National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing — Eleventh Informational Supplement. NCCLS Document MlOO-Sl 1, Vol. 21, No. 1, NCCLS, Wayne, PA, January, 2001.

In order to inhibit the growth of a strain of bacteria in vivo, a tetracycline compound achieves a plasma concentration in excess of the MIC for the strain. Plasma concentration refers to the concentration of a tetracycline compound measured in an individual's blood sample taken at steady state. Steady state is generally achieved after dosing for five to seven terminal half lives. The half lives of different tetracycline compounds vary from hours to days.

In one embodiment of the present invention, an antibacterial tetracycline compound is administered in an amount that is effective, as described above, and that results in a plasma concentration which is significantly below the MIC for commonly- occurring bacteria. Such amounts are considered to have no, or substantially no, antibacterial activity. Examples of commonly-occurring bacteria that are susceptible to tetracyclines are Escherichia coli (e.g., ATCC 25922 and 25922); Neisseria gonorrhoeae {e.g., ATCC 49226); Staphylococcus aureus (e.g., ATCC 29213 and 25213); and Streptococcus pneumoniae (e.g., ATCC 49619).

In one embodiment, an antibacterial tetracycline compound is administered in an amount that results in a plasma concentration which is less than approximately 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,

10%, 5%, 1% or 0.5% of the MIC for the commonly-occurring bacteria mentioned above. A skilled artisan can readily determine the amount of a particular antibacterial tetracycline compound to administer to achieve such concentrations.

For example, doxycycline is administered in an amount that results in a minimum steady state plasma concentration of about 0.1 μg/ml, 0.2 μg/ml, or 0.3 μg/ml, and a maximum steady state plasma concentration of about 0.7 μg/ml, 0.8 μg/ml, or 0.9 μg/ml.

The sub-antibacterial amount of an antibacterial tetracycline compound can also be expressed by daily dose. The daily dose of an antibacterial tetracycline compound is any amount that is sufficient to produce the effective, sub-antibacterial plasma concentrations described above. Such dose can, for example, be expressed as a percentage of a minimum antibacterial daily dose.

A skilled artisan knows, or is able routinely to determine, the minimum antibacterial daily dose for antibacterial tetracycline compounds. Examples of suitable sub-antibacterial doses of antibacterial tetracycline compounds for the methods of the present invention include less than approximately: 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% and 0.5% of a minimum antibacterial daily dose.

Some examples of non-antibacterial daily doses of antibacterial tetracycline compounds include about 20 mg/twice a day of doxycycline; about 38 mg of minocycline one, two, three or four times a day; and about 60 mg of tetracycline one, two, three or four times a day.

There is no necessary minimum effective amount of the antibacterial tetracycline compound, as long as the amount administered is capable of inhibiting tooth displacement. For example, when the amount is expressed as a percentage of the MIC plasma concentration, suitable minimum plasma concentrations include approximately 0.1%, 0.5%, 0.8% and 1% of the MIC plasma concentration. When the amount is expressed as a minimum actual plasma concentration, suitable actual plasma concentrations include approximately 0.01 μg/ml, 0.05 μg/ml, 0.1 μg/ml, 0.15 μg/ml, 0.2 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.55 μg/ml, 0.6 μg/ml, 0.65 μg/ml, 0.7 μg/ml, 0.75 μg/ml, 0.8 μg/ml, 0.85 μg/ml, 0.9 μg/ml, 0.95 μg/ml, and 1.0 μg/ml. When the dose is expressed as a percentage of a minimum antibacterial daily dose, the percentage is approximately 0.1%, 0.2%, 0.5%, 1%, 1.5% and 2% of the minimum antibacterial dose.

In a preferred embodiment, any form of doxycycline (e.g., doxycycline salts, such as doxycycline hyclate; and doxycycline hydrates, such as doxycycline monohydrate) is administered in a daily amount of, or equivalent to, from about 10 to about 60 milligrams of doxycycline, while maintaining a concentration in human plasma below the MIC.

