Download FIG 7: gingival index

Dottorato in Biomateriali Ciclo XVIII
Facoltà di Chimica Industriale
Università degli Studi di Pisa
President: Prof. Emo Chiellini
Third year 2004-2005
Treatment of the gingival hyperplasia with
metronidazole gel (Elyzol) in heart transplant
patients treated with cyclosporine A.
Dora Servidio
INTRODUCTION
The cyclosporine (CsA)-induced gingival overgrowth (GO) is a widely
documented side effect (Montebugnoli et al. 1996, Seymour et al. 2000).
Among the methods used to treat this clinical manifestation, the nonsurgical periodontal treatment associated with plaque control provided
controversial results (McGaw et al. 1987, Thomason et al.1993, Thomason
et al. 1995, Thomason et al. 1996, Somacarrera et al.1994, King et al. 1993,
Kantarci et al. 1999, Pernu et al.1992, Ellis et al. 1999, Daley et al 1991,
Stone et al 1991, Montebugnoli et al. 2000, Seymour et al.1987,
Montebugnoli et al. 1996, Hefti et al 1994, Schultz et al. 1990, Wondimu et
al 1993).
Interesting results have been obtained using metronidazole systemically.
(Wong et al. 1994, Cecchin et al. 1997); this drug, however, interferes with
the cyclosporine metabolism, thus contra-indicating the simultaneous
administration of the two molecules.
At present metronidazole is also available as mucoadhesive gel, which
allows to reach appropriate local concentrations without the above
mentioned systemic side effects.
The aim of this research is to evaluate the efficacy of locally-delivered
metronidazole used in association with the therapy of GO in heart
transplant patients treated with cyclosporine.
2
CHAPTER 1
1.1 Cyclosporine-induced gingival overgrowth
Cyclosporine is a cyclical hydrophobic endecaptide which derives from the
metabolic products of two species of fungi: the Trichoderma polysporum
and the Cylindrocarpon lucidum. The antimicrobial activity of the
cyclosporine is extremely weak but, as it had been shown since the first
studies, the compound had a remarkable inhibitory effect on the
lymphocytic proliferation (Borel et al. 1976). Since the discovery of its
immunosuppressive
properties
several
studies
have
shown
that
cyclosporine affects T lymphocytes selectively, while it has a minimal or
even no influence on B lymphocytes (Britton & Palacios 1982, Hess &
Colombani 1987).
The main use of cyclosporine is the prevention of the rejection of the organ
transplantation; moreover, cyclosporine is used in the treatment of several
autoimmune disorders. It is currently still proved that the therapy with
cyclosporine is associated with various side effects and, particularly, with
gingival overgrowth.
It is still unclear why not all the patients treated with cyclosporine A
develop gingival overgrowth, therefore it is possible to distinguish between
“responders” and “non-responders” patients according to the manifestation
or not of gingival overgrowth (McGaw et al. 1987, Seymour et al. 1991,
Montebugnoli et al.1996).
This individual variability in showing the gingival modifications seems to
be connected with a genetic predisposition, which is linked to certain HLA
phenotypes (Cebeci et al.1996, Thomason et al. 1996).
3
1.2 CYCLOSPORINE A
1.2.1 Pharmacokinetics
Cyclosporine can be taken orally, intramuscularly or intravenously. After
oral administration the drug is absorbed by the gastrointestinal tract in
quantities which may vary according to the patient.
The haematic peak is reached after 3-4 hours after the administration and
its half-life lasts approximately 17-40 hours (Beveridge et al. 1981).
Cyclosporine is totally hepatically metabolized by the enzymatic system
P450 mono-oxygenase (Maurer 1985). The metabolism entails the Ndemethylation, the hydroxylation
and the cyclization of the drug. 14
metabolites of the cyclosporine have been identified and the structures of 9
of them have been codified. In human beings the first metabolites produced
by cyclosporine are the 1,17 and the 21. Many of these metabolites are
excreted through the bile in the feces and only 10% of them are eliminated.
An oral dose of about 10-20 mg/Kg/die of cyclosporine is necessary to
obtain a plasmatic concentration of approximately 100-400 ng/ml in order
to maintain the immunosuppression.
4
1.2.2 Pharmacodynamics
Cyclosporine inhibits many of the processes involved in the immune
response given by the T lymphocytes; in particular, a concentration of
10/20 ng/ml inhibits the synthesis of interleukin-2 and this inhibition
limits the clonal amplification of the cytotoxic T lymphocytes. At even
higher concentrations (approx. A 100 ng/ml) the drug can inhibit the
response of the cytotoxic T lymphocytes to the interleukin-2. The
mechanism through which this effect occurs is not clear yet but it is
believed that the cyclosporine causes a block as regards receptors for the
interleukin-2 located on the T lymphocytes. On the contrary, the drug does
not seem to have a significant selective action against the T lymphocytes
(Hess et al. 1982). In this way the cyclosporine seems to have a selective
action against T lymphocytes: the T suppressor lymphocytes do not
undergo any effect, while the cytotoxic T lymphocytes and the T helper are
sensitive to the drug.
Such action selectivity might be due to the capacity of the cyclosporine to
bind to these cells in a specific way (Hess & Colombani 1987) in order to
be introduced inside the structure of the cell. At this level the drug binds to
numerous proteins, particularly to the calmodulin and the cyclophilin. The
former is involved in activating the T lymphocytes, while the function of
the latter is still unknown. The bond between cyclosporine and these
proteins is calcium-dependant (Colombani et al. 1985). The resistance or
sensitivity of the T lymphocytes towards the cyclosporine may depend on
the different intracellular concentration of such proteins (Hess &
