United States Patent [t9J
`Golub et al.
`
`(ttl Patent Number:
`(45] Date of Patent:
`
`4,666,897
`May 19, 1987
`
`[54]
`
`[75]
`
`INHIBITION OF MAMMALIAN
`COLLAGENOLYTIC ENZYMES BY
`TETRACYCLINES
`Inventors: Lorne M. Golub, Smithtown; Thomas
`F. McNamara, Port Jefferson; N. S.
`Ramamurthy, Smithtown, all of N.Y.
`(73] Assignee: Research Foundation of State
`University, Albany, N.Y.
`(21] Appl. No.: 566,517
`[22] Filed:
`Dec. 29, 1983
`Int. CJ,4 .............................................. A61K 31/65
`[51]
`[52) u.s. Cl ..................................................... 514/152
`[58] Field of Search ......................... 424/227; 514/152
`[56]
`References Cited
`U.S. PATENT DOCUMENTS
`2,895,880 7/1959 Rosenthal ........................... 424/227
`4,248,892 2/1981 Kanamaru eta!. ................. 424/317
`4,371,465 2/1983 McGregor .......................... 424/177
`4,457, 936 7/1984 Draeger et a!. ..................... 424/270
`OTHER PUBLICATIONS
`Bauer, et al., 1983, J. Invest. Dermatol., vol. 81, pp. 162,
`163 168.
`Harris, eta!., 1984, Collagen Rei. Res., vol. 4, p. 493.
`Berman and Manabe, 1973, Ann. Ophthalmol., Nov., p.
`1193.
`Golub, et a!., 1985, J. Periodontal Res., vol. 20, pp.
`12-23.
`Welgus and Stricklin, 1983, J. Bioi. Chern., vol. 258, p.
`12259.
`Uitto, eta!., 1984, J. Oral. Path., vol. 13, p. 412.
`
`Narayanan and Page., 1983, Collagen Rei. Res., vol. 3,
`p. 54.
`Golub, eta!., 1985, J. Periodontal. Res., Special Issue, p.
`93.
`Vater, et al., 1978, J. Clin. Invest., vol. 62, p. 987.
`Harris, et al., 1984, Collagen Rei. Res., vol. 4, pp.
`504-505.
`Cowen, eta!., 1985, Biochem. International, vol. 11, p.
`273.
`Belaisse, eta!., 1985, Biochem. Biophys. Res. Commun.,
`vol. 133, p. 483.
`Golub, et al., 1983, J. Periodontal Res., vol. 18, pp.
`516-526.
`Chemical Abstracts 100:96203a (1983).
`Chemical Abstracts, 93:142977t (1980).
`Chemical Abstracts, 76:30596k (1969).
`Dreisbach et al., Induction of Collagenase Production
`in Vibrio B-30, J. Bacterial, vol. 135, No.2 (1978) pp.
`521-527.
`Goodman and Gilman, The Pharmacological Basis of
`Therapeutics, 6th ed., pp. 961-962 (1980).
`Primary Examiner-Albert T. Meyers
`Assistant Examiner-John M. Kilcoyne
`Attorney, Agent, or Firm-Omri M. Behr
`[57]
`ABSTRACT
`A method of reducing pathologically excessive levels of
`activity of collagenolytic enzymes in mammals to sub-
`stantially normal levels by administering 10-100% of
`the normal antibiotic therapeutic dose of a tetracycline
`is disclosed.
`
`10 Claims, 8 Drawing Figures
`
`Dr. Reddy's Laboratories, Ltd., et al.
`v.
`Galderma Laboratories, Inc.
`IPR2015-_ _
`Exhibit 1050
`
`Exh. 1050
`
`

`
`U.S. Patent May 19, 1987
`
`Sheet 1 of4
`
`4,666,897
`
`14
`
`12
`
`m INTERPROXIMAL
`D LINGUAL
`
`FIG.Ib
`
`FIG.Ia
`
`Exh. 1050
`
`

`
`U. S. Patent May 19, 1987
`
`Sheet2 of4
`
`4,666,897
`
`II INTERPROXIMAL
`D LINGUAL
`
`10
`
`e 12
`:::;;:: = = .....__
`U? if.i
`U?!:= == _J=
`=<...::> == ~
`
`LJ..J-
`ZLJ..J
`
`U')
`0
`
`6~~~~~~~~~=~
`CONTROL
`DIABETES
`DIABEl +
`MINOCYCL.
`
`FIG.2b
`
`80
`
`40
`
`:;_ ~ 60
`1- =
`~~
`1- <..!)
`<...::> LJ..J
`<C =
`<...::> =
`i=~
`~:5 =_J
`r5 8
`~I
`=1st:,
`8cfi. o~-L--~-L--~~--~
`DIABEl +
`CONTROL
`DIABETES
`MINOCYCL.