In an especially preferred embodiment, doxycycline, a doxycycline salt, or a doxycycline hydrate is administered at a dose of, or equivalent to, 20 milligram of doxycycline twice daily. Such a formulation is sold for the treatment of periodontal disease by CollaGenex Pharmaceuticals, Mc. of Newtown, Pennsylvania under the trademark Periόstat®.

Non-antibacterial tetracycline compounds have no, or substantially no, antibacterial activity. Therefore, there is reduced indiscriminate inhibition of growth of bacteria, and the resulting threat of developing resistant bacteria. Accordingly, a non-antibacterial tetracycline formulation comprising a non-antibacterial tetracycline compound, such as the COLs discussed above, is administered at any effective dose at which side effects, if any, are acceptable.

For example, suitable maximum plasma concentrations of the COLs mentioned above include up to about 10 μg/ml, about 20 μg/ml, about 30 μg/ml, and even up to about 100 μg/ml, about 200 μg/ml and about 300 μg/ml. Suitable maximum daily doses of COLs include about 18 mg/kg/day, about 40 mg/kg/day, about 60 mg/kg/day and about 80 mg/kg/day.

A preferred COL is 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (COL-3). COL-3 is suitably administered in doses of up to about 200 mg/day; preferably about 150 mg/day, more preferably about 100 mg/day, even more preferably about 10 mg/day, or in amounts that result in plasma concentrations of up to about 50 μg/ml, about 40 μg/ ml, or about 30 μg/ml. For example, a dose of about 10 to about 20 mg/day of COL-3 produces plasma concentrations in humans of about 1.0 μg/ml.

There is no necessary minimum effective dose of COLs. Some typical minimum plasma concentrations of COLs include, for example, about 0.01 μg/ml, 0.1 μg/ml, 0.8 μg/ml, and 1.0 μg/ml. Some typical minimum daily doses of COLs include about 0.05 mg/day, about 0.1 mg/day, about 0.5 mg/day, about 1 mg/day, about 5 mg/day, or about 10 mg/day.

An advantage of the non-antibacterial tetracycline formulation useful in the method of the present invention is that they are administered at a dose which avoids side effects associated with high doses and/or long term administration of antibacterial formulations of tetracyclines. Examples of such side effects include the development of antibiotic resistant bacteria and the overgrowth of fungi and yeast. In order to avoid such side effects, antibiotics are normally administered to humans for a period of about eight to twelve days, and usually not more than about two weeks.

The non-antibacterial tetracycline formulations can more safely be administered for periods longer than antibiotic compounds. For example, the non- antibacterial tetracycline formulations can be administered for at least about three weeks, preferably at least about six weeks, more preferably at least about two months, and most preferably at least about six months. Optimally, the non-antibacterial tetracycline formulations can be administered for at least about one year.

Preparation of Tetracycline Compounds

Tetracycline compounds are either isolated from natural sources, or are prepared by any method known in the art. For example, natural tetracyclines may be modified without losing their antibacterial properties, although certain elements of the structure must be retained. The modifications that may and may not be made to the basic tetracycline structure have been reviewed by Mitscher in The Chemistry of Tetracyclines, Chapter 6, Marcel Dekker, Publishers, New York (1978). According to Mitscher, the substituents at positions 5-9 of the tetracycline ring system may be modified without the complete loss of antibacterial properties. Changes to the basic ring system or replacement of the substituents at positions 1-4 and 10-12, however, generally lead to tetracyclines with substantially less or effectively no antibacterial activity.

Admin istr ation

The non-antibacterial tetracycline formulations useful in the method of the present invention are administered to a mammal in need of inhibiting tooth displacement.

The non-antibacterial tetracycline formulation can be administered to the mammal at any time. If the mammal has undergone orthodontic treatment, administration occurs most preferably after the active phase. For example- administration of the non-antibacterial tetracycline formulation can occur within one month, preferably within two weeks, more preferably within one week, even more preferably within 48 hours, yet even more preferably within 24 after the active phase.