Colombani 1987).
5
An increase of cyclophilin corresponds to an increase of its bond with
cyclosporine, thus diminishing the interaction between the drug and the
calmodulin; on the contrary, when the levels of cyclophilin decrease, the
bond between cyclosporine and calmodulin gets stronger, thus inhibiting
the action of stimulus for the activation of T lymphocytes.
6
1.2.3 Side effects
Besides the gingival overgrowth, cyclosporine is associated with other
numerous side effects. Precisely, it has been noticed: nephrotoxicity (Calne
et al. 1979a, Hamilton et al. 1982a, Graffenried et al. 1986), which is
reversible with drug interruption (Palestine et al. 1984, Hows et al. 1981);
hyperuricemia and hyperkaliemia (Adu et al. 1983, Chapman et al. 1985);
hypertension (Graffenried et al. 1986) which does not seem to be dosedependant (Hamilton et al. 1982b); hepatotoxicity (Calne et al. 1979b) also
reversible
with
therapy
interruption
(Laupacis
et
al.
1981);
lymphoproliferative disorders (Nagington & Gray 1980); increased
predisposition to bacterial, viral and fungal infections (Calne et al. 1978,
Fernando et al. 1980); increased incidence of thrombus-hemolytic
phenomena
(Vanrenterghem
et
al.
1985);
neurotoxicity
whose
manifestation are hand tremor, hyperesthesia e paresthesia; hypertrichosis
(Atkinson et al. 1984).
7
CHAPTER 2
2.1 Role of antibiotics in periodontology
At present there is no unanimous opinion concerning antibiotics efficacy in
the treatment of denstruens periodontal disease; very often chemiotherapics
are used when the response to conventional mechanical therapy is
inadequate and after periodontal surgery. The choice of a chemiotherapic,
as well as its posology and duration of assumption had been lately based
only on an empirical criterion, i.e. the clinical evaluation (Ellen et al 1996).
The first antibiotics chosen to treat the high-grade periodontitis were at first
the penicilline, then the tetracyclines, thanks to their broad spectrum of
antibacterial action and their remarkable tropism towards the crevicular
fluid. More recently new information have been acquired about the
bacterial specificity in certain forms of periodontitis (Loesche
1979,
Socransky 1977, Slots 1986, Christersson et al 1989), which allow a more
rational approach to the aim, which is to reduce or eliminate the specific
periodontal pathogens (Renvert et al 1990a, Renvert et al 1990b, van
Winkelhoff & de Graff 1991). As a result, thanks to the improvements in
cultural techniques, important differences have emerged, particularly from
the qualitative point of view, in the composition of the subgingival
microflora during destruens periodontitis. Such variability can be detected
both among different individuals, and also among samples taken by
different sites of the same subject.
It is therefore possible to conclude that periodontal disease can be
considered as a combination of diseases, which are all infective, but with a
different bacterial etiology, requiring different types of
treatments
(Socransky et al 1988, Haffajee et al 1988a, Haffejee et al 1988b).
8
A rational choice among the different therapeutic options implies the
knowledge of the microflora implicated in the etiology of the various forms
of periodontitis.
2.2 Assumptions of the antibiotic therapy
There are several reasons which favour the use of chemiotherapics in
periodontology as therapeutic aid: among these, the frequent confirmation
of patients who do not respond adequately to the therapy, showing a
progressive decrease of attack or a low tendency to recovery, despite both
domiciliary and professional maintenance therapy are good.
In 1983 Slots e Rosling firstly discovered that in many patients affected by
juvenile
periodontitis
associated
with
Actinobacillus
actinomycetemcomitans, the response to the mechanical conventional
treatment is very often negative, probably because the microorganism is
able to localize not only in the pocket epithelium, but also in the gingival
connective tissue and in the alveolar bone (Slots et al 1984, Gillett &
Johnson 1982, Christersson et al 1987).
Even in adults periodontitis, the Actinobacillus actinomycetemcomitans
seems to considerably withstand both the mechanical and the surgical
therapy (Renvert et al 1990a, Renvert et al 1990b). The instrumental
therapy probably favours the selection of periodontogenic bacteria, so that
the
Actinobacillus
actinomycetemcomitans
or
the
Porphiromonas
gingivalis persist in the lesions; this phenomenon can explain the low
predictability of the therapeutic results in these patients.
Moreover, some forms of periodontitis need to repeat subgingival
instrumentations to control the advancing of the disease ( Gordon et al
1990, Loesche et al 1991, Loesche et al 1992).
9
The role of antibiotics in preventing the transmission of periodontogenic
bacteria is not to be underestimated (van Steenbergen et al 1993, Preus et al
1993).
In order to have an efficient antibiotic therapy it is necessary to choose the
chemiotherapic drug according to the cultural test and the in vitro
sensitivity tests; moreover
the chemiotherapic must not cause early
resistance, furthermore the doses must be sufficient to maintain efficient
concentrations for an adequate period of time and, lastly, it must not
determine relevant side effects. In periodontology it is extremely important
that very high doses are used for a short time in order to control pathogens
and prevent the virulence of saprophyte species.
10
2.3 Systemic vs topic antibiotic therapy
In periodontology, both topic and systemic therapy have specific
advantages and disadvantages.
Systemically administered antibiotics reach more easily not only the
microorganisms in the sick sites but also the ones hidden in the periodontal
tissues and in the oral cavity. (Slots & Rosling 1983). This undoubtedly
