WO1997034616A1

WO1997034616A1 - A method for periodontal disease treatment

Info

Publication number: WO1997034616A1
Application number: PCT/US1997/004147
Authority: WO
Inventors: Kenneth S. Kornman; Pamela K. Fink; Hwa-Ying Wang
Original assignee: Medical Science Systems, Inc.
Priority date: 1996-03-18
Filing date: 1997-03-18
Publication date: 1997-09-25
Also published as: AU2213697A

Abstract

A method for the treatment of periodontal disease is disclosed. The method includes the steps of identifying a patient with the periodontal disease and administering to the patient an antagonist of Tumor Necrosis Factor α (TNFα).

Description

A METHOD FOR PERIODONTAL DISEASE TREATMENT

BACKGROUND OF THF. INVENTION

1. Field of the Invention

This invention relates to a method of treating periodontal disease. More specifically the invention relates to a method of treating periodontal disease using an antagonist of TNFα. 2. Description of the Prior Art

Periodontal disease is a disease ofthe hard and soft tissues that support the teeth and is initiated by oral bacteria. Gingivitis is an early stage ofthe periodontal disease where the gums may become red. swollen and bleed easily. Gingivitis is usually painless and, if not treated, can advance to periodontitis. which may be classified by the magnitude of tissue destruction as mild, moderate, or severe. Periodontitis is primarily a disease of adults and is usually not detectable until after the age of 35. Bacteria that are present in dental plaque initiate periodontal disease. Toxins produced by the bacteria in the plaque activate the body's inflammatory and other immune mechanisms which ultimately leads to the destruction ofthe bone and gum tissue that support the teeth. As the disease progresses, the gums pull away from the teeth and periodontal pockets are formed which provide a protected environment for the bacteria, thereby causing the cycle to continue. However, some sites do not continue to be active. United States patent 5,328,829 discloses a method for determination of active periodontal disease sites within the oral cavity by measuring interleukin IL-1 β at the site. Smoking has been associated with an increased prevalence and severity of periodontitis. However, a significant number of individuals with periodontitis have never smoked. For the past 15 years, there has been evidence that certain forms of periodontitis that affect young children and teenagers are genetically determined. These diseases, which are of extremely low prevalence in the population, produce severe periodontitis in some individuals before the age of puberty and in other individuals between puberty and age 18. The genetic factors that were identified in those cases involved very overt biologic mechanisms that most likely would predispose the individual to multiple health problems. For several years, efforts to find the same type of genetic factors in adult forms of periodontitis were not successful.

In spite ofthe above failures, new evidence emerged beginning in 1990 from studies of identical twins that indicated that genetics play a significant role in the clinical presentation of disease in adult forms of periodontitis (Michalowicz et al., J Periodontol 62:293-299, 1991). While the twin studies indicated that there was a genetic component, it was not identified. It would be useful to determine patients who are susceptible to severe adult periodontitis.

The early detection of a predisposition to genetic diseases presents the best opportunity for medical intervention. Early genetic diagnosis may improve the prognosis for a patient through supervision and early intervention before the clinically detectable disorder occurs. In cases where patients with similar symptoms are treated with variable success, sophisticated genetic testing can differentiate individual patients with subtle or undetectable differences and can lead to more suitable individual treatments. It is even possible that early intervention may one day involve methods such as gene therapy. With the development of genetic testing, it is now possible to identify gene mutations which indicate a propensity to develop disease, even when the disease is of polygenic origin. The number of diseases that can be diagnosed by molecular biological methods continues to grow with increased understanding of the genetic basis of multifactorial disorders (see e.g.. United States Patent Nos. 4.582,788; 5,1 10,920; 4,801.531 ; 4,666,828; and 5,268,267). As with any infection, once initiated, inflammatory and other immune mechanisms ofthe body come into play (see United States Patent 5,328,829, column 1 , for a review). In general, research on inflammatory markers has had very limited success at differentiating periodontitis disease severity and there have been limited and unsuccessful efforts directed to the genetic aspects of the inflammatory response of periodontal disease. Genetic variation at the multiple loci controlling the inflammatory and other immune responses in selected diseases with inflammatory components has been a factor in determining susceptibility to, or severity of, disease.

Applicants investigated and determined that genetic factors that are associated with inflammatory and other immune responses are correlated with periodontal disease severity. In their analysis of periodontal disease applicants found a correlation with the presence of a DNA polymorphism in the gene sequence for interleukins IL-lα and IL-lβ and disease severity.

