Malgorzata Kozak

Mateusz Kurzawski

Anna Wajda

Joanna Lapczuk

Mariusz Lipski

Krzysztof Dziewanowski

MD Marek ; Drozdzik
email: drozdzik@sci.pam.szczecin.pl

TGF-β1 gene polymorphism in renal

BACKGROUND: The incidence of gingival overgrowth among renal transplant patients treated with cyclosporine A ranges from 13 to 84.6%, and the overgrowth is not only esthetic but also medical problem. The study was aimed at determination of association between TGF-β1 gene (TGFB1) polymorphism and gingival overgrowth in kidney transplant patients medicated with cyclosporin A.

METHODS: 84 kidney transplant patients with gingival overgrowth and 140 control transplant patients without overgrowth were enrolled into the case control study. TGFB1 polymorphism was determined using the PCR-RFLP assay for +869T>C in codon 10 and +915G>C in codon 25 as well as TaqMan real-time PCR assays for promoter -800G>A and -509C>T SNPs.

RESULTS:

In kidney transplant patients suffering from gingival overgrowth mean score of gingival overgrowth was 1.38±0.60, whereas in control subjects was 0.0. The patients with gingival overgrowth were characterized by similar distribution of TGFB1 genotypes and allele in comparison to subjects without gingival overgrowth. Among 16 potentially possible haplotypes of TGFB1 gene, only four were observed in the studied sample of kidney transplant patients: G_C_T_G, G_T_C_G, G_C_C_C, and A_C_T_G, with similar frequency in patients with and without gingival overgrowth.

CONCLUSION:

No association between the TGFB1 gene polymorphism and gingival overgrowth was revealed in kidney transplant patients administered cyclosporine A. (INN ciclosporin), which has been widely used since 1970s. The incidence of gingival overgrowth among renal transplant patients treated with cyclosporine A ranges from 13 to 84.6% [START_REF] Somacarrera | Factors relating to the incidence and severity of cyclosporine-induced gingival overgrowth in transplant patients. A longitudinal study[END_REF][START_REF] Margiotta | Cyclosporine-and nifedipine-induced gingival overgrowth in renal transplant patients: correlations with periodontal and pharmacological parameters, and HLA-antigens[END_REF][START_REF] Pernu | Gingival overgrowth among renal transplant recipients related to immunosuppressive medication and possible local background factors[END_REF][START_REF] Afonso | Cyclosporine A-induced gingival overgrowth in renal transplant recipients[END_REF].

Gingival overgrowth induced by cyclosporine A is probably the consequence of alterations in the turnover of epithelial and connective tissues, resulting in an increase in structural proteins sometimes associated with epithelial thickening. Numerous studies have investigated potential risk factors in the development of the condition, including the extent of renal disease, interval since transplantation, duration of renal replacement therapy, dose of cyclosporine A, gingival inflammation, plaque indices and recipient HLA-phenotype. Whilst some studies have suggested some associations between the incidence and severity of gingival overgrowth and sex, pre-transplant diagnosis, age at transplantation, duration of therapy and cyclosporine dosage, others have failed to find any significant drug-related risk factors for the development of the disease as previously reported [START_REF] Seymour | The pathogenesis of drug-induced gingival overgrowth[END_REF][START_REF] Thomas | Risk factors in the development of cyclosporineinduced gingival overgrowth[END_REF][START_REF] Thomas | Cyclosporine A-induced gingival overgrowth is unrelated to allograft function in renal transplant recipients[END_REF][START_REF] Afonso | Cyclosporine A-induced gingival overgrowth in renal transplant recipients[END_REF].

However, it remains unclear why a proportion of patients are susceptible to gingival overgrowth while others remain unaffected.