means an advantage because it allows to prevent reinfection from
exogenous sources. On the other hand, the concentration of antibiotic
locally released in the periodontal pockets is about 100 times bigger than
the one which can be obtained through systemic administration.
In 1985 Goodson et al noticed, following to the application of monolithic
fibres, a subgingival concentration of tetracyclines equal to 643g/ml,
which is maintained for 10 days thanks to the affinity of this antibiotic with
the radicular surface (Baker et al. 1983, Bjornvatn et al.1985). After
systemic administration, on the contrary, the highest concentration of
tetracyclines in the crevicular fluid is equal to 8g/ml.
Moreover, the topical antibiotic therapy reduces the appearance of side
effects, of interaction among the possible drugs and requires less
cooperation from the patient.
However, it is a rather difficult process when the therapeutic agent has to
be applied in deep pockets and requires a remarkable expenditure of time,
particularly if the site to be treated are numerous. Further limits to the
topical antibiotic therapy are the impossibility to kill the microorganisms
located on the oral mucosa or the scarcely deep penetration in the tissues.
Furthermore, the ability to eliminate the key pathogens is scarcely
11
predictable, above all because in patients affected by juvenile periodontitis
they are located in the subgingival sites which do not show a clinically
relevant grade of periodontal destruction. These sites can therefore act as
reinfection sites.
12
CHAPTER 3
3.1 Metronidazole
Metronidazole is the result of an efficient tricomonicidal research done by
French researches in the 50s’ (Scully et al. 1988).
In 1962 Schinn observed that the administration of the drug to patients
affected by Trichimonas vaginalis infection and ulcerative gingivitis could
solve both pathologies.
After that Davies et al.(1964) realized that metronidazole was efficient
against the infections sustained by the spirochete and in the early 70s’ the
bactericidal power of the drug against the anaerobic bacteria was confirmed
(Tally et al. 1972).
More recent studies indicate a large use of the drug particularly in nonspecific vaginal infections and in the ones supported by a anaerobes and
parasites (Scully et al. 1988, Smilack et al. 1991, Rosenblatt et al 1987,
McEvoy et al. 1991, Finegold et al. 1990).
The use of metronidazole has been recently introduced in periodontology,
particularly in case of deep pockets or infections sustained by anaerobes
which do not respond to conventional therapy.
13
3.2 Chemistry and Pharmacokinetics
Metronidazole is a low molecular weight compound and it is not ionized at
physiologic pH. The antibiotic is rapidly and completely absorbed after an
oral administration: an hour after the assumption about the 80% of an oral
dose is absorbed. The speed of
absorption may be reduced by the
simultaneous food intake (McEvoy 1991, Bergen et al. 1984), which does
not influence the drug bioavailability, though.
The passage of the metronidazole inside the cells occurs through simple
diffusion: in this way the intercellular concentration rapidly approaches the
extracellular one.
The half-life of the unaltered metronidazole is about 7 hours and a half
and this half-life can, however, be altered in patients affected by liver
failure, though it remains unchanged in patients suffering from renal
failure.
The antibiotic is metabolized in the liver and mainly eliminated through
urines and feces. (Plaissance et al. 1988, Lau et al. 1987).
Thanks to its low molecular weight it reaches all tissues and fluids of the
body, including the cerebrospinal fluid, the saliva and the crevicular fluid
(Giedrys-Leeper et al.1985, Bergen et al. 1984, Lippincott 1990, Goodman
and Gilman 1990, Amon et al 1978). The concentration of metronidazole in
the creviculare fluid depends on the dose and the number of
administrations; normally the seric concentration of the drug and the
crevicular concentration are equivalent, however, Britt and Pohlod have
demonstrated that the crevicular concentration is half the seric one.
14
3.3 Mechanism of Action
There are electron-transport proteins inside the anaerobe bacteria and the
sensitive protozoa; they are similar to the ferredoxin, at low redox
potential, and they take parts in the reactions of the electrons’ metabolic
removal. The nitrogen group of the metronidazole is able to function as an
electron acceptor, by forming a reduced cytotoxic compound which bonds
with proteins and DNA, causing the cell’s death. Therefore, it is obvious
that the reduction is the main mechanism of the selective toxicity on
anaerobe cells.
On sensitive microorganisms metronidazole acts as a bactericide and its
activity spectrum, which is mostly limited to anaerobe bacteria and some
protozoa, does not facilitate the uncontrolled growth of resistant antibiotic
aerobes and facultative anaerobes. Moreover, metronidazole determines a
radiosensitive effect on the malignant cells which has been demonstrated
both in vitro, and, on rats, in vivo.
15
3.4 Antimicrobial Spectrum
Metronidazole is the select drug for the treatment of infections caused by
Entamoeba histolytica, Giardia lamblia and Trichimonas vaginalis, both in
men and women.
The antibiotic is widely used to treat infections provoked by anaerobes,
since already at low concentrations metronidazole acts as a bactericide for
some anaerobes, such as bacteroides, fusobacteria and treponema (Sutter et
al. 1983, Wade et al 1987, Walker 1985); moreover, it is efficient against
most negative G anaerobe bacilli (A).
The drug seems not to be active against aerobes, facultative
microorganisms and microaerophilic ones, while its efficacy against
Actinobacillus actinomycetemcomitans and the Gram-positive coccus is