The IL-1 polymoφhism associated with severe disease has the potential to result in increased IL-1 biologic activity. Polymorphisms in the IL-1 A gene have not been associated with functional differences in IL-1 biologic activity, however, the +3953 IL-IB (alternative name is Taql) polymoφhism is associated with altered function. Monocytes from individuals homozygous for the IL-I B +3953 allele 2 produce more IL-l β than cells from individuals homozygous for allele 1 [Pociot et al, Eur J Clin Invest 22:396-402, 1992; di Giovine, et ύ7.. Cvtokine 7:606 (Abstract). 1995). Table

IL-1B+3953 Alleles¹ IL-lβ²

1/1 5.2 ng/ml

1/2 12.4 ng/ml

2/2 19.9 ng/ml

' Monocytes isolated from individuals with the indicated allelic pattern at locus +3953 ofthe IL-IB gene " IL-l β quantity in supernatant of a standard number of monocytes stimulated with LPS

Elevated tissue or gingival fluid levels of IL- 1 β have been previously associated with periodontitis (Yavuzyilmaz, et al.. Australian Dental J 40(1) :46-49, 1995; Stashenko et ai, J Clin Periodontol 18:548-554, 1991a; Stashenko et al, J Periodontol 62:504-509, 1991b), and various biologic mechanisms attributable to IL-1 , such as induction of bone resoφtion, have been proposed as an indictment of IL- 1 in the etiology of periodontitis (Stashenko 1991). However, when applicants modeled the disease using the BIOFUSION protocol, severe periodontitis could not be shown to be the result of over-production of IL-1 only. The influence of increased IL-1 on periodontal disease was observed to be mediated in large part through effects of other inflammatory mediators. Therefore, in computer simulations (BioFusion) of the biology the severe tissue destruction initiated by increased levels of IL-1 could be blocked by blocking IL-1, but could also be blocked by inhibiting other "downstream" mediators. Applicants therefore have investigated other possible associations and treatments.

The pathology of periodontitis involves both collagen loss and destruction of bone. TNFα generally produces a net loss of collagen, whereas IL-1 generally produces a net increase in collagen (Bartold, et al.. Fundamental of Periodontics. Chicago IL: Quintessence Publishing Co., 61-87, 1996). TNFα is known to inhibit collagen synthesis (Rapala, et al , Experientia 52(l):70-4, 1996; Chung KY, et al.. Journal of Biological Chemistry 271(6):3272-8. 1996) and to upregulate matrix metalloproteinases that degrade collagen. In addition TNFα, but not IL-1 , appears to lead to fibrosis and impaired healing (Chou, et al, Journal of Immunology 156(1 1 ):4354-62, 1996; Mohamed-Ali, et al, Cel) & Tissue Research 284(3):509-15, 1996; Buck, et al, American Journal of Pathology 149(1): 195-204. 1996) which results in a non-functional connective tissue matrix. Collagen expression by lung fibroblasts was reduced more by TNFα than by IL-lβ (Diaz, Journal of Biological Chemistry 268(14): 10364-71, 1993), and although TNF inhibits collagen -RNA transcription. IL-1 appears to act at post-transcriptional levels (Armendariz-Borunda, et al. , Journal of Biological Chemistry 267(20): 14316-21 , 1992). TNFα is also known to induce bone resoφtion, as well as block bone formation (Panagakos, et al. , Inflammation 18(3):267-84, 1994). For review of TNFα and its potential role in periodontal disease see Offenbacher 1996 (Offenbacher, Ann Periodontal 1 :821-878. 1996). Polymoφhisms in the TNFα gene were not found to be associated with severity of periodontitis (Kornman, et al, J Clin Periodontal 24:72-77, 1997). We therefore propose that the genetic predisposition for severe periodontitis, as determined by the IL-1 genotype, is the result of a genetically-determined increase in IL-1 biologic activity that then upregulates the TNFα and its pathologic effects. We propose that the clinical disease expression is the direct result of an increase in TNFα. The ΪL-1 genotype that is associated with periodontitis should therefore display increases in both IL-1 and TNFα. Although the two cytokines have many overlapping actions and synergintic effects, there are distinct and subtle differences, as noted above.

The ideal therapy for preventing or treating severe periodontitis therefore appears to involve the blockage or antagonism of TNFα. Blockage of TNFα secondarily, by primarily blocking IL-1 , is not a desirable approach, because IL-1 has many favorable attributes that are involved in protection of the host (Dinarello, e/ o/. , _AMΔ 269(14):1829-1835, 1993). Blocking IL- 1 in some animal models has actually increased mortality (Dinarello et al. , JAMA 269( 14): 1829- 1835, 1993). It therefore would not be advisable to block both cytokines for prolonged periods of time, as would be necessary to treat periodontitis. The potential to treat or prevent periodontitis by blocking biologic mechanisms initiated by IL-1 and/or TNFα have recently been demonstrated. It has been reported that local injections of combinations of IL-1 and TNF blocking agents directly into the periodontal tissues of monkeys reduced osteoclasts associated with the periodontal bone by 75% and reduced measurements of bone loss by 40% (Assuma, J Dent Res 76 (IADR Abstracts): #1298, 1997). These and other advantages ofthe present invention will become apparent from the following detailed description. SUMMARY OF THE INVENTION

According to the present invention a method for treating periodontal disease is disclosed. The method includes identifying a patient with periodontal disease and administering to the patient an antagonist of Tumor Necrosis Factor α (TNFα).