It was hypothesized, that gingival overgrowth induced by cyclosporine results in a disturbance in the homeostatic balance which is characterized by an increase in both the number of fibroblasts and an increase in the volume of the extracellular matrix. This loss of growth control results in an accumulation of redundant tissue of relatively normal composition [START_REF] Mcgaw | Cyclosporin-induced gingival overgrowth: an ultrastructural 10 stereologic study[END_REF]. There is considerable information supporting the role for transforming growth factor-β1 (TGF-β1) in mediation the dysregulated fibroblast proliferation and extracellular matrix synthesis. Evidence from experimental studies demonstrate that gingival fibroblasts in culture exhibit a proliferative response to TGF-β1 [START_REF] Anderson | Effects of transforming growth factorbeta and platelet derived growth factor on human gingival fibroblasts grown in serum-containing and serum-free medium[END_REF][START_REF] James | Gingival fibroblast response to cyclosporine A and transforming factor beta 1[END_REF]. It was also reported that TGF-β1 suppresses extracellular matrix degradation via downregulation of metalloproteinases (MMP) production and inducing tissue inhibitor of MMP synthesis [START_REF] Edwards | Transforming growth factor beta modulates the expression of collagenase nad metalloproteinase inhibitor[END_REF][START_REF] Overall | Independent regulation of collagenaze 72-kDa progelatinase and metalloproteinase inhibitor expression in human fibroblasts by transforming growth factor β[END_REF]. Also, administration of TGF-β1 antisense oligonucleotide increase the expression of collagen degrading enzymes in human gingival fibroblasts [START_REF] Cotrim | Expression of matrix metalloproteinases in cyclosporine-related gingival fibroblasts is regulated by transforming growth factor (TGF)-β1 autocrine stimulation[END_REF]. Experimental data also revealed that proliferation of rat gingival cells under cyclosporine A depended on TGF-β1 mRNA induction by the drug (Yoshida et al, 2005). Clinical studies demonstrated that circulating and gingival crevicular fluid levels of TGF-β1 were increased in cyclosporin-medicated transplant patients [START_REF] Buduneli | Evaluation of transforming growth factor-beta1 level in crevicular fluid of cyclosporin A-treated patients[END_REF], and TGF-β1 was found to be an independent risk factor for gingival overgrowth in immunosuppressed patients, also medicated with cyclosporine A ( Gurkan et al, 2008). Recently, an accumulation of extracellular matrix related to TGF-β1 overexpression in cultured fibroblasts from patients medicated with cyclosporine A has been reported by [START_REF] Dreyfuss | Differences in the expression of glycosaminoglycans in human fibroblasts derived from gingival overgrowths is related to TGFbeta up-regulation[END_REF].

It was reported that expression of TGF-β1 gene (TGFB1) may depend on genetic constitution, i.e.

within the TGFB1 some functional SNPs were defined. Clinical studies in kidney transplant patients medicated with cyclosporine A suggested an association between TGFB1 gene polymorphism located in coding regions (codon 10 -+869T>C Pro10Leu and codon 25 +915G>C Arg25Pro) and gingival overgrowth [START_REF] Linden | The influence of transforming growth factor-β1 gene polymorphisms on the severity of gingival overgrowth associated with concomitant use of cyclosporine A and a calsium channel blocker[END_REF][START_REF] Radwan-Oczko | The relationship of transforming growth factor-β1 gene polymorphism, its plasma level, and gingival overgrowth in renal transplant recipients receiving different immunosuppressive regimens[END_REF]. However, contradictory results in kidney transplant patients with respect of the same as aforementioned SNPs are also available [START_REF] Radwan-Oczko | Transforming growth factor-β1 gene expression and cyclosporine A-induced gingival overgrowth: a pilot study[END_REF].

Therefore, it was decided to investigate the role of four SNPs within TGFB1 gene, previously associated with alterations in gene expression level, in gingival overgrowth in kidney transplant patients medicated with cyclosporine A. Two of the SNPs were previously evaluated, and lead to aminoacid substitution: +869T>C in codon 10 (rs1800470, Pro10Leu) and +915G>C in codon 25 (rs1800471, Arg25Pro). The other two SNPs are located in promoter region of TGFB1: -800G>A (rs1800468), and -509C>T (rs1800469), and thus can affect promoter function and gene expression (Grainger et al., 1999;[START_REF] Grainger | TGF-β in blood: a complex problem[END_REF].

Materials and Methods

The protocol of the study was approved by the ethics committee of the Pomeranian Medical University, Szczecin, Poland, i.e. the experiments were undertaken with the understanding and written consent of each subject and according to ethical principles, including the World Medical Association Declaration of Helsinki (version, 2002). Patients of Polish origin, Caucasians, Control samples were from 140 kidney transplant patients (91 males, 49 females), aged from 17 to 66 years (mean 39.7±11.4 years), who were free from gingival overgrowth signs at 6 months after transplantation, as evaluated by consultant periodontal specialists. Patients' characteristics are shown in table 1.

During the study period all subjects were administered cyclosporin A, azathioprine, prednisone and one of two calcium channel blockers, i.e. diltiazem or verapamil; eight patients without gingival overgrowth were given atenolol or prazosine. The medication regimens administered in both groups of the study, including cyclosporin A dose and concentrations, dosages of verapamil, diltiazem and prednisone evaluated at monthly intervals during 6 months were comparable, i.e. all calculated pvalues were >0.05 (Table 2). The serum concentrations of cyclosporin A were measured by fluorescence polarization immunoassay using TDx analyzer (Abbott) in all patients.

