Inhibition of the degradation of connective tissue matrix protein components in mammals

Bis-phosphonates such as clodronate, etidronate, pamidronate and alendronate are found to inhibit the degradation of connective tissue matrix protein components in mammals, including humans, and therefore are useful in the therapeutic and prophylactic treatment of mammals against a variety of physiological and pathological connective tissue disorders or extracellular protein degradation disorders including wounds, burns, fractures, lesions, ulcers, cancer and metastasis progression in connective tissues, rheumatoid arthritis and other arthitides, periodontitis, peri-implantitis, cysts, root canal treatment, AIDS, ulceration of the cornea, gastric ulceration, aftae, trauma, acne, psoriasis, loosening of end-osseal hip-protheses.

FIELD OF INVENTION 
The present invention relates to treatments for reducing degradation of 
connective tissue matrix protein components, and more particularly to the 
use of bis-phosphonates in the treatment or prophylaxis of connective 
tissue degradation disorders or extracellular protein degradation 
disorders in mammals, especially humans. 
BACKGROUND OF THE INVENTION 
Matrix metalloproteinases (MMPs) constitute a family of genetically related 
proteolytic enzymes which are capable of cleaving almost all protein 
constituents of the extracellular matrix. MMPs take part in the 
extracellular matrix destruction and remodeling in both physiological and 
pathological conditions, e.g. in wound healing, cancer and metastasis 
progression, rheumatoid arthritis, periodontitis, etc. (Krane, S. M., Ann. 
N.Y. Acad. Sci. 732:1-10, 1994, Woessner, J. F. Jr., Ann. N.Y. Acad. Sci. 
732:11-21, 1994). 
Nine MMPs have been disclosed so far in the literature: fibroblast-type 
collagenase (MMP-1), 72K gelatinase (MMP-2), stromelysin-1 (MMP-3), 
putative metalloproteinase-1 (MMP-7), PMN-type collagenase (MMP-8), 92K 
gelatinase (MMP-9), stromelysin-2 (MMP-10), stromelysin-3 (MMP-11) and 
macrophage metallo-elastase. Each of the MMP enzymes contains a putative 
tridentate Zn.sup.2+ -binding site which is believed to constitute the 
active site in the enzyme (Birkedal-Hansen, H., J. Periodontol. 
64:474-484, 1993). 
A comprehensive review of the MMPs, their known modes of action, their 
inhibition by various compounds as well as details of the involvement of 
MMPs in various pathological conditions and diseases is given in the above 
mentioned Ann. N.Y. Acad. Sci. 732 and also by Birkedal-Hansen et al. in 
Crit. Rev. Oral Biol. Med. 4:197-250, 1993, the relevant disclosures of 
which are incorporated herein by reference. 
MMPs and/or their endogenous inhibitors are frequently found in cells, 
tissue and interstitial fluids, and it is believed that they play an 
important role in the remodeling of the extracellular matrix. They are 
associated with rapid cell movements and in the reshaping of the 
extracellular matrix during growth. Increased amounts of MMPs are 
expressed also during the invasive growth of primary tumors and 
metastases, and they seem also to induce expression of MMPs in adjacent 
stromal cells (DeClerck, Y. A. et al., Ann. N.Y. Acad. Sci. 732:222-231). 
A link is believed to exist between destruction of joints in rheumatoid 
arthritis and the expression and action of MMPs and proteolytic cascades 
related to them (Sorsa, T. et al., Semin. Arth. Rheum. 22:44-53, 1992). 
Also loosening of hip-prostheses has been shown to involve the expression 
and action of MMPs, especially MMP-1 (Santavirta et al., Clin. Orthoped. 
Res. 290:206-215, 1993). MMP-8 seems to be predominant in gingiva, 
gingival crevicular fluid and saliva in periodontal diseases (Sorsa et 
al., Ann. N.Y. Acad. Sci. 732:112-131, 1994) and peri-implant sulcular 
fluid (Ingman et al., J. Clin. Periodontol. 21:301-307, 1994). 