Also provided in the present invention is a method of treating periodontal disease comprising administering to a patient having periodontal disease a pharmaceutically acceptable amount of an antagonist of Tumor Necrosis Factor α (TNFα).

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, patients are identified with mild, moderate or severe periodontal disease. The patients are then evaluated and where appropriate as described herein below treated with an antagonist of tumor necrosis factor α (TNFα).

The term antagonist or antagonizing is used in its broadest sense. Antagonism can include any mechanism or treatment which results in inhibition, inactivation, blocking or reduction in TNFα levels or TNFα activity. Additional antagonists include agents that block upregulation of TNFα production (Allison et al , Ann NY Acad Sci 762:331 -340, 1995), agents that block release of membrane-bound TNFα (Williams g. a... J Clin Invest 97O2V2833-2841. 1996; McGeehan et al, Nature 370(6490):558-561, 1994; Gearing et al, Nature 370(6490):555-557, 1994). and/or agents that regulate the levels of soluble receptors for TNFα (Mullberg et al., J Immunol 155(1 1):5198-5205, 1995). TNFα activity may be blocked at several steps, including signals that initiate transcription ofthe genes for TNF, such as cytokines that activate the cells to produce TNF, receptors for the activating signals, signal transduction pathways, and modifiers of those pathways, within the cell that result in transcription. There are also post-transcriptional events, including enzymatic cleavage ofthe cell-bound proenzyme to release TNFα into the environment. It is also possible to block TNF activity by blocking or interfering with cell-bound receptors for TNF on effector cells, or by enhancing the presence or activity of soluble TNF receptors that interfere with TNF binding to effector cells. Many of these mechanisms have been reviewed recently (Stewart et al , Am J Kidnev Dis 25(6):954-966, 1995; Arend, Advances in Internal Medicine 40:365-394, 1995; Burger et al, Neurology 45(6):S39-43, 1995). Some of the blocking agents do not affect transcription or steady state levels of m-RNA, but result in less protein. Some of these compounds also block the cyclooxygenase and lipoxygenase pathways for arachidonic acid metabolites, however their effects on IL-1 and TNF synthesis may be unrelated to those activities (Sirko 1991 ). These agents have been called cytokine suppressing anti-inflammatory drugs, or CSAIDs (Young 1994). The CSAIDs reduce TNF by inhibiting translation (Olivera 1993). For example, the antagonizing step can include blocking cellular receptors for TNFα, or utilizing antibodies against TNFα and soluble receptors for TNFα to reduce the TNFα levels in the circulation. Additionally. methods which interfere with IL-1 production so that IL-1 modulation of TNFα, as contemplated by the present invention, is not available. For example treatment with IL-lra has been shown to reduce IL-1 levels, and to secondarily reduce TNFα levels. Periodontal diseases are bacterial ly-induced inflammatory conditions of the soft tissues that surround and support the teeth. The inflammation can affect the superficial gingival tissues (gingivitis) or can destroy the connective tissue and bone that support the teeth

(periodontitis). Periodontitis is graded in severity based on the amount of bone and connective tissue destruction that is clinically measurable.

In a further embodiment of the present invention the patients are screened to determine if they have a genetic predisposition to periodontal disease thereby requiring more rigorous treatment and earlier intervention with rigorous treatment. Alleles associated with severe disease were identified as IL-1 A allele 2 together with IL-IB (Taql) allele 2 [alternative designation is IL-IB (+3953) allele 2]. It was determined that the Odds Ratio (OR) for severe periodontitis is

4.3 for patients carrying at least one copy of IL-1A allele 2 and IL-IB (Taql) allele 2 among nonsmokers. In a population of smokers and nonsmokers the OR for a smoker or patients carrying at least one copy of IL-1 A allele 2 and IL-I B (Taql) allele 2 is 10.06 for having severe disease.

Although the most direct conclusion from the IL-1 genetic polymoφhism data would be that severe periodontitis is the result of over-production of IL-1 , applicants attempts to mechanistically explain the tissue destruction that is characteristic of periodontitis were successful only when the IL-1 induction and amplification of TNFα was included in the process. In particular,

TNFα activates PMNs within the inflamed tissue and leads to a net loss of collagen. IL-1 by itself does not lead to a net decrease in collagen and may actually increase net collagen. IL-lβ in the tissue of severe periodontitis patients had no correlation with collagen loss in the area (Feldner et al , J. Periodontal Res. 29:54-61 , 1994).

Polymoφhisms in the TNFα gene were not found to be associated with severity of periodontitis. Applicants, therefore, show that the genetic predisposition for severe periodontitis, as determined by the compound IL-1 genotype, is the result of a genetically-determined increase in IL-1 biologic activity that then amplifies TNFα and its pathologic affects. The IL- 1 amplification of TNFα is well established.