controversial (Sutter et al. 1983, Wade et al.1987, Walker et al. 1985).
The antibiotic resistance does not represent a therapeutic problem
(Dubreuil et al.1989, Bourgault et al.1986, Tally et al 1984), although
strains of T. vaginalis and B. fragilis resistant to metronidazole after longduration therapies have been described (Sprott et al. 1983, Musial et al.
1987, Dombrowsky et al. 1987).
16
3.5 Side effects
The less important side effects related to the use of metronidazole include
metallic taste, glossitis, oral candidiasis, nausea, vomit and the urticaria
(Mc Evoy et al. 1987, Lippincott 1991, Goodman and Gilman 1990).
The serious toxic effects are uncommon; however, neurotoxic effects, such
as vertigo, ataxy and leucopenia are reported.
In general, short–duration therapies with metronidazole are well tolerated;
however, some side effects which are completely reversible to the drug
suspension might occur.
The assumption of antibiotic together with alcohol, may cause a disulfiramlike effect characterized by nausea and vomit.
Moreover, it has been demonstrated that metronidazole potentiates the
anticoagulant effects of the coumarin-like substances.
Besides, the drug and its metabolites have shown to be mutagenic on
certain strains of Salmonella typhimurium (Ames test). The oral chronic
administration of very high doses has produced in rats a statistically
significant increase of liver and lungs tumor. In human beings, on the
contrary, no congenital anomalies, intrauterine deaths or births of under
weight babies have been detected.
The cultures on human lymphocytes in presence of metronidazole up to
10.000 micro g/ml have not highlighted any toxic activity and, as regards
patients who have received a high dose of drug for an amebic hepatitis, no
increase of the frequency of chromosome aberrations has been detected.
However, it is good practice to avoid the administration of the drug for
long periods of time.
17
CHAPTER 4
4.1 Pharmacological properties of Elyzol 25%
4.2 Preparations
Local use of antibiotics in periodontal treatment has attracted wide interest.
The drug has been applied directly into the periodontal pockets either by a
non-degradable or a degradable carrier.
However, a non degradable carrier is less acceptable for clinical use,
because it has to be removed, after release of the active agent.
A metronidazole 25% dental gel (Elyzol ), which will vanish from the
pockets has been manufactured and developed by CABON s.p.a.
The gel consist of a semi-solid suspension of metronidazole benzoate in a
mixture of glyceryl mono-oleate (GMO) and triglyceride (sesame oil).
It will flow freely when applied to the pockets. In contact with the gingival
crevicular fluid (GCF) highly viscous liquid crystals are spontaneously
formed in the gel.
This prevents the gel from being easily expelled from the pockets. The
sparingly soluble metronidazole benzoate is released by break down of the
gel matrix (by lipases) and by diffusion of dissolved metronidazole
benzoate.
Metronidazole benzoate is subsequently hydrolyses into metronidazole by
esterases that are present in the GCF.
The free metronidazole may be absorbed directly from the pockets or be
discharged and then absorbed from the gastro-intestinal tract.
The time to reach peak plasma concentration varies from 2 to 8 h, the
concentration at 48 h is very low. Metronidazole can found in plasma
18
samples 30 h after application of gel, in 93% after 36 h, in 33% after 48 h
and finally in 9% after 72 h.
The bioavailability of oral metronidazole approaches 100% but values of
bioavailability as low as 56% were observed by Rabin et al. (1980).
The systemic load after one application of gel is not likely to exceed that
seen after a 250 mg metronidazole tablet. Further, high concentration of
metronidazole can be obtained in the periodontal pockets without inducing
high plasma concentrations.
ELYZOL25% dental gel contains metronidazole in the form of benzoate as
active principle and it is a proprietary medicine conceived to be applied in
the periodontal pocket.
After the application the preparation becomes more fluid by filling the
pocket homogeneously and, when in touch with the crevicular fluid, it
forms a highly viscous gel which gradually releases metronidazole.
Metronidazole is an active antibiotic against the gram-microorganisms
which are mainly located in the sub-gingival flora in the adult periodontitis.
It has an antibacterial effect against Bacteroides spp., Fusobacteria,
Selenomonas, Wolinella, Spirochetes and other obligate anaerobic
organisms, while it is still not active against the aerobic bacteria.
Also
some
anaerobic
bacteria,
such
as
Actinobacillus
actinomycetencomitans are sensitive to concentrations of metronidazole
obtained after the local application of ELYZOL25% dental gel.
19
4.3 Pharmacokinetic Properties
After the application ELYZOL 25% dental gel concentrations of
metronidazole of about 100mg/ml have been measured in the periodontal
pocket for at least 8 hours and bigger concentrations than 1mg/ml after 36
hours. The mucosa absorbs the metronidazole which is gradually released
by the dental gel: the bioavailability is about 70%. The plasmatic
concentration reaches the maximum value after about 4 hours.
Systemic concentrations bigger than 1.3mg/ml have not been measured.
4.4 Pre-clinical safety data
Metronidazole, as well as benzoate metronidazole, is a well known active
principle, and it has already been used for a long time for systemic use.
For this reason the preclinical studies of the product ELYZOL25% dental
gel were mainly focused on the aspects connected with the local application
of the formulation.
The studies regarding the irritation of the oral mucosa have shown, on the
whole, no reactions of local sensitization.
4.5 Pharmaceutical information
1g of gel contains
Excipients:

Glyceryl monooleate 518mg

Sesame oil
71mg
20
4.6 Incompatibility.
No cases of chemical-physical incompatibility with other substances are
known.
4.7 Term.
36 months. The use-by date indicated on the packet refers to the intact, well
preserved product.
21
SPERIMENTAL PART
5.1 Material and method
A prospectic intra-subject double-blind longitudinal study was performed
with placebo, as regards the effects on periodontal area of metronidazole
associated with detartrasis. 6 patients (5 male, 1 female) aged between 14
and 67 (average age 45.3+20.5) who had undergone a heart transplant in a
period ranging from 22 to 77 months (average time 56.5 + 25.9) were
studied.
All patients were chosen at random in the group of patients with enhanced
gingival volume and with hyperplastic index (HI) >30 (RESPONDERS).
The evaluation of this index had been used in a previous study
(Montebugnoli et al. 1996, Seymour at al. 1991).
In order to define the hyperplastic index, full-arches alginate impressions
were taken; the study samples of the 12 front elements were divided into 10
vestibular units and 10 lingual units (fig. 1). The hyperplastic index is
formed by two components: vertical (McGaw et al. 1987) and horizontal
(Seymour et al. 1985). The vertical component measures the increase of
gingival volume in apico-coronal sense in each gingival unit and it is
graduated in 4 points; the horizontal component measures the thickening
both in vestibular and in lingual direction of each gingival unit according to
3 points.
The maximum score which can be obtained by adding the two components
and all the 20 gingival units is 100 and the HI has been expressed as a
percentage on an arbitrary basis (Fig. 10).
Each subject has undergone two kinds of treatments: 1) metronidazole +
detartrasis and 2) placebo + detartrasis. The two front (upper and lower)
sextants were divided into four hemi-sextants: upper right and upper left
22
and lower right and lower left. Each treatment was performed in two
contralateral hemi-sextants (es: upper right and lower left, upper left and
lower right) and a balanced random pre-programmed list assigned each
patient a hemi-sextants and each kind of treatment (detartrasis +
metronidazole or detartrasis + placebo).
At first all patients underwent a detartrasis performed by same operator on
both upper and lower sextants; at the end of the session ELYZOL 25%
dental gel or placebo substance was applied for each patient on the gingival
sulci with a syringe originally designed by CABON in the hemi-sextants
assigned by the random list.
The pockets were filled until gel could be seen at the gingival margin.
The same gels were applied by the same operator and in the same hemisextants 7 days after the first application, as proposed by Klinge et
al.(1992).
Any gel above the gingival margin and overflow from the applicator was
carefully collected and transferred to a plastic container.
After this procedere, the patiens were asked to spit in the container to
remove any further excess gel from the oral cavity.
The total amount of metronidazole used during applications varied from 72
to 256 mg. Between 35 and 153 mg of metronidazole was collected after
application resulting in an actual dose of metronidazole of 29-103 mg per
patient.
The average actual dose of metronidazole per tooth varied between 2 and 5
mg.
The peak plasma concentration (c max)after gel administration ranges from
223 to 1303 ng/ml.
After each application each patient was warned not to drink and eat for at
least two hours.
23
The patients were recalled 1,2,3,4 months after the second application and
an hour before each detartrasis session each patient received
2 g of
amoxicillin as antibiotical prophylaxis against the bacterial endocarditis.
During the first appointment before the detartrasis session and during the
next appointments another operator, different from the one who had
performed the applications, determined what follows:
 Plaque index (PI) according to the system set by Silness & Loe (1964)
 Bleeding index according to the system set by Ainamo & Bay (1975)
 Gingival index (GI) according to the system set by Silness & Loe (1963)
 Periodontal survey performed using a periodontal probe (P.C.P. Hu friedy). All the measuring had been performed starting from the gingival
ridge in 6 points around each dental element.
 Hyperplastic index (HI)
During the first and the next appointments patients were given dental care
instructions.
24
5.2 Statistical evaluation
For the statistical analysis ANOVA of multiple regression for repeated
measures with split plot design was used to evaluate difference between
treatments, plaque index, gingival index between the two groups
(detartrasis + placebo e detartrasis + metronidazole), in relation to the time
inside each group and the interaction between groups and time. The
Bonferroni t-test was applied as a multiple comparison t-test.
As far as bleeding index is concerned, Chi square
analysis of the
contingency tables was used to evaluate significant differences between the
two groups and the Mc Nemar test was used to evaluate the differences as
regards time.
25
5.3 Results
Fig. 1 shows the time profile of the periodontal probing expressed in mm
(average + DS) in the two groups of dental elements (one treated with
detartrasis + metronidazole
and the other with detartrasis + placebo)
belonging to the 6 patients who completed the study after 2 months. Table
1 refers to the relevant statistical analysis of the differences.
Significant F values were detected with reference to the “patient” variable
(the variability of periodontal probings is wide within each group) and to
the “moment” variable (the values of the periodontal values after 1 month