There is a variety of other disorders in which extracellular protein 
degradation plays a prominent role. Examples of such diseases include 
arthritides, acquired immune deficiency syndrome (AIDS), burns, wounds 
such as bed sores and varicose ulcers, fractures, trauma, gastric 
ulceration, skin diseases such as acne and psoriasis, lichenoid lesions, 
epidermolysis bollosa, aftae (reactive oral ulcer), dental diseases such 
as periodontal diseases, peri-implantitis, cysts and root canal treatment 
or endodontic treatment related diseases, etc. 
Although MMPs are believed to play a major role in the degradation of 
interconnective tissues and their components, so far there is little 
evidence of the specific role and mechanism of the separate enzymes in the 
biological environment. Since the actual mechanism of the MMPs is not 
known, finding inhibitors for the various MMPs is based more on 
experimental testing than on theoretical considerations. 
A number of synthetic and natural inhibitors for MMPs have been found and 
tested for a possible usefulness as described by Vincenti et al. in Arth. 
Rheum. 37:1115-1126, 1994. Thus, chelating agents and moieties including 
EDTA, hydroxamate, thiol, phosphonamidate, phosphinate and phosphoramidate 
groups have been tested (Birkedal-Hansen et al. Crit. Rev. Oral Biol. Med. 
4:197-250, 1993). Various sulfur-based inhibitors have also been tested 
but no very good results have been obtained (Vincenti et al. Arth. Rheum. 
37:1115-1126, 1994). Tetracyclines have been found to inhibit MMPs, 
especially MMP-8 although the inhibition mechanism is not known (Golub et 
al., Curr. Op. Dent. 2:80-87, 1992; Suomalainen et al., Antimicrobial, 
Agents & Chemother. 36:227-229, 1992; Sorsa et al., Ann. N.Y. Acad. Sci. 
732:112-131, 1994; Lauhio et al., Clin. Exp. Immunol. 98:21-28, 1994) 
As disclosed for instance in the above mentioned Ann. N.Y. Acad. Sci. 732, 
it is envisaged that specific inhibitors could be produced by polyclonal 
and monoclonal antibodies to various MMP-enzymes. .alpha.-Macroglobulins 
are also known to inactivate susceptible proteinases and may function as 
MMP inhibitors. Tissue inhibitors of metalloproteinases (TIMPs) are known 
to inhibit the activity of the active forms of MMPs. 
Although the above discussion shows that inhibitors for MMPs do exist and 
have been investigated, the tests are still mostly at the experimentation 
stage and no clinically acceptable inhibitor for MMPs exists as a 
therapeutic or prophylactic drug for any of the pathological states and 
diseases potentially connected with MMPs. Adverse side effects, which have 
been detected in the above described MMP inhibitors include, for instance, 
toxicities (synthetic peptides), antimicrobial activities (tetracyclines), 
etc. 
While the MMP-dependent reactions provide the mechanism for at least one of 
the key pathways by which the structural macromolecules of interstitial 
connective tissues are degraded, it is to be observed that mineralized 
matrices are believed to be degraded by a totally different reaction 
mechanism. This is the osteoclastic pathway which is based, at least in 
part, on a release of acidic thiol proteinases to a specific area on the 
mineralized matrix surface. 
Bisphosphonates constitute a group of compounds known to have an effect on 
the mineralized matrices of the body. Bisphosphonates are compounds 
characterized by a P-C-P bond. Several bisphosphonates have been 
investigated in humans and animals with respect to their effect on bone 
and bone derived cells. They are especially known for their ability to 
inhibit bone resorption (Fleisch, H., Drugs 42:919-944, 1991). 
No effect of bisphosphonates on other tissues than bone has been reported. 
Despite the fact that bisphosphonates have been studied on human 
neutrophil myeloperoxidase (Kawolick et al., Arch. Oral Biol. 