Therefore the ideal therapy for preventing or treating severe periodontitis would be blocking (or antagonism) of TNFα. Blocking of TNFα secondarily by primarily blocking IL-1 is not a preferred embodiment, because IL-1 has many favorable attributes that are involved in protection of the host. (Dinarello et al, JAJvlA 269(14):! 829-35, 1993). Blocking IL-1 in some animal models has actually increased mortality. An embodiment which would primarily block IL-1 with secondary effect of TNFα blocking, is not preferred for prolonged periods of time for this reason. However some of the protective attributes of IL- 1 are redundant with TNFα so that a blocking of TNFα activity in a patient can be tolerated for longer periods of time which would be necessary for treatment of periodontal disease.

The relative contributions of TNF and IL-1 to chronic inflammatory conditions is just beginning to be clarified. Transgenic mice have been developed that over-produce TNF. In these mice, abnormal TNF production has been shown to contribute to disease initiation and progression of rheumatoid arthritis, systemic inflammatory response syndrome, diabetes, and other inflammatory or immune conditions (Probert et al, J Leukocyte Biol 59(4):518-525, 1996). In addition, it has been shown in the transgenic mouse that the arthritis resulting from excess TNF can be blocked by inhibiting IL-1 activitv (Probert et al. Eur J Immunol. 25(6): 1794- 1797, 1995), indicating that the pathogenic load of TNF in arthritis is carried primarily through IL-1. We are not aware of any work showing the reverse, i.e. that pathogenic effects of excess IL-1 may be blocked by blocking TNF activity.

The DNA sample is obtained from blood or tissue samples. In a preferred embodiment, the DNA will be obtained from blood cells obtained from a finger prick of the patient with the blood collected on absorbent paper. In a further preferred embodiment, the blood will be collected on an AmpliCard™ (University of Sheffield, Department of Medicine and Pharmacology, Royal Hallamshire Hospital, Sheffield, England SIO 2JF), also described in Tarlow JW, et al. Journal of Investigative Dermatology 1994: 103: 387-389, incoφorated by reference herein. The DNA is then isolated from the dried blood spots and then target sequences amplified using the polymerase chain reaction (PCR). Oligonucleotide DNA primers that target the specific polymorhic DNA region within the genes of interest are prepared so that in the PCR reaction amplification of the target sequences is achieved. This embodiment has the advantage of requiring only a small amount of blood and avoids the necessity for venipuncture or a tissue biopsy. However, other means for collecting DNA and determining polymoφhism patterns as known in the art can be used. The amplified DNA sequences from the template DNA are then analyzed using restriction enzymes to determine the genetic polymoφhisms present in the amplified sequences and thereby provide a genetic polymoφhism profile ofthe patient.

Polymoφhisms are variants in the gene sequence. They can be sequence shifts found between different ethnic and geographic locations which, while having a different sequence, produce functionally equivalent gene products. Polymorphisms also encompass variations which can be classified as alleles and/or mutations which can produce gene products which may have an altered function. Polymoφhisms also encompass variations which can be classified as alleles and/or mutations which either produce no gene product, an inactive gene product or increased levels of gene product.

Some diseases have prominent inflammatory and other immune components. One ofthe primary components ofthe inflammatory and other immune responses is cytokine production. (Larsen et al. Ann. Rev. Immunol 1 :335-359, 1983) Cytokines are peptide/protein immunomodulators that are produced by activated immune cells including thymus-derived T lymphocytes (T-cells), B lymphocytes and monocyte/macrophages. The cytokines include interleukins (IL-1 through IL-15), colony stimulating factors (CSFs) for granulocytes and/or macrophages (CSF-G, CSF-M, CSF-GM), tumor necrosis factors (TNFs α & β), and interferons (IFN α, β & γ). The basic activity of IL-1 includes the combined activities of IL-lα, IL-lβ and IL-1 receptor antagonist (IL- Ira). (For a review, see Duff. Br. J. Rheumatol 32 (Suppl 1) : 15-20. 1993: and Basic and Clinical Immunology, 8th Ed., 1994, Stites, Terr & Parslow, editors, Chapter 9. pgs. 105-123). United States patent 5,328,829 found IL-l β at active sites in periodontal disease but did not report any correlation with disease state. Association of a single cytokine polymoφhism and disease states have been found as, for example, in Systemic Lupus Erythematosus, Ulcerative Colitis and Juvenile rheumatoid arthritis (Mansfield et al.. Gastroenterology 106:637-642, 1994; Verjans et al. Rheum Dis Clin North Am 18: 177-186, 1992; Blakemore et al. Arthritis and Rheumatism 37(9) :1380-1385, 1994; McGuire et al, Nature 371 :508-51 1, 1994; McDowell et al. Arthritis & Rheumatism (in press 1995). As stated above, applicants have found an association between IL-1 polymoφhisms and severe periodontal disease. 11-1 , by itself, does appear capable of explaining severe periodontitis, yet TNFα has many of the attributes required for severe disease, yet unexpectedly TNFα gene polymoφhisms were not associated with severe periodontitis.