`
`20
`
`FIG.2a
`
`Exh. 1050
`
`

`
`U.S. Patent May 19, 1987
`
`Sheet 3 of4
`
`4,666,897
`
`"0>
`_§
`= <C
`
`<..!)
`
`0:: = --LL.J = ---' = 0::
`>->< = 0:: = >-=
`
`0...
`
`1200
`
`1000
`
`800
`
`-.... ' ' ' '
`
`'-- C (CONVENTIONALl
`
`',"
`D(CONVENTIONALl
`·----,(
`'--.,
`
`(GERM-FREE)
`
`O 0
`
`6
`5
`I
`TIME (WEEKS AFTER STREPTOZ.OTOCIN ADMIN.)
`
`FIG.3
`
`---' <C = ::2
`1-= LL.J > = = <...>
`
`Exh. 1050
`
`

`
`V. S. Patent May 19, 1987
`
`Sheet4 of4
`
`4,666,897
`
`__:
`<:.9
`
`3
`
`2
`
`0
`
`PEN.
`+
`
`MIN.
`
`•
`
`120
`
`w... 80
`<...>
`(.!)
`
`PD
`
`KD
`
`40
`
`0
`
`PEN.
`+
`
`f+
`PD
`
`MIN.
`
`•
`
`K.~
`
`6
`
`~ 4
`E
`E
`
`0
`0....
`
`2
`
`0
`
`PEN.
`+
`
`MIN.
`~
`
`PD
`
`KD
`
`FIG.4
`
`MIN.
`J +
`
`PEN .
`
`•
`
`FIG.4b
`MIN.
`
`PD
`
`· KD ·
`
`ACTIVE ENZYME
`
`D TRYPSIN-ACTIVED ENZYME
`
`/TRYPSIN CONTROL
`
`~~~~~==~~~~===-
`
`PO
`
`KD
`
`FIG.4a
`
`Exh. 1050
`
`

`
`4,666,897
`
`1
`INHIBITION OF MAMMALIAN
`COLLAGENOLYTIC ENZYMES BY
`TETRACYCLINES
`
`BACKGROUND OF THE INVENTION
`Collagenase is an enzyme normally present in mam-
`mals. The enzyme, which is produced in a number of
`body tissues and cells, degrades collagen, (the major
`structural protein of connective tissues, such as those in 10
`bone, skin, tendon and gingiva) normally during con-
`nective tissue remodeling. However, when collagenase,
`is produced in excessive amounts, then the pathologic
`destruction of these tissues is the result. This excessive
`collagenase production has been observed to occur in a 15
`number of disease states such as hyperparathyroidism,
`diabetes, periodontal disease and rheumatoid arthritis.
`The results of excess collagenolysis are serious and
`debilitating, such as excessive resorption of bone associ-
`ated with hyperparathyroidism, the ulceration of the 20
`cornea, the destruction of joint tissue associated with
`rheumatoid arthritis, and the breakdown of the gingival
`collagen fibers and the bony socket associated with
`periodontal disease.
`In recent years, tetracycline has been advocated as an 25
`adjunct in the treatment ofperiodontal diseases, includ-
`ing chronic periodontitis in the adult (Fasciano and
`Fazio, Quintessence
`International, October, #10,
`1081-1088 (I 981)) and more often, for rapidly progres-
`singjuvenile periodontitis (Slots, et al., J. Peridontol, 50, 30
`405-509 (1979)); Genco, et al., J. Dent. Res. 60, Special
`lssue A, Abstract 872 (1981). lts therapeutic efficacy
`has been attributed solely to the drug's antimicrobial
`activity particularly against specific Gram-negative
`organisms believed to be the cause of these diseases 35
`(Genco, J. Periodontol, 52, 545-558 (1981)). Recently,
`Williams, et al., J. Peridontal Res. , 16, 666-674 (1981)
`described a significant improvement in periodontal dis-
`ease in dogs on Iang-term tetracycline therapy, an effect
`that did not appear to correlate with expected shifts in 40
`the crevicular microflora. Williams, et. al. concluded
`that the beneficial effect of tetracycline must have re-
`sulted from the Suppression of a strain of bacteria that
`was not measured in their study. Similar clinical
`changes were observed in a limited number of human 45
`subjects. Tetracycline therapy in periodontal disease
`has also been evaluated by Ciancio, J. Periodontol., 43,
`155-159 (1976) and Ciancio, et al., J. Periodontol, 51,
`531-534 (1980). Kornman & Kar!, (1982, J. Periodontol.,
`53, 604-610) reported that the Iang-term use of tetracy- 50
`cline was clinically beneficial in patients who did not
`respond to routine periodontal therapy (debridement by
`instrumentation). In all of these studies, the only per-
`ceived value of tetracyclines is an antibacterial drug.