35:2015-2035, 1990), on enzymes capable of producing reactive oxygen 
species and on non-specific hydroxylases and acidic serine proteinases 
(Felix et al., Biochem. Biophys. Arch. 429:429-438, 1979), no effects of 
bisphosphonates on collagenase, gelatinase or other MMP activities have 
been noted. In a round table discussion reported in said Ann. N.Y. Acad. 
Sci. 732: 273-279, 1994, bisphosphonates are mentioned only in connection 
with bone resorption and osteoclasts. The effects of bisphosphonates on 
matrix metalloproteinases participating in connective tissue protein 
matrix destruction has until now been unknown. 
SUMMARY OF THE INVENTION 
The present inventors have now surprisingly found that bisphosphonates have 
a marked inhibitive effect on matrix metalloproteinases (MMPs) and that 
they consequently are capable of inhibiting the degradation of connective 
tissue matrix protein components in mammals. 
It is therefore an object of the present invention to provide a method 
useful in the treatment of connective tissue degradation disorders by 
inhibiting the activity of MMPs in a mammalian system. 
The present invention relates to a method for reducing a pathologically 
excessive degradation of connective tissue matrix protein components in 
mammals, e.g. humans, by administering to said mammal a bisphosphonate the 
presence of which is effective in reducing the matrix metalloproteinase 
(MMP) activity in said mammal. More particularly the method comprises 
administering to the mammal an effective amount of bisphosphonate which 
results in a significant reduction of the MMP dependent protein 
degradation in said mammal. 
The present invention also relates to pharmaceutical preparations of a wide 
variety, which preparations include an amount of bisphosphonate effective 
to reduce the activity of one or more MMPs. 
The present invention also includes the use of bisphosphonates for the 
preparation of a composition to be administered to mammals in the 
treatment or prophylaxis of connective tissue degradation disorders or 
extracellular protein degradation disorders. 
The bisphosphonates which are useful in the method of the present invention 
include bisphosphonates which are active as inhibitors against matrix 
metalloproteinases (MMPs), especially against one or both of the following 
MMPs: MMP-1 and MMP-8, both of which have a significant impact on the 
protein degradation system in mammals in inflammatory and other diseases 
(Krane, Ann. N.Y. Acad. Sci. 732: 1-10, 1994). 
Examples of suitable bisphosphonates include commercially available 
bisphosphonates such as clodronate, etidronate, pamidronate, tiludronate, 
etc. An especially preferred bisphosphonate for use in the present 
invention is clodronate which has been shown to inhibit the activity of 
MMP-1 and MMP-8. 
The amount of bisphosphonate to be used in the method of the present 
invention varies depending on the specific bisphosphonate used, the 
patient to be treated as well as on the route of administration. According 
to the invention it is even possible to use, without substantial changes, 
the various bisphosphonates in the dosage forms which are commercially 
available for the treatment of bone disorders. Thus, the present invention 
provides the practitioner instantly with a new way of treating patients 
suffering from connective tissue matrix protein degradation while using a 
drug which is known to be safe, non-toxic and well tolerated. 
As a result of the present invention, mammals suffering from an excessive 
matrix metalloproteinase activity in the connective tissue system may now 
be treated to prevent or reduce matrix protein degradation. Mammals with 
diseases such as ulceration of the cornea, cancer invasion and metastasis, 
AIDS, rheumatoid arthritis, gastric ulceration, burns, wounds such as bed 
sores and varicose ulcers, fractures, trauma, gastric ulceration, skin 
diseases such as acne and psoriasis, lichenoid lesions, epidermolysis 
bollosa, aftae (reactive oral ulcer), dental diseases such as periodontal 
disease, periodontitis, peri-implantitis and root canal treatment 
(endodontic treatment) related disorders, loosening of end osseal 
hip-protheses, and other diseases and disorders which have extracellular 
matrix protein degradation as a part of the disease process, may be 
treated either with bisphosphonates alone or in combination with other 
drugs normally used in connection with the disease or disorder in 
question. 
The present anti-collagenase therapy by bisphosphonates provides a valuable 
supplementary aid for the use by a medical, dental and veterinary 
practitioner in the control and treatment of, among others, such diseases 
as those mentioned above. 