and after 2 months are significantly inferior to the basal ones; no significant
difference exists between the values detected after the 1° month and the
ones detected after the 2° month).
Non-significant F values were detected as regards the “treatment” variable
and the “treatment x moment” interaction (no significant difference exists
between the two groups as regards the decrease of periodontal probing and
between basline condition after 1 month condition).
Similar results were assessed by the statistical analysis relevant to the
variation of plaque index in the two groups of elements in the same 6
patients analyzed after two months (fig. 2 and table 2).
Different results were recorded as regards the evaluation of gingival index
(Fig. 3, Table 3). Significant F values were detected not only for the
“patients” and “moment” variables but also for the “treatment X moment”
variable (the decrease of values concerning periodontal index between
baseline conditions and after 1 month conditions is significantly higher in
the elements treated with detartrasis + metronidazole compared to the
elements treated with detartrasis + placebo).
Fig. 4 and Table 4 show the percentage of elements with positive bleeding
index in the two treatment groups. Statistically significant values were
26
detected after one month with respect to the baseline values; no significant
difference exists between the values recorded after 1° month and the one
recorded after 2° month. Moreover, no significant difference was detected
between the two treatment groups.
Fig. 5 shows the time profile of the periodontal probing in the two groups
of dental elements of the 4 patients who completed the study after 4
months (two out of the 6 patients had to abandon the study due to heart
complications). Table 5 makes reference to the statistical analysis of the
differences.
Once again, significant F values were recorded as regards the “patient” and
“moment” variables, while, with respect to the analysis performed on 6
patients after 2 months, a significant value appears to be also for the
“treatment x moment” interaction (after 4 months,
the values of the
periodontal probing in the group of elements treated with detartrasis +
placebo significantly increase compared to the values recorded after 3
months and they are not more different than the baseline ones; After 4
months there is a significant difference between the probings recorded in
the group of elements treated with detartrasis + placebo compared to the
group treated with detartrasis + metronidazole ).
As regards the other periodontal evaluation indexes, plaque index (Fig. 6
and Table 6), gingival index (Fig. 7 and Table 7) and bleeding index (Fig. 8
and Table 8) the statistical analysis reported the following profile: in all
indexes a significant decrease, statistically not different between the two
groups with similar values 2,3,4 months after the treatment, was detected.
As regards the hyperplastic index, it never fell under 30% during the whole
study (Table 9).
27
5.4 Discussion
In the course of GO treatment the most used procedures make reference to
the periodontal non-surgical therapy (scaling e root planing) associated
with plaque control and aiming to reduce the gingival inflammation
(Somacarrera et al. 1994, Kantarci et al 1999).
The results of our study are in accordance with Somacarrera and Kantarci’s
ones: after one month from periodontal treatment the gingival volume has
already decreased significantly together with all the parameters connected
with the inflammation. The gingival conditions reached after the first
month have been unaltered for another month in the group with 6 patients
studied for 2 months and for at least other 2 months in the group of 4
patients followed for 4 months.
These results highlight that the gingival inflammation plays an important
role in the gingival overgrowth which occurs during therapy with
cyclosporine and probably enters the mechanism of pathogenesis of this
pathology, as proposed by some authors (Montebugnoli et al. 2000,
McGaw et al. 1987, Thomason etal.1993, Thomason et al. 1995,
Thomason et al. 1996, Ellis et al1999, Kantarci et al. 1999, Pernu et al.
1992, Daley et al.1991, Stone et al. 1991, Somacarrera et al. 1994, King et
al. 1993). What is, however, underlined by the present study is that the
decrease of the inflammation parameter significantly influences the grade
of gingival volume.
In addition to the non-surgical periodontal therapy procedures, the use of
metronidazole has been as well introduced among the strategies set in the
last years to control this parameter in the GO therapy; Wong et al (1994)
and Cecchin et al. (1997) showed that metronidazole assumed systemically
(400 mg/die x 7 days in the first study and 750 mg/3die x 14 days in the
second study) is efficient as regard the control of cyclolsporineinduced GO,
28
thus underlining once more that the reduction of the inflammation through
the antimicrobial action of the drug plays an important role in controlling
the gingival volume.
However, systemically taken metronidazole has an unpleasant side effect: it
inhibits the hepatic enzyme P-450, which is essential for the metabolism of
cyclosporine. Therefore, the attendant assumption of metronidazole and
cyclosporine might cause the increase of the plasmatic concentrations of
the latter, thus causing big problems connected with CsA’s side effects.
In recent years antibiotic has been available in dental gel form, thus
allowing to reach adequate local concentrations without the above
mentioned systemic effects.
The efficacy of metronidazole applied locally in patients affected by
periodontitis has often turned out to be contradictory.