`Diabetes in rats and humans has been found to in- 55
`crease tissue collagenase activity. Evidence for this
`effect was seen in extracts of gingiva and skin (Rama-
`murthy and Golub, J. Periodontal Res., 18., 31-39
`(1983)) andin cultures of gingival tissue (Ramamurthy,
`et al., J. Periodontal Res., 9, 199-206 (1974); Golub, et 60
`al., J. Dent. Res., 57, 520-525, 1978; Kaplan, et al., J.
`Dent. Res., 61, Special Issue, 275 (1982)). Unusually
`severe periodontal disease which occurs during diabetes
`in man (Finestone and Boorujy, Diabetes, 16, 336-340,
`1967; Cianciola, et al., J.A.D.A., 104, 653-660 (I 982)) 65
`and experimental animals (Bissada, et al., Periodontics.,
`4, 223 (1966)), reflects accelerated collagen breakdown
`which could be mediated by the excessive collagenase
`
`2
`generated during this systemic disease. lt has been sug-
`gested that the overgrowth of Gram-negative organ-
`isms in the gingival crevice of the diabetic rat could, by
`generating excessive endotoxin in the area, be the cause
`5 of the abnormally high collagenase Ievels in the gingiva
`(McNamara et al., Archs. Oral Bio/., 27, 217-223 (1982)).
`Additionally, it has been reported that tetracycline is
`effective in
`the treatment of rheumatoid arthritis
`(Brown, et al., Comp. Pathol. ZooAnimals, (eds. Montali
`& Migaki), Smithsonian Institution Press, 259-266
`(1980)). Brown's solerationale for the use of these anti-
`biotics is to eliminate infection of the joint tissues with
`the "mycoplasma group of microorganisms which he
`believes is the cause of arthritis. However, there has
`been heretofore no recognition of the ability of tetracy-
`cline to reduce pathologically excessive Ievels of collag-
`enolytic enzyme(s) (such as collagenase) activity to
`substantially normal Ievels.
`SUMMARY OF THE INVENTION
`The present invention relates to a method of reducing
`pathologically excessive amounts of collagenolytic en-
`zyme(s) activity in a mammalian system. More particu-
`larly, this invention relates to a method of reducing
`such excess collagenase or other collagenolytic en-
`zymes such as elastase or gelatinase to substantially
`normal Ievels which comprises administering to mam-
`mals in need of anti-collagenase therapy an anti-collage-
`nase effective amount of a tetracycline.
`For convenience sake, the presently marketed dosage
`forms of the various tetracyclines may be utilized in the
`method of the present invention without substantial
`change. This is especially facilitative to the practice of
`the invention. Tetracyclines and their various dosage
`forms have been utilized for many years and the side
`effects from such dosage forms are weil studied, weil
`recognized, and minimized by the said dosage forms.
`The method ofthe present invention involving reduc-
`ing excessive collagenolytic enzyme activity Ievels to
`normal Ievels can be used to treat conditions in a num-
`ber of diseases, including rheumatoid arthritis, peri-
`odontal disease and ulcerated corneae. These disease
`states (and others) all exhibit the pathological effects of
`excessive collagenase Ievels which include excessive
`resorption of bone, destruction of joint tissue, break-
`down of the gingival collagen fibers and the bony sock-
`ets, and ulceration of the cornea, as weil as other de-
`struction of collagen containing tissue. While other
`manifestations of these disease states may be treated
`with supplementary drugs, i.e., inflammation in rheuma-
`toid arthritis, or infection in periodontal disease, the use
`of the tetracycline will serve to reduce the excessive
`collagenase Ievels and collagenolytic activity to a nor-
`mal Ievel. This anti-collagenase thereapy is valuable
`supplementary aid for use by a medical practitioner in
`the treatment and control of such diseases as described
`above.
`BRIEF DESCRIPTION OF THE DRA WINGS
`FIG. I (a & b ) are block graphs of a) Collagenolytic
`activity, and b) bone lass in conventional diabetic rats.
`FIG. II (a & b) are similar to FIG. I, a & b except that
`the rats arealso germ-free.
`FIG. 111 shows the effect of minocycline on the skin
`collagen Ievels of streptozotocin diabetic rats.
`
`Exh. 1050
`
`

`
`4,666,897
`
`3
`FIG. IV shows the effects of penicillin and minocy-
`cline on gingival disease and on GCF collagenolytic
`activity in twin young diabetic males.