For better understanding of the present invention, together with other and 
further objects and the nature and advantages of the present invention, 
reference is made to the following detailed description of specific 
embodiments.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, a method for treating mammals 
suffering from excessive matrix protein component degradation in the 
connective tissue system is disclosed. The method comprises administering 
to the mammal an amount of a bisphosphonate that results in a significant 
reduction of said protein degradation. 
The bisphosphonates useful in carrying out the method of the present 
invention are so called geminal bisphosphonates having the general formula 
##STR1## 
In the bisphosphonates which have so far been studied, the substituents R' 
and R" independently stand for a hydrogen or a halogen atom, a hydroxy, 
optionally substituted amino or optionally substituted thio group or an 
optionally substituted hydrocarbon residue; preferably one of R' and R" is 
hydroxy, hydrogen or Cl. Other substituents which provide effective 
bisphosphonates are not intended to be excluded from the present 
invention. 
Said geminal bisphosphonates are generally called merely bisphosphonates 
and it should be understood that a reference in the present disclosure to 
bisphosphonates is to be understood as referring to geminal 
bisphosphonates. 
Several bisphosphonates have already been investigated on mammals. Thus, 
the bisphosphonates useful in practicing the present invention may be 
selected from the commercially available bisphosphonates used in humans, 
which include (1-hydroxyethylidene)bis-phosphonate (etidronate), 
(dichloromethylene)bis-phosphonate (clodronate), 
(3-amino-1-hydroxypropylidene)bis-phosphonate (pamidronate), and 
(4-amino-1-hydroxybutylidene)bis-phosphonate (alendronate). 
Other bisphosphonates useful in carrying out the present method further 
include the following bisphosphonates investigated for their effect on 
bone in humans: {[4-chlorophenyl)thio]methylene}bis-phosphonate 
(tiludronate), (6-amino-1-hydroxyhexylidene)bis-phosphonate, 
[1-hydroxy-2-(3-pyridinyl)-ethylidene]bis-phosphonate (risedronate), 
[3-(dimethylamino)-1-hydroxypropylidene]bis-phosphonate, 
[1-hydroxy-3-(methylpentylamino)propylidene]bis-phosphonate (BM 21.0955). 
There are also a number of bisphosphonates which have been used in animals 
and these are also contemplated as being useful in the present invention, 
e.g. [(cycloheptylamino)methylene]bis-phosphonate (YM 175), 
[1-hydroxy-3-(1-pyrrolidinyl)-propylidene]bis-phosphanate (EB-1053), 
[1-hydroxy-2-(1H-imidazol-1-yl) ethylidene]bis-phosphonate (CGP 42'446), 
and (1-hydroxy-2-imidazo-[1,2-a]pyridin-3-yl-ethylidene) bis-phosphonate 
(YM 529). The bisphosphonates useful in carrying out the present invention 
are, however, not intended to be limited to the above mentioned compounds. 
A comprehensive study of known bisphosphonates is given by Fleisch, H., 
Drugs 42(6):919-944, 1991, the disclosure of which is incorporated herein 
by reference. 
Bisphosphonates are commercially available for clinical applications 
focused on four areas. They are used as (a) skeletal markers in the form 
of .sup.99m technetium derivatives for diagnostic purposes in nuclear 
medicine; (b) antiosteolytic agents in patients with increased bone 
destruction, especially Paget's disease, tumor bone disease and, recently, 
osteoporosis; (c) inhibitors of calcification in patients with ectopic 
calcification and ossification; (d) antitartar agents added to 
toothpastes. 
The bisphosphonates used or investigated for use in bone disease have, in 
general the following properties: they bind strongly onto bone mineral, 
inhibit calcium phosphate crystal formation and dissolution, inhibit 
normal and ectopic mineralization (suggested mechanism being 
physicochemical inhibition of crystal growth), and inhibit bone 
resorption. The exact mechanism in bone resorption has not been 
elucidated. 