Stelzel and Flores de Jacoby (1996,1997) showed that metronidazole alone
does not produce higher results compared to scaling; these data are
confirmed by other studies (Klinge et al.1992, Pedrazzoli et al.1992,
Ainamo et al.1992).
But other authors highlighted that administration of metronidazole
associated with detartrasis gives better results compared to detartrasis
alone.
Noyan et al.(1997) showed that scaling and root planing associated with the
systemic assumption of metronidazole or the topical application of gel are
more efficient than only scaling or than the gel application alone in
determining a significant clinical and microbiological improvement after
42 days from the treatment. These results have been confirmed also by
other authors (Radvar et al. 1996, Hitzig et al. 1994, Loesche et al. 1984,
Loesche et al. 1991, Korman et al. 1989, Van Winkelhoff et al. 1989, Van
Winkelhoff et al. 1992, Pavicic et al.1994, Berglundh et al 1998).
29
On the other hand, other studies showed that metronidazole, when
associated with detartrasis, does not produce results which are better than
detartrasis alone.
Awartani et al. (1998) showed that in presence of pockets with probing
higher than 4,5 mm both detartrasis and metronidazole 25%, and detartrasis
associated with
metronidazolo 25% determine a significant probing
decrease after 4 weeks with respect to the baseline values, with no
significant difference among the three types of treatments. Similar results
were obtained also by other authors (Rudhart et al.1998, Palmer et al.
1998, Lie et al. 1998, Linden- Newman et al. 1991).
The results of the present study are in accordance with Awartani’s ones and
point out that the local therapy with metronidazole associated with
detartrasis in the GO treatment does not produce significant advantages in
the reduction of gingival volume with respect to detartrasis alone.
The decrease of gingival volume and of the parameters connected with the
periodontal inflammation shows a similar trend both in the group of
patients followed for two months and treated with metronidazole associated
with detartrasis and in the group treated with detartrasis only, if we exclude
a slight difference in favour of metronidazole as regards the IG parameter
after one month.
However, the results obtained in the small group of patients followed for 4
months show a significant difference in the control of gingival volume
between the two kinds of treatments: in the group with dental elements
treated with only detartrasis gingival volume shows a significant increase 3
months after the treatment and the values recorded after 4 months seem to
be significantly higher than the ones recorded in the group of the elements
treated with metronidazole associated with detartrasis.
This datum may mean that metronidazole determines an effect which lasts
long, even if in the short term its effect is equal to the one which is
30
obtained through detartrasis; however, it is necessary to consider that this
datum might also be influenced by the small number of patients considered.
Stelzel and Flores de Jacoby (1997) showed that the effect of
metronidazole alone in periodontopatic patients does not produce results
which are higher than the long-term scaling.
On the other hand, Berglundh et al. (1998) pointed out that the systemic
assumption of metronidazole and amoxicillin associated to detartrasis is
more efficient compared to detartrasis alone when there are deep pockets
because it brings in the long term a higher improvement of clinical attack.
A similar result was assessed also by Hitzig et al. (1994) who highlighted
with time a better efficacy of metronidazole compared to detartrasis in
reducing the bleeding, the probing and improving the clinical
attack.
Literature reports that other studies show a higher efficacy in the long term
than other chemiotherapics, different from metronidazole and clorexidine,
with respect to the detartrasis (Killoy et al. 1995a, Killoy et al.1996,
Stabholtz et al. 1991).
In conclusion, the results of the present study show that the reduction of
gingival inflammation reduces significantly after one month already the
extent of gingival overgrowth in patients who underwent a therapy with
cyclosporine.
Similar results have been obtained through treatment of patients with
detartrasis or with detartrasis associated with application of metronidazole
gel. After both treatments the gingival volume remains unaltered for at
least 3 months.
After 4 months, however, it seems that the treatments with metronidazole
associated with detartrasis are more efficient in controlling GO compared
to the treatment with only detartrasis.
31
TABLES
32
Table 1: periodontal probing
2 months
6 patients
Detartrasis+elyzol
Basal
3.87 
1.5
NS
Detartrasis+placebo 3.82 
1.6
Treatment
Patients
Moments
Treatment x moment
1 month
P<.01 3.21
1.1
NS
P<.01 3.35 
1.2
F
1.5
14.1
15.9
1.7
NS
2 months
3.381.1
NS
NS
3.55  1.1
P
NS
P<.01
P<.01
NS
33
Table 2: plax index
2 months
6 patients
Detartrasis+elyzol
Detartrasis+placebo
Basal
.62 
.70
NS
.66 
.71
Treatment
Patients
Moments
Treatment x moment
1 month
P<.05 .51 .73
NS
.43 
.67
P<.05
F
.8
6.9
5.3
.3
NS
2 momths
.52.74
NS
NS
.50  .71
P
NS
P<.01
P<.05
NS
34
Tab 3: gingival index
2 months
6 patients
Detartrasis+elyzol
Detartrasis+placebo
Basal
1.65 
.50
NS
1.55 
.51
Treatment
Patients
Moment
Treatment x moment
1 month
P<.01 .83 .73
P<.01
P<.05
1.01 
.67
F
.4
6.9
106.9
4.4
NS
2 months
1.03.74
NS
NS
1.09  .71
P
NS
P<.01
P<.01
P<.05
35
Tab 4: bleeding index
2 months
6 patients
Detartrasis+elyzol
Detartrasis+placebo
Basal
64%
NS
53%
1 month
P<.01
18%
NS
P<.01
18%
NS
NS
2 months
18%
NS
24%
36
Tab 5: periodontal probing
4 months
4 patients
Basal
Detartrasis
+ elyzol
3.96
P<.01