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`The tetracycline utilized in the present invention may
`be any of the readily available, pharmaceutically ac-
`ceptable tetracycline known in the medical art. In-
`cluded in this group of tetracyclines are those such as 10
`chlortetracycline, which is marketed under the trade-
`name Acronize, Aureocina, Aureomycin, Biomitsin,
`Biomycin and Chrysomykine; Demeclyeycline mar-
`keted as Ledermycin, Detravis, Meciclin, and Mexo-
`cine; Doxycycline marketed as Vibramycin, Vibra- 15
`mycin Hyclace, Liomycin Vibradox, Panamycin, Titra-
`dox, Hydramycin, Tecacin; Lymecycline which is mar-
`keted as Armyl, Mucomycin, Tetramyl, Tetralysal;
`Methacycline which is marketed as Adriamicina, Cy-
`clobiotic, Germicilclin, Globociclina, Megamycine, 20
`Pindex, Londomycin, Optimycin, Rondomycin; Mino-
`cycline which is marketed as Minocin, Klinomycin,
`Vectrin; Oxytetracycline which is marketed Biostat,
`Oxacycline, Oxatets, Oxydon, Oxymycin, Oxytan, Ox-
`ytetracid, Ryomycin, Stezazin, Tetraject, Terramycin, 25
`Tetramel, Tetran, Dendarcin, Dendrcin; Rolitetracy-
`cline marketed as Bristacin, Reverin, Superciclin, Syn-
`tetrex, Syntetrin, Synotodecin, Tetraverin, Transcy-
`cline, Velacicline, Velacycline; and Tetracycline mar-
`keted as Achromycin, Ambramycin, Cyclomycin, Pol- 30
`ycycline, Tetrabon, and Tetracyn.
`The active salts, which are formed through proton-
`ation of the dimethylamino group on carbon atom 4,
`exist as crystalline compounds and are very viable in
`water. However, these amphoteric antibiotics will crys- 35
`tallize out of aqueous solutions of their salts unless stabi-
`lized by an excess of acid. The hydrochloride salts are
`used most commonly for oral administration and are
`usually encapsulated because of their bitter taste. Water
`soluble salts may be obtained also from bases such as 40
`sodium or potassium hydroxides but are not stable in
`aqueous solution, they are also formed with divalent
`and polyvalent metals, these forms, though operative
`are not preferred.
`The tetracyclines used in the method of the present 45
`invention are preferably administered at a dosage Ievel
`of from 10 to 100%, suitably 20-80%, of the normal
`antibiotic therapeutic dose of the particular tetracycline
`compound being employed. By normal antibiotic thera-
`peutic dose is meant the dosage of the particular tetra- 50
`cycline compound which is commonly used and recom-
`mended for the treatment of bacterial infection. More
`than 100% of the normal antibiotic therapeutic dose
`may be utilized in the method of the present invention
`but there is no particular advantage in doing so and such 55
`over-dosage may in fact Iead to complications.
`The present invention is intended for use as long term
`(as weil as short-term, or even episodic) therapy. The
`long term administration of tetracyclines at antibioti-
`cally effective dose Ievels may have a negative effect on 60
`healthy flora such as intestinal flora and may also Iead
`to the production of resistant organisms. These disad-
`vantages are substantially reduced by the use of sub-
`antibiotic dosages.
`The normal antibiotic therapeutic dose of the tetracy- 65
`cline is, for the most part, weil studied and weil docu-
`mented so that few, if any, side effects other than those
`mentioned above can be anticipated in using this safe
`
`4
`dosage Ievel for the reduction of excess collagenase to a
`normal collagenase Ievel. For instance, the normal and
`usual dose of tetracycline is, orally, the equiva1ent of
`250 mg. of tetracycline hydrochloride 4 times daily;
`5 intramuscular, the equivalent of 100 mg. of tetracycline
`hydrochloride 2 or 3 times daily; intravenous infusion,
`the equivalent of 250-500 mg. of tetracycline hydro-
`chloride over a period of! to 1 hour, twice daily. And
`the usual dosage range, orally, is the equivalent of 1-4 g.
`of tetracycline hydrochloride daily; intramuscularly,
`the equivalent of 200-500 mg. of tetracycline hydro-
`chloride daily; and intravenously, the equivalent of 500
`mg. to 2 g. of tetracycline hydrochloride daily.
`For rolitetracycline, the usual dose range is intramus-
`cularly, 150-350 mg. every 12 hours; and intravenously,
`350-700 mg. every 12 hours. For chlortetracycline the
`usual daily dose range is 250-500 mg.
`When oxytetracycline is utilized the usual dose is,
`orally, 250 mg. 4 times daily; intra-muscularly, 100 mg.