The toxicity of bisphosphonates is in general low and they are well 
tolerated. The relatively few adverse effects of bisphosphonates include 
gastrointestinal intolerability, especially with some amino derivatives 
and inhibition of normal mineralization. One significant negative side 
effect of bis-phosphonates, especially etidronate, in prolonged 
administration is generally related to their activity on bone, i.e. they 
not only stop the resorption of bone, which is a desired effect as 
anti-osteolytic agent, but they may also prevent mineralization entirely, 
and this is the reason that especially etidronate is normally administered 
only for a short term followed by a delay of three months. 
The amount of bisphosphonate administered to reduce mammalian matrix 
protein degradation in the connective tissue system according to the 
present invention is an amount that significantly reduces MMP activity. In 
the preferred embodiment of the present invention, the bisphosphonate is 
administered in an amount sufficient to significantly reduce the activity 
of the collagenases MMP-1 and MMP-8. 
It is obvious that the dosages of the various bisphosphonate analogs will 
vary somewhat from each other and the ranges set forth below are only 
illustrative and should not be taken as limiting the range of doses. The 
person skilled in the art will be able to determine an optimal dosage of 
the selected bisphosphonate on the basis of his or her experience. 
For illustrative purposes, a suitable amount of the preferred 
bisphosphonate, clodronate, is about 100 to 5000, and even up to 10,000 
mg/kg/day in oral therapy and 0.1 to 10 mg/kg/day in infusion, such as at 
2.5 mg/kg/day i.v. for a week or up to 6400 mg/kg/day p.o. for 5-300 days. 
The preferred method of treatment includes clodronate compositions 
administered in suitable pharmaceutical carriers or devices. 
Etidronate may be used at the usual dosage amounts of from 5 mg/kg/day p.o. 
or i.v. for three days up to 400 mg/kg p.o. for six months, preferably for 
only 3-14 days and then repeated after three months. Pamidronate may be 
administered at the usual dosage of 1 mg/kg i.v. for 24 hours to 1200 
mg/kg/day p.o. for six days or 300 mg/kg/day p.o. for 13 months. 
Alendronate and BM 21.0955 are reported to be effective in the treatment 
of calcemia at a single dose of 10 mg and 2-4 mg, respectively, and can be 
so used in the present invention, although preferably for a longer term of 
at least two weeks. 
The pharmaceutical dosage forms may be in the form of capsules, tablets, 
solutions or suspensions suitable for oral administration of the 
bisphosphonate to the affected mammal. The bisphosphonates may also be 
intravenously administered in the form of infusions in suitable solvents 
such as a saline solution, or as injectable solutions. In some situations 
a topical treatment is preferred and the pharmaceutical composition may be 
formed into creams, pastes, gums, jellies, ointments, liquids, drops, 
aerosols, plasters, membranes, etc. 
The pharmaceutical preparation according to the present invention may also 
incorporate the bisphosphonate in a polymeric carrier delivery system for 
topical or local delivery to the afflicted area. 
A topical administration is especially suitable for treating locally 
affected areas of the mammal such as the gingiva in the case of 
periodontitis, peri-implantitis or root canal treatment, the cornea in the 
case of an ulceration of the cornea, the lungs in the case of lung cancer, 
the skin in the case of acne or psoriasis or skin diseases involving 
tissue destruction such as bed sores, varicose ulcers, etc. 
Specific dental preparations include mouth rinse or mouth wash liquids, 
tooth pastes, dental floss, guided tissue regeneration (GTR) membranes, 
chewing gums, periodontal or surgical pastes, laquers, etc. Especially in 
the case of periodontal diseases it may be preferable to incorporate the 
bisphosphonate into a polymeric carrier which may be delivered therein 
directly to the afflicted area. The periodontal pockets may also be 
topically treated with bisphosphonate preparations by various techniques 
either at home by home care devices or at the dentist by chair-side 
devices such as ultrasonic systems. 