1.6
NS
3.97
P<.01

1.7
Detartrasis
+ placebo
Treatment
Patients
Moment
Treatment x moment
1
month
3.20

1.1
NS
3.39

1.2
NS
NS
2
month
s
3.23

1.1
NS
3.44

1.2
F
2.06
35.9
9.9
5.5
NS
NS
3
month
s
3.16

1.1
NS
3.42

1.2
NS
NS
4
month
s
3.32

1.2
P<.05
3.65*

1.3
P
NS
P<.01
P<.01
P<.05
37
Tab 6: plax index
4 months
4 patients
Detartrasis
+ elyzol
Detartrasis
+ placebo
Basal
.75
P<.05

.76
NS
.79
P<.05

.74
Treatment
Patients
Moment
Treatment x moment
1
month
.60

.81
NS
.52

.74
NS
NS
2
month
s
.60

.73
NS
.44

.68
F
.7
9.7
3.6
0.6
NS
NS
3
month
s
.56

.76
NS
.44

.74
4
months
NS
NS
.52

.65
NS
.50

.71
P
NS
P<.01
P<.05
NS
38
Tab 7: gingival index
4 months
4 patients
Basal
Detartrasis
+ elyzol
1.70
P<.01

.51
NS
1.60
P<.01

.64
Detartrasis
+ placebo
Treatment
Patients
Moment
Treatment x moment
1
month
.70

.61
NS
.89

.74
NS
NS
2
month
s
.83

.53
NS
.92

.60
F
.8
10.9
73.4
0.5
NS
NS
3
month
s
.75

.60
NS
.83

.64
NS
NS
4
month
s
.79

.65
NS
.77

.71
P
NS
P<.01
P<.01
NS
39
Tab 8: bleeding index
4 months
4 patients
Basal
Detartrasis
+ elyzol
69% P<.01
10%
NS
NS
Detartrasis
+ placebo
56%
1
month
P<.01
10%
2
month
s
NS
4%
3
month
s
NS
NS
NS
10%
10%
4
month
s
NS
NS
NS
10%
10%
NS
NS
15%
40
Tab 9: hyperplastic index
Patients
1
2
3
4
5
6
Basal
64
42
56
59
84
49
1 month
58
30
49
43
64
45
2 months
57
30
53
44
71
53
3 months
4 months
37
47
46
62
32
42
52
71
41
FIGURES
42
mm
5
4,5
**
4
3,5
3
**
2,5
2
0
Basal
Metronidazole+ Detartrasis
1 Month
2 Months
.
Placebo + Detartrasis
Fig. 1: periodontal probing expressed in mm (average + DS) in
the two groups of dental elements (one treated with detartrasis +
metronidazole and the other with detartrasis + placebo) belonging
to the 6 patients who completed the study after 2 months.
** P<.01
43
1
0,8
*
0,6
0,4
*
0,2
0
0
Basal
1Month
Metronidazole+detartrasis
2Month
s
.
Placebo+detartrasis
FIG. 2: plaque index in the two groups of elements in the same 6
patients analyzed after two months
*P<.05
44
2
1,5
**
1
**
*
0,5
0
0
Basal
1 Month
Metronidazole + Detartrasis
2 Months
.
Placebo + Detartrasis
FIG 3: gingival index in the two groups of elements in the same 6
patients analyzed after two months
* P<.05
** P<.01
45
70
60
%
50
40
30
ns
**
20
10
0
BASALE
1 MESE
Metronidazole +detartrasis
2 MESE
Placebo+detartrasis
FIG 4: the percentage of elements with positive bleeding index in
the two treatment
**P<.01
NS: no significant difference
46
6
*
5
**
4
*
3
**
2
1
0
0
BASAL
1 Month
Metronidazole + detartrasis
2 Months
3 Months
4 Months
.
Placebo + detartrasis
FIG 5: time profile of the periodontal probing in the two groups
of dental elements of the 4 patients who completed the study after
4 months
*P<.05
47
1,2
1
*
0,8
0,6
*
0,4
0,2
0
0
BASAL
1
2
E
moME
MESE
Metronidazole+detartrasisI
SE
3
4
MESE
MESE
Placebo+detartrasis
.
FIG 6: plaque index
*P<.05
48
2
**
1,5
1
**
0,5
0
0
BASAL
1month
Metronidazole+placebo
2months
3months
4months
.
Placebo+detartrasis
FIG 7: gingival index
** P<.01
49
80
70
60
%
50
**
40
30
20
10
0
BASALE
BASAL
1 MESE
Metronidazole+detartrasis
2 MESE
3 MESE
4 MESE
Placebo+detartrasis
FIGRA 8: bleeding index
**P<.01
50
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SUMMARY
INTRODUCTION ……………………………………………………………..pag 2
CHAPTER 1
1.1 Cyclosporine-induced gingival overgrowth …………………………………pag 3
1.2 Cyclosporine A
1.2.1 Pharmacokinetics ……………………………………………………….pag 4
1.2.2 Pharmacodinamics ……………………………………………………...pag 5
1.2.3 Side effects ……………………………………………………………...pag 7
CHAPTER 2
2.1 Role of antibiotics in periodontology ………………………………………pag 8
2.2 Assumption of the antibiotic therapy ……………………………………….pag 9
2.3 Systemic vs topic antibiotic therapy ………………………………………..pag 11
CHAPTER 3
3.1 Metronidazole ………………………………………………………………pag 13
3.2 Chemistry and pharmacokinetics …………………………………………...pag 14
3.3 Mechanism of action ………………………………………………………..pag 15
3.4 Antimicrobial spectrum …………………………………………………….pag 16
3.5 Side effects ………………………………………………………………….pag 17
CHAPTER 4
4.1 Pharmacological properties of Elyzol 25% ………………………………pag 18
4.2 Preparations ………………………………………………………………pag 18
4.3 Pharmacokinetics properties ………………………………………………pag 20
4.4 Pre-clinical safety data …………………………………………………….pag 20
4.5 Pharmaceutical information ……………………………………………….pag 20
4.6 Incompatibility …………………………………………………………….pag 21
4.7 Term ……………………………………………………………………….pag 21
63
SPERIMENTAL PART
CHAPTER 5
5.1 Material and method ……………………………………………………….pag 22
5.2 Statistical evaluation ……………………………………………………….pag 25
5.3 Results ……………………………………………………………………...pag 26
5.4 Discussion ………………………………………………………………….pag 28
TABLES ………………………………………………………………………..pag 32
FIGURES ………………………………………………………………………pag 42
REFERENCES ………………………………………………………………pag 51
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