`2 or 3 times daily; intravenously, 250-500 mg. over a
`period of! to 1 hour twice daily. The usual dose range
`is orally, 1 to 4 g. daily; intramuscularly, 200-500 mg.
`daily; intravenously 500 mg. to 2 g. daily.
`For methacycline the usual dose is 600 mg. of the
`hydrochloride salt or 560 mg. of methacycline base,
`daily in divided doses. When demeclocycline is utilized
`the usual dose is 600 mg. daily in 4 divided doses of 150
`mg. or 2 divided doses of 300 mg. each, and the usual
`dose range is from 150-900 mg. per day. Doxycycline is
`typically utilized in a dosage of 100 mg. every 12 hours
`during the first day of treatment followed by a mainte-
`nance dose of 100 mg. daily.
`The method of the present invention utilizes pharma-
`ceutical compositions which incorporate the particular
`tetracycline being utilized. For oral administration the
`tetracyclines utilized in this invention may be formu-
`lated in the form of tablets, capsules, elixirs or the like.
`For parenteral administration they may be formulated
`into solutions or suspensions or intramuscular injec-
`tions, or additionally, the tetracyclines of the present
`invention may be reasonably incorporated into a poly-
`mer carrier delivery system for use topically or locally,
`specifically in cases of periodontal diseases or to be
`delivered topically directly into the periodantat packet
`by some other technique (e.g. by the patient using the
`Water-Pik harne care device or by the dentist using an
`uhrasonie (cavitron) system. The dosage of tetracy-
`clines administered in the present invention is also addi-
`tionally dependent upon the age and weight of the ani-
`mal species being treated, the mode of administration,
`and the type and severity of the excess collagenase
`induced disease being treated.
`The following discussion describes in detail composi-
`tions and methods illustrative of the present invention.
`It will be apparent to those skilled in the art that many
`modifications, both of materials and methods, may be
`practiced without departing from the purpose and in-
`tent of this disclosure.
`EXPERIMENT AL
`EXAMPLE I a)
`The reduction of collagenolytic enzyme(s) Activity in
`Test Animals
`(a) Conventional Rats
`Five month old (mean weight, 445g) male Sprague
`Dawley rats were made diabetic by I.V. injection of
`streptozotocin (70 mg/kg body weight), after a 12 hour
`
`Exh. 1050
`
`

`
`5
`fasting period, (as described by Golub, et al., Biochim.
`Biophys. Acta., 534, 73-81 (1978) and Golub, et al., In-
`feet. Immun., 37, 1013-1020 (1982). Age matched unin-
`jected rats served as controls. Some of the diabetic rats
`received the tetracycline, minocycline (20 mg. Mino-
`cin ® per day; Lederle Laboratories, Pearl River,
`N.Y.), by gavage on a daily basis beginning 3 days after
`diabetes is induced. After a 25 day experimental period,
`blood samples were taken for glucose analysis, and
`subgingival plaque samples were taken for assessment of 10
`the microflora in the region (McNamara, et. al., Archs.
`Oral. Bio/., 27, 217-223 (1982)). The animals were then
`sacrificed, gingival samples were taken for tissue cul-
`ture and the jaw bones dissected. The results are set
`forth below and illustrated in FIG. I.
`To test the effect ofphysiologic Ievels ofminocycline
`(the concentration in human serum, during a therapeu-
`tic regimen, is approximately 2 ug/ml; that in the gingi-
`val crevicular fluid is 4-5 times greater; Ciancio, et. al.,
`1980, J. Periodontol., 51, 531-534) on mammalian collag- 20
`enolytic activity in vitro, peritonealexudate polymor-
`phomuclear leucocytes (PMNLs) were collected from
`15 mole (non-diabetic) rats 4 hours after I.P. injection of
`0.15% glycogen. An extract of PMNLs was prepared,
`250 fLI aliquots of this extract were incubated with 14C- 25
`glycine labeled collagen fibrils for 14 hours at 35" C. (in
`the presence or absence of minocycline, or EDT A, or
`additional CaCh), and collagenolytic activity was mea-
`sured as described by Nicoll, et al., (Experientia., 37,
`315-317).
`(b) Germ-free rats
`In a second separate procedure, germfree rats (pur-
`chased from Taconic Farms, Germantown, N.Y.), were
`maintained in inflatable vinyl isolators (Standard Safety
`Equipment Co., Palatine, Ill ), until a weight of 360 35
`g± 15 was reached. At this time (6 weeks after pur-
`chase), diabetes was induced in some of the animals, as
`described above in a), and the control and diabetic ani-
`mals maintained under germfree conditions for an addi-
`tional 6 weeks prior to sacrifice. Half of the diabetic 40
`animals
`received minocycline
`(Minocin @)
`(20
`mg/day) by gavage for the last 4 weeks of the protocol.