While bis-phosphonates have been added to toothpaste as indicated above for 
removing tartar already formed on the teeth, bis-phosphonates have never 
been proposed for the treatment of periodontal disease. Diabetes patients 
are especially prone to suffer from periodontitis but are not known to 
suffer from osteoporosis any more than the general public. Dental 
preparations according to the present invention are therefore particularly 
suitable for the treatment of periodontitis and the prophylactic treatment 
of periodontitis in diabetes patients. 
Eye drops will be suitable for application to the eyes. Aerosols or sprays 
may be used for damaged lungs caused by burns, wounds or cancer and for 
preventing metastases. The person skilled in the art will be able to 
select the form of preparation which is best suited for the disease or 
disorder in question. 
For prophylactic administration, the patients should preferably be selected 
from at-risk populations, such as diabetics and health care workers. 
The following Examples illustrate the invention without, however, limiting 
it in any way. 
EXAMPLE 1 
Inhibition of Purified Human Fibroblast collagenase (MMP-1) by clodronate 
Purified trypsin-activated human fibroblast-type MMP-1 (Konttinen et al., 
Matrix, 11:395-403; for trypsin-activation of latent pro-MMPs, see Sorsa 
et al., Med. Biol. 63:66-72, 1985) was incubated with purified 1.5 .mu.M 
type I collagen in different indicated clodronate concentrations and 
buffer for 20 hours at 22.degree. C. Preincubations of MMP-1 with the 
buffer and clodronate before adding the substrate lasted 1 hour at 
22.degree. C. After addition of Laemmli's sample buffer, the samples were 
immediately heated to 100.degree. C. for 5 minutes. Subsequently the 
reaction products were separated on sodium dodecyl sulfate polyacrylamide 
gel electroforesis (SDS-PAGE) (Sorsa et al., Med. Biol. 63:66-72, 1985) 
using 8-10% crosslinked gels. The gels were stained with Coomassie 
brilliant blue and the stain intensity of collagen .alpha.- and .alpha.A 
chains was quantified using LKB Ultroscan Densitometer model 2202 coupled 
with Axxiom chromatography computer program. The value representing the 
.alpha.A chains (the 3/4 cleavage fragments) was multiplied by 4/3 and its 
proportion of total collagen in the sample was measured. Four separate 
incubations were used to obtain the mean values and the standard 
deviations presented in FIG. 1. 
FIG. 1 shows a diagram presenting the effect of clodronate on 
trypsin-activated purified MMP-1. The columns present mean values with 
standard deviations of four separate incubations. The concentrations of 
clodronate are indicated. 
EXAMPLE 2 
Inhibition of Collagenase Activity in Jaw Cyst Tissue Samples by Clodronate 
Extracts from jaw cyst tissue containing collagenase activity were prepared 
according to the methods used previously by the inventors (Teronen et al., 
Eur. J. Oral. Sci., in press, 1994; Teronen et al., J. Oral. Pathol. Med., 
in press, 1994). The extracts were preincubated with the concentrations of 
clodronate indicated in FIG. 2, at 22.degree. C. for 1 hour before 
addition of type I collagen and incubation for 60 hours at 22.degree. C. 
The protocol for the measurement of collagenase activity was the same as 
in Example 1. The effects of clodronate at different concentrations on the 
collagenolytic activity present in the cyst extracts are presented in FIG. 
2. 
FIG. 2 shows a diagram of the effect of clodronate on autoactive cyst 
collagenase activity at different concentrations presented below each 
column. Two series are shown. 
EXAMPLE 3 
Inhibition of Purified Human Neutrophil Collagenase (MMP-8) by Clodronate 
Purified human MMP-8 activated by aminophenyl mercuric acetate (APMA) 
(Sorsa et al., Med. Biol. 63:66-72, 1985) was incubated with purified 1.5 
.mu.M type I collagen in different clodronate concentrations and buffer 
for 20 hours at 22.degree. C. Preincubations of MMP-8 with the buffer and 
clodronate before adding the substrate lasted 1 hour at 22.degree. C. 