Applicants have reviewed this unexpected finding in Iight of the following: 1. IL1 has as one of its key effects amplifying the action of TNFα. [Michie. et al.. Arch Surq 125:531-536, 1990: Larsen et al. Ann. Rev. Immunol 1 :335-359, 1983]. Therefore, argue that this interaction affects disease severity in periodontal disease.

2. Applicants data shows that the IL1 polymoφhism appears to regulate how effective IL1 is on amplifying TNFα. 3. Review of relevant prior art literature and BioFusion modeling indicates that the effect of increased IL1 in causing the tissue damage seen in periodontal disease, may be blocked by blocking either IL 1 or TNFα. From this, the present invention provides for the treatment of periodontal disease by blocking the action of TNFα, even though there is no direct association of TNFα polymorphisms and severe disease. In a preferred embodiment, direct blockers of TNFα will be used, as for example Tumor necrosis factor soluble receptor (TNFsr), which is a cytokine cascade blocker or the recombinant molecule (rTNFsr) which has been used clinically. Soluble forms of cytokine receptors containing the extra-cellular domain of the cytokine receptor may act as binding proteins that modulate cytokine activity. There are two soluble TNF receptors. Soluble receptors to TNF have been shown to block septic shock responses in rodents, under certain conditions, whereas protection required simultaneous blockage of both IL- 1 and TNF under other conditions (Russell et al., J Infectious Dis 171(6): 1528-1538, 1995). Septic shock patients have been recently treated with a recombinant soluble fusion protein that is a dimer of the soluble TNF receptor and the Fc portion of IgGl (Fisher et al., N Eng J Med 334(26): 1697-1702, 1996). The fusion protein to block TNF activity did not reduce mortality, and in higher doses there was a trend to increased mortality, as compared to placebo treated patients. This same dimer did show promise however, in improving renal function and speeding recovery in renal transplant patients given OKT3 therapy (Eason et al , Transplantation 61 (2):224-228, 1996). Additionally, antibodies to TNFα and other cytokine inhibitors are available. [Cytokines and Cytokine Inhibitors Workshop; Dinarello et al, JAMA 269(14): 1829-1835, 1993; Grewal et al, Amer J Surq 167 :214-19, 1994a; Grewal et al, Gastroenterology 106 : 163, 1994b) IL-lra has also been shown to reduce circulating levels of TNF (Aiura, et al.. Infect Immun 61 (8) 3342-3350, 1993).

In the method of the present invention, patients presenting with an IL-1 polymoφhism that predisposes to severe periodontal disease, as well as smokers, will be aggressively treated with a TNFα antagonist. Further patients with severe disease, no matter what their IL-1 polymorphism will also be treated aggressively. Those having ordinary skill in the art will be able to ascertain the most effective dose and times for administering the compounds ofthe present invention, considering route of delivery, metabol^ _j ofthe compound, and other pharmacokinetic parameters such as volume of distribution, clearance, age of the subject, etc.

The compounds may be administered along with a pharmaceutical carrier and/or diluent. The compounds of the present invention may also be administered in combination with other agents. The compounds utilized in the present invention, are administered in combination with other drugs or singly consistent with good medical practice. The composition is administered and dosed in accordance with good medical practice taking into account the clinical condition ofthe individual patient, the site and method of administration, scheduling of administration, and other factors know to medical practitioners. The "effective amount" for puφoses herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved gum condition and other indicators as are selected as appropriate measures by those skilled in the art.

The following examples serve to illustrate specific embodiments of the invention, but should not be considered as a limitation on the scope of the invention.

Example 1. Identification of Patients

Reactions and manipulations involving DNA techniques, unless stated otherwise, were performed as described in Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, incoφorated herein by reference. Methodology as set forth in United States patents 4,666.828; 4.801 ,531 ; and 5,272,057 and McDowell el al , 1995 are also used unless stated otherwise. Enzymes used in PCR were from GIBCO BRL, thermocyclers were either Perkin-Elmer or Biometra. Restriction enzymes Ncol and Taql were from Promega (US). Restriction enzymes Aval and Bsu36I were from NEB (USA).

Genetic polymoφhisms associated with periodontal disease in adults was detenriined using the protocol of McDowell et al. (1995). Because of the masking effect of smoking, genetic factors associated with severe disease were determined in nonsmokers. A group of otherwise healthy adults were screened at a dental clinic for the presence of periodontal disease. The study included primarily individuals of Northern European descent. Each patient was screened for the absence of disease or, if the disease was present, its degree in each of four parameters. The four variables of interest are clinical attachment loss (CAL), pocket depth, gingivitis and inteφroximal bone loss. A blood sample is taken, DNA isolated and the genetic polymoφhism at IL-1 A and IL- IB genetic loci determined. In addition, a dental history of each patient was obtained including specific questions on family history of diabetes, cardiovascular disease or early tooth loss as well as whether they were smokers. In order to determine periodontal disease status, each patient underwent an examination including a full mouth measurement of pocket depth (PD), recession (R), plaque (Pl) and bleeding on probing (BOP). Clinical attachment loss (CAL) is computed from pocket depth and recession. Radiographs assess bone loss. Based on these measurements, the patient was classified as either healthy, mild to moderate periodontitis or severe periodontitis.