The non-antibacterial tetracycline formulation may be administered by any method known in the art. The actual preferred amounts of a non-antibacterial tetracycline formulation in a specified case will vary according to the particular tetracycline compound used, the mode of application, the particular sites of application, and the subject being treated (e.g. age, gender, size, tolerance to drug, etc.)

The non-antibacterial tetracycline formulation may be administered systemically. For the purposes of this specification, "systemic administration" means administration to a human by a method that causes the compounds to be absorbed into the bloodstream.

Preferably, the non-antibacterial tetracycline formulation is administered orally by any method known in the art. For example, the non-antibacterial tetracycline formulation can be administered in the form of tablets, capsules, pills, troches, elixirs, suspensions, syrups, wafers, chewing gum and the like. Additionally, the non-antibacterial tetracycline formulations can be administered enterally or parenterally, e.g., intravenously; intramuscularly; subcutaneously, as injectable solutions or suspensions; intraperitoneally; or rectally. Administration can also be intranasally, in the form of, for example, an intranasal spray; or transdermally, in the form of, for example, a patch.

For the pharmaceutical purposes described above, the non-antibacterial tetracycline formulations useful in the methods of the invention can be formulated per se in pharmaceutical preparations optionally with a suitable pharmaceutical carrier (vehicle) or excipient as understood by practitioners in the art. These preparations can be made according to conventional chemical methods.

In the case of tablets for oral use, carriers commonly used include lactose and corn starch, and lubricating agents such as magnesium stearate are commonly added. For oral administration in capsule form, useful carriers include lactose and corn starch. Further examples of carriers and excipients include milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, calcium stearate, talc, vegetable fats or oils, gums and glycols.

When aqueous suspensions are used for oral administration, emulsifying and/or suspending agents are commonly added. In addition, sweetening and/or flavoring agents may be added to the oral compositions.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the non-antibacterial tetracycline formulations can be employed, and the pH of the solutions can be suitably adjusted and buffered. For intravenous use, the total concentration of the solute(s) can be controlled in order to render the preparation isotonic.

The non-antibacterial tetracycline formulation can further comprise one or more pharmaceutically acceptable additional ingredient(s) such as alum, stabilizers, buffers, coloring agents, flavoring agents, and the like. The non-antibacterial tetracycline formulation may be administered at intervals. For example, the tetracycline formulation may be administered one to six times a day, preferably one to four times a day, more preferably twice a day, and even more preferably once a day.

In an embodiment, the non-antibacterial tetracycline formulation containing any of the above described doses of any antibacterial tetracycline compounds or non- antibacterial tetracycline compounds, such as those mentioned above, e.g. , doxycycline and COL-3, is administered by controlled release over a particular period of time, such as a 24 hour period. The level of tetracycline compound over a particular period of time is typically measured by plasma concentration, such as discussed above. Suitable controlled release formulations include delayed, sustained, and immediate (i.e., instantaneous) release.

For example, doxycycline is preferably administered in an amount of about 40 milligrams over the 24 hour period. The controlled-release 40 mg doxycycline can, for example, be formulated to contain 30 mg of doxycycline for instantaneous release and 10 mg of doxycycline for delayed release.

Methods for controlled release of drugs are well known in the art, and are described in, for example, international patent application PCT/US02/ 10748, which is assigned to CollaGenex Pharmaceuticals, Inc. of Newtown, Pennsylvania and U.S. Patent Nos. 5,567,439; 6,838,094; 6,863,902; and 6,905,708.

In one embodiment, the non-antibacterial tetracycline formulation is administered to a mammal in need of inhibiting tooth displacement as a pharmaceutical composition comprising an antibacterial tetracycline compound or a non-antibacterial tetracycline compound in an amount that is effective to achieve its purpose but has substantially no antibacterial activity.

A mammal in need of inhibiting tooth displacement is any mammal that has unwanted tooth displacement. For example, a mammal that has previously undergone orthodontic treatment will have post-treatment tooth displacement. A mammal that can benefit from the methods of the present invention may be any mammal. Categories of mammals include, for example, humans, farm animals, domestic animals, laboratory animals, etc. Some examples of farm animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include rats, mice, rabbits, guinea pigs, etc.