`Blood and tissues were then obtained to assess gingival
`collagenolytic enzyme activity and the Ioss of skin col-
`lagen and bone. The results are set forth below and 45
`illustrated in FIG. II.
`
`4,666,897
`6
`treated with minocycline was measured. The tech-
`niques used were those described by Schneir and
`Golub, (Streptozotocin: Fundamentals and Therapy (Ed.
`M. K. Agarwal, Elsevier/North-Holland Biomed Press,
`Chapter 12, pp 161-182, (1981)).
`The techniques used to measure the collagenolytic
`enzyme activity of gingival tissue in culture, and in
`polymorphonuclear leucocytes (PMNLs), are those
`described by Golub, et. al., (J. Dent. Res., 57, 520-525
`(197)8) and Nicoll, et. al., (Experimentia, 37, 315-317,
`(1981)) with minor modifications.
`The conventional and germfree control rats were
`found to exhibit relatively low blood glucose concen-
`trations ranging from 85 to 186 mg% (mean=l41 mg
`15 %±14). As expected, the diabetic conventional and
`germfree rats, with or without minocycline therapy,
`exhibited severe hyperglycemia having blood glucose
`vales ranging from 310 to 832 mg%, with a mean of 503
`mg %±42. The germfree rats and isolators showed
`negative results for aerobic and anaerobic cultures
`throughout the experimental period, and treatment of
`the conventional diabetic rats with the tetracycline
`minocycline eliminated detectable signs of a Gram-neg-
`ative microflora in the gingival crevices.
`The gingiva from the conventional and germfree
`control rats produced minimal collagenolytic activity in
`tissue culture; less than 10% of the 14C-collagen fibrils
`were found to be degraded after 3 days in culture (see
`FIGS. Ia and Ila). In contrast, collagenolytic activity
`30 was markedly increased in gingiva from the diabetic
`rats, under both conventional (see FIG. Ia) and germ-
`free (see FIG. Ila) conditions. Administering the tetra-
`cycline minocycline orally on a daily basis reduced the
`gingival collagenolytic activity in both diabetic conven-
`tional and germfree rats by 62% and 70% respectively
`(see FIG. Ia and Ila).
`Diabetes in the conventional rats produced a slight,
`but significant, increase (p>0.05) in bone loss in the
`interproximal region, compared to controls; this effect
`was completely reversed by the tetracycline administra-
`tion (see FIG. Ib). The patterns of change on the lingual
`surfaces of the mandibles were similar; however, these
`effects arenot found tobe statistically significant. In the
`germfree rats, no differences in bone Ievels could be
`detected between the three groups (see FIG. Ilb), how-
`ever significant changes are seen in skin (see FIG. 111).
`Diabetes in the germ-free rats (6 weeks after inducing
`(c) Results
`diabetes with streptozotocin) resulted in a 48% reduc-
`tion inskincollagen content compared to the germ-free
`Alveolar bone Ioss was assessed in the right and left
`mandibles of both the conventional and germfree rats 50
`control rats (p > 0.01 ), and minocycline treatment of the
`germ-free diabetics increased the collagen Ievels in this
`(control and diabetic) using a modification of a previ-
`tissue by 45% (p>0.05). Similar patterns of change in
`ously described technique (Stralfors, et.al" Archs. Oral.
`skin collagen content were seen in conventional con-
`Bio/., 12, 1213-1216 (1967); Heijl, et. al., J. Periodonta/
`trol, conventional diabetic, and conventional minocy-
`Res., 15, 405-419 (1980)). The mandibles were defleshed
`(autoclaving followed by soaking in 2N NaOH for 2-3 55 cline-treated diabetic rats (see modified FIG. 111).
`The collagenolytic activity in the leucocyte (or
`h), dried, and the distance (expressed as units; I
`unit=0.05 mm) between the cementoenaniel junction
`PMNL) extract was completely inihibited by EDT A
`(CEJ) and the crest of the alveolar bone is measured in
`(mammalian collagenase is a calcium dependent neutral
`the long axis of the root surface at 10 specified sites on
`protease; (see Seilers and Murphy, Int. Rev. Conn. Tiss.
`lingual surfaces of the 3 molar teeth (see Crawford,
`60 Res., 9, 151-189 (1981)), and the collagensubstratewas
`et.al., J. Periodontal Res., 13, 316-325 (1978)) using an
`not susceptible to non-specific proteolysis by trypsin
`eye-piece micrometer in a dissecting microscope (20X
`(see Table 1). Minocycline, in concentrations approxi-
`magnification ).