All clinical variables were calculated on six surfaces (distal buccal. buccal. mesial buccal, mesial lingual, lingual and distal lingual) on each tooth (excluding third molars) for up to 168 sites. All radiographic variables were calculated on two surfaces on each tooth for up to 56 sites.

Disease severity classification was as follows:

Periodontally Healthy: Patients presented with all pocket depths <4mm, unlimited facial CAL, inteφroximal CAL of <2mm and <15% radiographic bone loss. Unlimited plaque and gingival inflammation and recession may be present.

Mild to Moderate Periodontitis: No history of disease onset prior to age 35. Patients presented with no more than two missing teeth, other than third molars, teeth extracted for orthodontic therapy and teeth lost as a result of extra-oral trauma. Patients also presented with a PD>6 mm on five to nine inteφroximal sites. At least two ofthe qualifying inteφroximal sites must occur in different quadrants. Gingival inflammation (as exemplified by bleeding on probing) was present in at least two quadrants. Full mouth radiographs must disclose less than four inteφroximal sites with > 50% bone loss. Radiographic total mouth mean bone lose must be less than 25%. There is no specifications for CAL in this classification.

Severe Periodontitis: Patients presented with of >10 inteφroximal sites that measure >7 mm, with PD of >7 mm occurring on at least eight teeth. CAL measured >5 mm on >1 1 sites. Full mouth radiographs taken within the last three years showed >7 interproximal sites with >50% bone loss on radiographs with a total mouth mean bone loss greater than 30%.

Statistical Analysis x¹ analysis was used. The Odds Ratio (relative risk) is calculated from a 2x2 contingency table as described by Woolf, 1955.

Sample Contingency Table

Genotype of Interest Phenotype 1 Phenotype 2

Present A B

Absent C D PCR Amplification and Restriction Enzyme Digestion Protocols for TNFA

TNFA: The single base variation (G/A) polymoφhism at TNFA base -308 was identified as follows:

SCREENING: PCR amplification of genomic templates. One mismatch inserted in a primer to complete an Ncol site (underlined).

PRIMERS: The following primers were produced in an ABI DNA synthesizer based on the genomic sequences (Nedospasov et al, 1986; GENBANK).

5' AGG CAA TAG GTT TTG AGG GCC AT 3' (SEQ ID No. 1)

5' TCC TCC CTG CTC CGA TTC CG 3' (SEQ ID No. 2)

PCR CONDITIONS:

Primers final concentration of 2mM and 1.5mM final concentration of MgCI _. 1 cycle [94°C (3 minutes), 60°C (1 minute), 72°C (1 minute)]; 35 cycles [94°C (1 minute), 60°C (1 minute), 72°C (1 minute)]; 1 cycle [94°C (1 minute), 60°C (1 minute), 72°C (5 minutes)].

RESTRICTION ENZYME DIGESTION: Digestion was with 6 Units per 30μl reaction mixture of Ncol at 37°C, for 8 hours. Sizing was by 8% PAGE or 2% agarose gels. PREDICTED RESULTS FROM DIGESTION:

Allele 1 Ncol digestion of PCR products of allele 1 will yield 87 and 20 basepair (bp) fragments. Allele 2 Ncol digestion of PCR products of allele 2 will be ineffective and yield a 107 basepair (bp) product.

Adults, smokers and nonsmokers, were screened for periodontal disease severity using a consensus clinical criteria as described herein above. The data are shown in Table 2.

Abbreviations used in Table 2 are as follows: PD (pocket depth). BOP (bleeding on probing). CAL (clinical attachment loss), #>49% (number of sites where bone loss is greater than 49%), %bl (percent bone loss), S.D. (standard deviation); H=healthy, M=mild/moderate, S=severe. * indicates significance at least at 95% confidence level. TABLE 2

In Table 3 the clinical data is displayed and compared between smokers and nonsmokers. Note that there is a significant difference in the overall clinical disease state between smokers and nonsmokers.

TABLE 3

Table 4 summarizes and compares the clinical findings for Allele 2 carriage rate in all subjects by disease severity and cytokine. No association is seen between disease severity/status and TNFα.