After addition of Laemmli's sample buffer, the samples were immediately 
heated to 100.degree. C. for 5 minutes. Subsequently the reaction products 
were separated on sodium dodecyl sulfate polyacrylamide gel electroforesis 
(SDS-PAGE) (Sorsa et al., Med. Biol. 63:66-72, 1985) using 8-10% 
crosslinked gels. The gels were stained with Coomassie brilliant blue and 
the stain intensity of collagen .alpha.- and .alpha.A chains was 
quantified using LKB Ultroscan Densitometer model 2202 coupled with Axxiom 
chromatography computer program. The value representing the .alpha.A 
chains (the 3/4 cleavage fragments) was multiplied by 4/3 and its 
proportion of total collagen in the sample was measured. Four separate 
incubations were used to obtain the mean values and the standard 
deviations presented in FIG. 3. 
FIG. 3 shows a diagram presenting the effect of clodronate on 
APMA-activated purified MMP-8. Three separate results of incubations are 
presented. The concentrations of clodronate are indicated. 
EXAMPLE 4 
Inhibition of Collagenase Activity in GCF from Periodontitis Patients by 
Clodronate 
Gingival crevicular fluid (GCF) from deep (&gt;6 mm) periodontal pockets of 
patients with adult periodontitis has been shown by Western-blotting to 
contain autoactive MMP-8 (Sorsa et al., Ann. N.Y. Acad. Sci. 732:112-131, 
1994) and was therefore used in this experiment. The samples were 
collected with filter strips placed gently on the opening of the gingival 
orifice approximately 1 mm into the sulcus for 2-5 minutes. The absorbed 
fluid was eluated with 75 .mu.l per strip of 50 mM Tris-HCl, pH 7.5, 
containing 0.15 M NaCl and 1 mM CaCl.sub.2. The samples were then 
centrifuged and aliquots of the supernatant were used for the enzyme 
assay. The GCF incubations with type I collagen and the effects of 
clodronate were performed using the same protocol as in Examples 1 and 2. 
The results of the five separate incubations are shown in FIG. 4. 
FIG. 4 shows a diagram of the effect of clodronate on the autoactive 
collagenase activity of GCF. The columns present mean values with standard 
deviations of five incubations. The concentrations of clodronate are 
indicated. 
EXAMPLE 5 
Inhibition of Collagenase in Peri-implant Sulcular Fluid (PISF) from 
Peri-implant Patients by Clodronate 
Peri-implant sulcular fluid (PISF) from peri-implant pockets of loosening 
dental implants of five patients were used. The samples were collected 
with filter strips placed gently on the opening of the implant margin 
approximately 1 mm into the sulcus for 2 to 5 minutes. The absorbed fluid 
was eluated with 50 .mu.l per strip of 50 mM Tris-HCl, pH 7.5, containing 
0.15 M NaCl and 1 mm CaCl.sub.2. The samples were then centrifuged and 
aliquots of the supernatant were used for the collagenase assay. The 
samples used in this experiment were also found to contain immunoreactive 
MMP-8 using specific polyclonal rabbit antiserum for human 
polymorphonuclear neutrophilic collagenase (MMP-8) by Western-blotting 
(Sorsa et al., Ann. N.Y. Acad. Sci. 732:112-131, 1994) as well as by 
functional characterization (Ingman et al., J. Clin. Periodontol. 
21:26-31, 1994). The incubations with type I collagen and clodronate were 
performed using the same protocol as in Example 1 and 2. The results of 
five double incubations (n=10) are shown in FIG. 5. 
FIG. 5 shows a diagram of the effect of clodronate on PISF collagenase. The 
columns present mean values with standard deviations of ten separate 
incubations. The concentrations of clodronate are presented below each 
column. 
As can be seen from the above Examples 1 to 5, the presence of 
bisphosphonates provides a significant inhibition/reduction of the 
activity of MMP-1 and MMP-8. 
It should be noted that the values in the specific Examples 1 to 5 are 
relative and that they are not intended to indicate general absolute 
values for the inhibition rates. The obtained values are specific for the 
tests performed, and other absolute values may be obtained under different 
conditions. The Examples do, however, clearly indicate the inhibitive 
effect of bisphosphonates on the activity of MMPs. This inhibitive effect, 
as such, is not dependent on the test conditions. 