`mately those found in human serum and gingival crevic-
`ular fluid (Ciancio, et. al., J. Periodonta/, 51, 531-534
`Because uncontrolled diabetes in the rat results in the
`massive resorption of skin collagen associated with
`65 (I 980)), produced almost complete inhibition of PMNL
`increased collagenase activity in this tissue, the collagen
`collagenase activity, an effect which was reversed by
`content of entire skins from the germfree and from the
`increasing concentrations of calcium (See Table 1).
`conventional control and diabetic rats, including those
`Two non-tetracycline antibiotics (penicillin - strepto-
`
`Exh. 1050
`
`

`
`6
`
`8.3
`
`38.5
`82.1
`90.3
`
`Conclusion
`Diabetes increased the collagenolytic enzyme activ-
`25 ity within the rat gingival tissue. Administering minocy-
`cline to the diabetic rats reduced the abnorrnally high
`Ievel of collagenolytic enzyme activity within the gingi-
`val tissue by 42.5, 72.7 and 77.2%, for rats that were
`diabetic and administered the drug for 15, 28 and 56
`30 days, respectively. (Again note that the drug did not
`TABLE 2
`reduce collagenase activity below normal Ievels). Also
`The Effect of Four Different Types of Tetracyclines
`note that the Ionger the duration of minocycline ther-
`on Rat PMNL Collagenolytic Activity in vitro
`apy, the greater the therapeutic effect in reducing the
`Collagenolytic Activity:
`Incubation
`collagenolytic activity.
`% 14c-collagen gel lyzed
`Mixture
`----------------------------~~~~---35
`EXAMPLE III
`PMNL extract•
`39.0
`PMNL extract + minocycline••
`4.0
`Human Gingival Crevicular Study
`PMNL extract + Achromycin..
`2.0
`PMNL extract + Terramycin••
`4.4
`(a) 19 year old males
`PMNL extract + Vibramycin••
`4.5
`40
`PMNL extract + ampicillin••
`35.7
`PMNL extract + Cefazolin••
`50.0
`_____ .,;_.;...._.....,._____________
`*Extract obtained from 30 X 10° PMNLs per ml.
`**All antibiotics were added to the reaction mixturein a final concentration or zo
`ug/ml; this Ievel or antibiotic approximates the concentration oF tetracycline Fa und
`in the periodantat pockets or humans treated with these drugs.
`
`4,666,897
`
`8
`diabetic (control) rats, and the diabetic rats, was incu-
`bated at 37" C. in vitro with radiolabeled collagen fibrils
`to assess collagenolytic ennzyme activity.
`
`Gingival
`Collagenolytic
`Activity
`(% ct 4-Collagen
`Degraded)
`9.1
`50.4
`
`% Inhibition of
`Collagenolytic
`Activity by
`Minocycline
`
`63.0
`
`47.8
`
`29.0
`
`17.2
`
`10.9
`
`42.5
`
`72.7
`
`77.2
`
`3
`4
`
`2.6
`88.7
`
`12.4
`
`7
`mycin) similarly tested were found to have no detect-
`able effect on collagenase (Table I).
`In a similar but separate experiment, the effect of four
`(4) different types oftetracyclines on PMNL collageno-
`lytic activity was tested (Table 2). All of the tetracy-
`clines inhibited this mammalian collagenolytic activity,
`whereas two additional non-tetracycline antibiotics
`(ampicillin and cefazolin) were again found to be inef-
`Experimental
`fective against this enzyrne(s).
`Group
`10 Control
`TABLE 1
`Diabetes-
`15 day duration
`The Effect of Minocycline on rat PMNL
`Diabetes _
`Collagenolytic Activity in vitro
`28 day duration
`Collagenolytic Activity:*
`Incubation
`Diabetes -
`% 14c-collagen gel lysed
`Mixture
`------------------...;;;.......;;;.......;;..._ 15 56 day duration
`PMNL extract ( control)**
`85.5
`I
`Diabetes+
`2
`PMNL extract +
`3.1
`Minocycline, 15 days
`EDTA (54 mM)
`Diabetes+
`Trypsin (I JJ.g)
`Minocycline, 28 days
`Bacterial
`Diabetes+
`collagenase (250 JJ.g)
`20 Minocycline, 56 days
`PMNL extract + Minocin
`(5 JJ.g/ml)
`PMNL extract + Minocin
`(20 JJ.g/ml)
`Above, (6.) + Caclz (10 mM)
`Above, (6.) + CaCiz (50 mM)
`Above, (!.) + Penicillin -
`Streptomycin (20 JJ.g/ml)
`*Each value is the mean or duplicate analysis arter background activity (3.5%) was
`subtracted.
`••Extract obtained from 38 X 106 cells/ml.