TABLE 4

Disease Status' IL-1A IL-IB +3953 IL-1B-51 1 IL-1RN TNFα

Mild 38.8- 40.8 55.1 59.2 26.5

Moderate 61.9^J 47.6 61.9 33.3" 38.1

Severe 55.8 46.5 62.7 48.8 20.9

¹ Subjects classified as to disease status as defined herein above

² % of individuals in the Mild group who carry at least one copy of allele 2 ofthe IL-IA gene ⁵ Moderate vs. Mild Odds ratio: 2.57( 1.10 - 6.00) />=0.028

^J Moderate vs. Mild Odds ration: 0.345 (0.146 - 0.813) p=0.0\ 4. Example 2. Determination of TNFα in gingival tissue.

This is a prophetic example. In order to demonstrate the role of TNFα in individuals susceptible to severe periodontitis, we perform the following experiment. Our objective is to determine if the IL-1 genotype results in elevated TNFα levels in gingival tissues. The levels of IL-l α, IL-lβ, IL-1 receptor antagonist (IL-lra), and TNFα are measured in gingival tissue from patients who are positive for the IL-1 genotype and from patients who are negative for the genotype. Patients who are referred to the Periodontics Clinic are selected based on the following criteria: 1) diagnosis of chronic adult periodontitis (this is defined as probing depth of ≥ 5mm at 2 sites in each quadrant with radiographic evidence of loss of alveolar bone); 2) between the ages of 35 and 55 years; 3) a minimum of 18 natural teeth; and 4) have never smoked or have quit smoking more than 5 years previous to the date of the examination and have a pack- year history of < 10 (pack-year history is calculated by multiplying the number of years smoked by the average number of packs smoked per day).

The exclusion criteria include: 1) pregnancy or lactation: 2) diabetes; 3) HIV infection; 4) bleeding disorders; 5) immunosuppressive chemotherapy; 6) severely compromised immune function; 7) any condition necessitating antibiotic pre-medication for dental appointments; 8) chronic usage for more than three months of more than an average of 325 mg of aspirin or non- steroidal anti-inflammatory drugs per day (the participants history is reviewed for a period of three years preceding the date ofthe clinical examination used to determined clinical criteria for entry into the trial); and 9) continual daily use of antibiotics for at least two month's duration within the five years preceding the date ofthe examination, or more than four episodes of antibiotic therapy in any one year period within the two years preceding the date of the examination. After meeting entry criteria, patients are selected.

Blood samples for each patient are collected on DNAase-free and RNAase-free blotting paper cards (Amplicard™) (University of Sheffield, Sheffield, England SI O 2JF), also described in Tarlow JW, et al. Journal of Investigative Dermatology 1994: 103: 387-389, incoφorated by reference herein. The samples are encoded with a number to maintain masking of the investigators.

Gingival crevicular fluid (GCF) samples are collected from 8 teeth in contralateral quadrants. GCF is collected following isolation of the teeth in the region to be sampled.

Supragingival plaque is gently removed, and small precut filter paper strips are inserted from the buccal and lingual aspects along the mesial surface of each of the teeth. These strips are angled to meet at the midpoint of the mesial surface, left in place for 30 seconds and removed to microcentrifuge tubes containing 50 μl of 1% bovine serum albumin in phosphate buffered saline/Tween20. The samples are eluted and analyzed for IL-1 β and TNF-α. The samples from the two strips from each tooth surface are combined and analyzed as the sample from that tooth.

After collection of GCF, local anesthesia is administered on the buccal and lingual/palatal surface in the area ofthe first molar - second bicuspid. An interdental tissue biopsy between the first molar and second bicuspid is taken by means of a horizontal incision coronal to the alveolar crest that is carried from the buccal surface through to the lingual/palatal surface. A solid triangular piece of gingival tissue is removed and placed into a formalin-containing plastic sample jar.

The gingival tissue samples from the biopsies are digested and total DNA is quantitated. Tissue proteins are also extracted and assayed by enzyme linked immunoabsoφtion (ELISA) for quantity of IL-lα. IL-lβ and TNFα. Quantity of each cytokine in each sample is expressed as nanograms protein per nanogram of total DNA in the tissue sample, to standardize the size of the samples.

The results are shown in Tables 5, 6 and 7. Table 5 shows the IL-1 genotypes and IL-β concentrations. Table 6 shows the IL-1 genotypes and IL-lα concentrations. Table 7 shows the IL-1 genotypes and TNFα production in gingival tissues. In the following tables, the "+" signs under the concentration column indicate the expected IL or TNF concentrations, with a single "+" indicating a low concentration and a plurality of "+" signs indicating higher concentrations.

TABLE 5 IL-1 genotypes and IL-lβ production in the gingival tissues

IL-IB + 3953 IL-1A IL-l β cone.

-889 (or + 4845)

22 any mix +++

12 12 or 22 ++

12 1 1 +

1 1 any mix + TABLE 6 IL-1 genotypes and IL-lα production in the gingival tissues

IL-IB + 3953 IL-IA IL- 1 α cone.