PROPHETIC EXAMPLE 1 
Treatment of Humans Suffering from Periodontitis 
Gingival crevicular fluid (GCF) is taken from the periodontal pockets of an 
adult patient suffering from periodontitis. The GCF is shown to contain an 
elevated amount of MMP-8. Clodronate is administered orally to said 
patient for two weeks at a dosage of 2400 mg/day (capsules taken twice 
daily), whereafter the treatment is continued for two months at a dosage 
level of 1600 mg/day. 
After the initial two weeks the enzyme activity in GCF has significantly 
decreased and after the whole treatment the enzyme activity is at a normal 
level indicating that the collagen degradation significantly decreases and 
the active tissue descruction associated with periodontitis also 
decreases. 
The patient is monitored in subsequent inspections and the treatment is 
renewed at need. 
PROPHETIC EXAMPLE 2 
Treatment of Humans Suffering from Periodontitis 
Gingival crevicular fluid (GCF) from the periodontal pockets of an adult 
patient is shown to contain a very high amount of MMP-8 indicating severe 
periodontitis. Etidronate is administered topically by a dentist to each 
afflicted tooth in a gel containing 1% etidronate. The treatment is 
repeated once or twice during five consecutive days. 
The treatment results in a significant decrease of MMP-8 activities in the 
GCF of said patient. The treatment is repeated after three months at need. 
PROPHETIC EXAMPLE 3 
Treatment of Humans Suffering from Rheumatoid Arthritis 
Arthritic synovial fluid is taken from the joints of an adult patient 
suffering from rheumatoid arthritis. The synovial fluid is shown to 
contain an elevated amount of MMP-1 indicating an active degradation of 
collagenase. Clodronate is administered as an infusion at a dosage of 4 
mg/kg in saline solution for five consecutive days, whereafter the 
treatment is continued by oral administration of 2400 mg clodronate/day 
for two months. 
After an initial two weeks the MMP-1 activity in the synovial fluid 
significantly decreases and after the whole treatment the enzyme activity 
is at a basically normal level indicating that the collagen degradation 
has stopped. 
The patient is monitored in subsequent inspections and the treatment is 
renewed at need. 
PROPHETIC EXAMPLE 4 
Prophylaxis of Periodontitis in Humans Suffering from Diabetes Mellitus 
A group of patients suffering from diabetes mellitus are monitored for 
periodontitis. Half of the group receive clodronate topically administered 
in a clodronate containing tooth paste used twice daily for several years. 
The other half of the group does not receive bis-phosphonate in any form. 
The MMP-8 activity of the treated patients remains at a steady normal 
level. None of the patients prophylactically treated with clodronate 
develops periodontitis. Several of the patients in the untreated group 
develop periodontitis showing elevated levels of MMP-8 activity. 
While there has been described what are presently believed to be the 
preferred embodiments of the present invention, those skilled in the art 
will realize that changes and modifications may be made thereto without 
departing from the spirit of the invention, and it is intended to claim 
all such changes and modifications as fall within the true scope of the 
invention. 
It is especially envisaged that the matrix metalloproteinases will be found 
to play an important role in several diseases and disorders, in which 
their action has not so far been discovered. The invention is intended to 
encompass the use of bisphosphonates also in the treatment and prophylaxis 
of such conditions, as well as the formulation of bisphosphonates into 
various dosage forms for said treatment and prophylaxis. 
The foregoing description of the specific embodiments will so fully reveal 
the general nature of the invention that others can, by applying current 
knowledge, readily modify and/or adapt for various applications such 
specific embodiments without departing from the generic concept, and, 
therefore, such adaptations and modifications should and are intended to 
be comprehended within the meaning and range of equivalents of the 
disclosed embodiments. It is to be understood that the phraseology or 
terminology employed herein is for the purpose of description and not of 
limitation.