Examples

The following exemplary data serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention.

Eighteen young adult male Wistar rats received an orthodontic appliance that delivered a mesially directed constant force of lOcN to the three molars together at the right side of the maxilla, according to the split-mouth rat model described by Ren et al., J. Dent. Res. 82:38-42 (2003).

The rats were divided into 3 groups of 6 rats. The first received a daily dose of ImL of vehicle only for 14 days. The vehicle consisted of 2% carboxymethylcellulose in physiological saline. Groups 2 and 3 were given a daily does of ImL vehicle with 6 and 30 mg of COL-3 per kg of bodyweight, respectively.

Orthodontic tooth displacement was measured with a digital caliper (Mitutoyo Co., Kawasaki, Japan) at days 0, 3, 7, 10 and 14. The distance between the most mesial point of the maxillary molar unit and the enamel-cementum border of the ipsilateral maxillary incisor of the gingival level was measured at both the experimental side and the control side.. The net amount of orthodontic tooth displacement was considered to be the change in these differences.

On day 14, the rats were anaesthetized and perfused. The right and left maxillae were dissected and fixated. Osteoclasts were identified by the ED-I mouse- anti-rat monoclonal antibody (Instruchenie, Delfzijl, The Netherlands).

Three sections with the interval of 20-25 sections were chosen for both the experimental and control teeth of each rat. Sections showing pulp of at least one root were selected for each study side. The counts from the three sections of each side (mesial or distal) were averaged to obtain the mean number of osteoclasts per side for each study root.

Figure 1 shows tooth displacement over time. After 14 days, the molars of the animals in the 0, 6 and 30 mg/kg group had moved over 1.72 ± 0.53 mm, 1.34 ± 0.18 mm and 1.13 ± 0.13 mm, respectively. The slopes of regression lines through the data points represent the rate of orthodontic tooth movement. A regression analysis showed a significant dose-dependent inhibition of tooth movement by COL-3.

Figure 2 A shows an overview of an orthodontically moved second molar. ED-I positive cells are visible as dark spots and are also exclusively located at the mesial bone surfaces and in the marrow spaces. In panel 2B, similar areas as indicated by the square in figure 2 A are represented. Figure 2Bl shows a control root of a rat that received no COL-3. Distally from the root, osteoclasts are present on the alveolar bone. Hardly any positive cells are present at the mesial side. Figure 2B2 shows a control root of a rat from the 30 mg/kg group. The distribution of ED-I positive cells is similar. At the mesial side of a representative experimental root of the 0 mg/kg group, many osteoclasts are lining the alveolar bone, while at the distal side only a few are present (Fig. 2B3). However, at an experimental root of the 30 mg/kg group (Fig. 2B4), less osteoclasts are present at the mesial side compared to the 0 mg/kg group. Figure 2C shows a larger magnification of an osteoclast stained with ED-I- The cell is close to the bone and has multiple nuclei.

Osteoclasts were counted at the mesial and distal sides of the roots (Fig. 3). At the mesial sides of the control teeth, 0.5 ± 0.5, 0.3 ± 0.2, and 0.3 ± 0.3 osteoclasts were found for the 0, 6, and 30 mg/kg COL-3 groups respectively (Fig. 3A). These differences were not significant. At the distal sides of the control teeth 14.3 + 2.8, 13.6 + 5.7 and 14.0 + 3.1 osteoclasts were present, respectively. The number of osteoclasts at the distal sides of the control roots did not differ significantly between the three COL groups. Significantly more osteoclasts were presented at the distal sides than at the mesial sides.

COL-3 significantly reduced the rate of tooth movement in a dose dependent manner. The present study showed that a larger number of osteoclasts were present at the distal than at the medial sides of the control roots. Not being bound by theory, the presence of osteoclasts at the distal sides is probably related to a physiological distal drift.