-889 (or + 4845) any mix 22 ++++

12 or 22 12 + to ++

1 1 12 + 10 ++ any mix 1 1 + to ++

TABLE 7 IL-1 genotypes and TNFα production in the gingival tissues

IL-IB + 3953 IL-IA TNFα cone.

-889 (or + 4845) any mix 22 ++

22 any mix ++

12 12 + to ++

1 1 or 12 1 1 +

1 1 1 1 or 12 +

It is to be expected that IL-1 tissue levels are higher in the IL- 1 genotype positive patients than in the IL-1 negatives. The fact that TNFα levels are also higher shows that the IL-1 genotype is also secondarily amplifying TNFα, most likely through paracrine mechanisms.

Example 3 (Prophetic). Effects of Blockage of TNFα on Periodontitis This is a prophetic example. To show that TNFα is the critical step in tissue destruction in periodontitis, we propose the following experiment. Our objective is to determine effects of specifically blocking TNFα on periodontitis.

Periodontitis is induced in a well-defined rat model system as previously described

(Golub et al. , Annals ofthe New York Academy of Sciences. 732 : 96- 1 1 1 , Sep 6 , 1994 and Chang et al, Research Communications in Molecular Pathology & Pharmacology. Mar: 9] (3):303-18,

1996). Sprague-Dawley "Specific Pathogen Free" rats are infected with the bacterium Porphyromonas gingivalis (Pg). After confirmation of successful infection, the animals are randomized to the following groups:

Group 1 : no Pg infection

Group 2: Pg infection + no treatment

Group 3: Pg infection + block IL-1

Group 4: Pg infection + block TNFα

Group 5: Pg infection + block IL-1 and TNFα Specific blocking agents for the rat cytokines, IL-l α and β, and TNFα are used (Kiaidi et al., American Surgeon. 61(7):569-72, 1995). Six weeks after Pg infection, gingival tissue samples are taken by biopsy and assayed for the quantitative expression of IL-1 and TNFα in the tissues. This analysis is performed by extracting the proteins from the tissue samples and assaying by enzyme linked immunoabsoφtion (ELISA) for quantity of IL-lα, IL-l β, and TNFα.

Quantity of each cytokine in each sample is expressed as nanograms protein per nanogram of total DNA in the tissue sample, to standarize the size ofthe samples. The animals are sacrificed and the mandibles are assessed by histomoφhometry for bone level and number of osteoclasts.

The following results are expected:

Tissue levels Experimental Group Bone loss IL-lα IL-lβ TNFα

Group 1 : no Pg infection Group 2: Pg infection + no treatment +++ +++ +++ +++ Group 3: Pg infection + block IL-1 ++ ++ ++ ++ Group 4: Pg infection + block TNFα +/- + + + Group 5: Pg infection + block IL-1 and TNFα +/- + + +

Throughout this application various publications and patents are referenced. Full citations for the referenced publications and patents not included herein above are listed below. The disclosures of these publications in their entireties are hereby incoφorated by reference into this application in order to more fully describe the state ofthe art to which this invention pertains. The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations ofthe present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Many other variations and modifications may be made in the methods herein described, by those having experience in this art, without departing from the concept of the present invention. Accordingly, it should be clearly understood that the methods described in the foregoing description are illustrative only, and not intended as a limitation on the scope of the invention.

Claims

CLAIMS What is claimed is:

1. A method of treating periodontal disease, comprising the steps of identifying a patient with periodontal disease; and administering to the patient an antagonist of Tumor Necrosis Factor α (TNFα).

2. The method of claim 1, wherein said periodontal disease is selected from the group consisting of periodontitis and gingivitis.

3. The method of claim 1 , wherein said antagonist of TNFα is an antibody which binds to TNFα.

4. The method of claim 1, wherein said antagonist of TNFα is a soluble receptor which binds to TNFα.

5. The method of claim 1 , wherein said soluble receptor is recombinant.

6. The method of claim 1, wherein said antagonist of TNFα is an agent which binds to the cellular receptor for TNFα, thereby blocking binding of TNFα to said cellular receptor.

7. The method of claim 1 , wherein said antagonist of TNF α is interlukin-1 receptor antagonist.

8. A method of treating periodontal disease comprising administering to a patient having periodontal disease a pharmaceutically acceptable amount of an antagonist of Tumor

Necrosis Factor α (TNFα).

9. The method of claim 8, wherein said periodontal disease is selected from the group consisting of periodontitis and gingivitis.

10. The method of claim 8, wherein said antagonist of TNFα is an antibody which binds to TNFα.

1 1. The method of claim 8, wherein said antagonist of TNFα is a soluble receptor which binds to TNFα.

12. The method of claim 1 1 , wherein said soluble receptor is recombinant.

13. The method of claim 8, wherein said antagonist of TNFα is an agent which binds to the cellular receptor for TNFα, thereby blocking binding of TNFα to said cellular receptor.

14. The method of claim 8, wherein said antagonist of TNF α is interlukin-1 receptor antagonist.