`
`Conclusion
`These results (FIGS. I-III and Tables 1-2) demon-
`strate that: (1) tetracyclines inhibit rnarnmalian collage-
`nolytic enzyme(s) activity and, as a result, inhibit the
`pathologic loss of collagen (e.g. skin & bone), and (2)
`tetracyclines have this therapeutic capability through a
`mechanism unrelated to the drug's antibacterial effi-
`cacy. Note that non-tetracycline antibiotics did not
`have this anti-collagenolytic capability.
`EXAMPLE II
`Effect of Minocycline Therapy on the Abnormally
`High Collagenolytic Enzyme Activity in Extracts of
`Gingiva from Diabetic Rats
`Rats were made diabetic chemically with the agent
`streptozotocin. Some of the diabetic rats (half were
`administered minocycline orally on a daily basis) were
`killed 15 days, or 28 days, or 56 days after inducing
`diabetes and the gingiva were dissected, the tissues were
`extracted, and the extracts partially purified by ammo-
`nium sulfate precipitation. The partially purified en-
`zyme preparation from the gingiva of a group of non-
`
`1. analysis
`h
`In a separate study involving umans, gingival cre-
`vicular fluid (GCF) samples were collected on filter
`paper strips inserted into 8 interproximal pockets of
`selected teeth in the maxillary arch of two 19 year-old
`45 twin brothers with juvenile-onset (insulin-dependent)
`diabetes mellitus. Fluid volume was immediately deter-
`mined on a modified Periotron (Model 6000, Harco
`Electronics Ltd., Winnipeg, Canada); then incubated
`with 10 JLI (3H-rnethyl) collagen (20,675 DPM) and 70
`50 JLI buffer (50 mM Tris-HCI, pH 7.8, containing 0.2 M
`sodium chloride and 5 mM calcium chloride). The GCF
`samples (or reagent blanks or trypsin controls) were
`incubated at 27" C. for 18 hours with gentle shaking.
`The reaction was stopped and undigested collagen pre-
`ss cipitated by adding 10 JLI 0.1 M phenanthroline in diox-
`ane/water (1:1, v/v), 10 JLI of nonradioactive methyl-
`ated carrier collagen (2 mg/ml) in 50 mM Tris-HCI
`buffer containing IM NaCI (pH 7.0), and 100 JLI diox-
`ane. After mixing, the radiolabeled collagen degrada-
`60 tion products were collected by filtration and counted
`in a liquid scintillation spectrorneter (Golub, et. al.,
`1976, J. Dent. Res., 55, 1049). Using this technique, the
`reaction blank (no enzyme or GCF added) released
`about 10% of the total substrate counts; 2-20 ng bacte-
`65 rial collagenase produced a linear increase in the release
`of radioactive counts from the 3H-collagen substrate
`(the 20 ng Ievel of enzyme degraded 60% of the colla-
`gen substrate); and 50 ng trypsin released Iess than I%
`
`Exh. 1050
`
`

`
`9
`of the counts above blank values. GCF collagenase
`activity was expressed as units of equivalent activity of
`vertebrate collagenase (obtained from New England
`Nuclear, Cat. No. NEK-016); 1 unit was operationally
`defined as the amount of enzyme that degrades 1 p,g of
`collagen per hour at 27' C.
`Four additional GCF samples were collected from
`other maxillary pockets of the same diabetic twin broth-
`ers on filter paper strips, their volume determined (see
`above), and collagenolytic activity assessed using the 10
`same procedure described above but with a different
`substrate (10 p,l of (3H-propionate) collagen, NET-660,
`New England Nuclear Corp., Boston, Mass., 302,000
`DPM).
`Half of the 12 GCF samples per subject were acti- 15
`vated by pretreatment with 1.04 uM trypsin followed
`by the addition of a 5-fold molar excess of soybean
`trypsin inhibitor. Sampies were selected to be treated or
`not treated with trypsin by matehing them for Gingival
`Index (Loe and Silness, Acta. Odont. Scand., 21, 533-551 20
`(1967)), GCF flow, and for packet depth; the clincal
`parameters (GI, PD) were measured immediately after
`GCF collection. All measurements were carried out
`immediately before antibiotic coverage and after 7 days
`of treatment with either pencillin G (lg. per day; patient 25
`P. D.) or the tetracycline minocycline (200 mg/day;
`patient K. D.).
`
`a ii) Treatment
`In the human study, the adolescent diabetic twin (K. 30
`D.) was treated with the tetracycline minocycline (See
`FIG. IV). Prior to treatment, he demonstrated a greater
`severity of gingival inflammation (a significantly higher
`G. I. and a tendency towards increased GCF flow) but
`similar packet depth compared to the other diabetic 35
`twin (P. D.) who in this study was treated with penicil-
`lin. Prior to antibiotic the