As expected, the mesially directed orthodontic force induced a strong interest in the number of osteoclasts at the mesial side of the roots and a reduction at the distal side. The reduction at the distal side was independent of COL-3 administration. However, the increase at the mesial side was COL-3 dependent. The increase was significantly less in the 30 mg/kg group, compared to the 0 and 6 mg/kg group.

The movement of teeth back to the original placement that is seen post orthodontic treatment involves the same remodeling processes as in orthodontic tooth movement. Since COLs are able to reduce the rate of orthodontic tooth movement they are also able to inhibit tooth displacement post-treatment.

Claims

We Claim:

1. A method for inhibiting tooth displacement in a mammal in need thereof, comprising administering to the mammal an effective amount of a non-antibacterial tetracycline formulation.

2. The method according to claim 1, wherein the tetracycline is administered after orthodontic treatment.

3. The method according to claim 1 , wherein the tetracycline formulation comprises a non-antibacterial amount of an antibacterial tetracycline.

4. The method according to claim 3, wherein the antibacterial tetracycline is selected from the group consisting of doxycycline, minocycline, tetracycline, oxytetracycline, chlortetracycline, demeclocycline, or lymecycline, or pharmaceutically acceptable salts thereof.

5. The method according to claim 3, wherein the antibacterial tetracycline is doxycycline.

6. The method according to claim 3, wherein the antibacterial tetracycline is minocycline.

7. The method according to claim 2, wherein the tetracycline formulation comprises a non-antibacterial tetracycline.

8. The method according to claim 7, wherein the non-antibacterial tetracycline is selected from the group consisting of COL-I, COL-2, COL-4, COL-6, COL-7 or COL-9, or pharmaceutically acceptable salts thereof.

9. The method according to claim 7, wherein the tetracycline is COL-3, or its analogs, or pharmaceutically acceptable salts thereof.

10. The method according to claim 7, wherein the tetracycline is COL-8, or its analogs, or pharmaceutically acceptable salts thereof.

11. The method according to claim 7, wherein the tetracycline is COL-10, or its analogs, or pharmaceutically acceptable salts thereof.

12. The method according to claim 7, wherein the tetracycline is selected from COL-306, COL-308, COL-311, COL-801, COL-802, COL-803, COL-804, COL-805, or COL-1002.

13. A method for reducing osteoclasts present on alveolar bone surfaces in a mammal in need thereof, comprising administering to the mammal an effective amount of a non-antibacterial tetracycline formulation.

14. The method according to claim 13, wherein the tetracycline is administered after orthodontic treatment.

15. The method according to claim 13, wherein the tetracycline formulation comprises a non-antibacterial amount of an antibacterial tetracycline.

16. The method according to claim 15, wherein the antibacterial tetracycline is selected from the group consisting of doxycycline, minocycline, tetracycline, oxytetracycline, chlortetracycline, demeclocycline, or lymecycline, or pharmaceutically acceptable salts thereof.

17. The method according to claim 15, wherein the antibacterial tetracycline is doxycycline.

18. The method according to claim 15, wherein the antibacterial tetracycline is minocycline.

19. The method according to claim 13, wherein the tetracycline formulation comprises a non-antibacterial tetracycline.

20. The method according to claim 19, wherein the non-antibacterial tetracycline is selected from the group consisting of COL-I, COL-2, COL-4, COL-6, COL-7 or COL-9, or pharmaceutically acceptable salts thereof.

21. The method according to claim 19, wherein the tetracycline is COL-3, or its analogs, or pharmaceutically acceptable salts thereof.

22. The method according to claim 19, wherein the tetracycline is COL-8, or its analogs, or pharmaceutically acceptable salts thereof.

23. The method according to claim 19, wherein the tetracycline is COL-10, or its analogs, or pharmaceutically acceptable salts thereof.

24. The method according to claim 19, wherein the tetracycline is selected from COL-306, COL-308, COL-311, COL-801, COL-802, COL-803, COL-804, COL-805, or COL-1002.