Genotyping

Four SNPs within TGFB1 gene, previously associated with alterations in gene expression level were selected for the purpose of this study. Two of them are located in promoter region of TGFB1:

-800G>A (rs1800468), and -509C>T (rs1800469), the others lead to aminoacid substitution: +869T>C (rs1800470, Pro10Leu) and +915G>C (rs1800471, Arg25Pro). Genomic DNA was extracted from 200 µL of whole blood samples using GeneMATRIX Quick Blood DNA Purification Kit (EURx, Poland). Each individual was genotyped for a presence of 4 SNPs in TGFB1 gene. The allelic discrimination TaqMan real-time PCR assays (Assay IDs: C_8708474_20, C_8708473_10, and C_22272997_10, Applied Biosystems, USA) were used for detection of -800G>A,-509C>T and +869T>C SNPs, respectively. Fluorescence data was captured using an ABI PRISM 7500 FAST Real-Time PCR System (AppliedBiosystems), after 40 cycles of PCR. For analysis of +915G>C polymorphism, a PCR-RFLP method was applied. Briefly, polymerase chain reaction was performed in 15µl of total volume, with a pair of primers: 5'-cgc tgc tgt ggc tac tgg t-3', and 5'-ctc cgg ttc tgc act ctc c-3', previously described by [START_REF] Pulleyn | TGFbeta1 allele association with asthma severity[END_REF]. Subsequently, a 254 bp PCR product was digested with the Cfr13I endonuclease (Fermentas, Lithuania), yielding two DNA fragments (171+83 bp) in case of wild-type G allele. The +915G>C substitution creates additional restriction site recognized by Cfr13I, yielding 3 fragments (141+83+30 bp), which was visualized after electrophoresis in 3.5% agarose gels stained with ethidium bromide [START_REF] Pulleyn | TGFbeta1 allele association with asthma severity[END_REF]. haplotype frequencies. Linkage disequilibrium (LD) was measured as followed -the D' was calculated using 2LD software and squared correlation coefficient (r 2 ) was evaluated. Odds ratios (OR) and 95% confidence interval (95% CI) were calculated using the Newcombe-Wilson method without the continuity correction. A p level of less than 0.05 was considered statistically significant.

Results

Out of 84 patients with gingival overgrowth, 57 subjects were classified as score 1 of gingival overgrowth, 22 patients were ascribed score 2 and 5 subjects score 3. Mean score of gingival overgrowth was 1.38±0.60 according to Pernu's scoring system [START_REF] Pernu | Gingival overgrowth among renal transplant recipients related to immunosuppressive medication and possible local background factors[END_REF]. Control transplant patients were characterized by healthy gingiva, i.e. were scored 0.

The genotype frequency distribution for all analyzed SNPs did not show a significant deviation from Hardy-Weinberg equilibrium in any of the study groups (p>0.1). The distribution of TGFB1 gene genotypes and alleles in kidney transplant patients is shown in Table 3. The patients with gingival overgrowth induced by immunosuppressive medication were characterized by similar distribution of TGFB1genotypes and allele to subjects without overgrowth, i.e. SNPs within the two coding regions: +869T>C in codon 10 (rs1800470, Pro10Leu) and +915G>C in codon 25 (rs1800471, Arg25Pro) as well as two promoter region SNPs: -800G>A (rs1800468), and -509C>T (rs1800469). In the next step of the study we subjected for analysis haplotypes of TGFB1 gene (Table 4). The results of linkage analysis showed that all analysed SNPs are in strong linkage in a Polish population (Figure 1). The EH program (Jurg Ott, Rockefeller University, New York) was used to estimate haplotype frequencies based on results of typing for the following studied SNPs: -800G>A, -509T>C, +869T>C, +915G>C. Among 16 potentially possible haplotypes derived from 4 SNPs, only four were observed in the studied sample of kidney transplant patients, with respective frequencies: G_C_T_G (0.545), G_T_C_G (0.324), G_C_C_C (0.071), and A_C_T_G (0.060). Similarly to distribution of the genotypes and allele, frequency of TGFB1 haplotypes were comparable in kidney transplant patients with and without gingival overgrowth.

Discussion

TGF-β1 patophysiological data as well as reports on functional polymorphism of its gene -TGFB1 2008) showed that the aforementioned polymorphisms are not associated with gingival overgrowth observer in kidney transplant patients medicated with cyclosporin A.

Therefore, it was decided to evaluate TGFB1 gene polymorphism in kidney transplant patients medicated with cyclosporine A. The former studies included polymorphisms in the coding regions of the gene resulting in aminoacid substitutions, namely in codon 10 -+869T>C Pro10Leu and codon 25 +915G>C Arg25Pro [START_REF] Linden | The influence of transforming growth factor-β1 gene polymorphisms on the severity of gingival overgrowth associated with concomitant use of cyclosporine A and a calsium channel blocker[END_REF][START_REF] Radwan-Oczko | The relationship of transforming growth factor-β1 gene polymorphism, its plasma level, and gingival overgrowth in renal transplant recipients receiving different immunosuppressive regimens[END_REF][START_REF] Radwan-Oczko | Transforming growth factor-β1 gene expression and cyclosporine A-induced gingival overgrowth: a pilot study[END_REF]. However, it is well known that polymorphisms located in promoter region of the gene can also be functional, affecting expression level of genes, hence in the present study polymorphisms in the promoter of TGFB1 gene were evaluated at positions -800G>A and -509C>T (Grainger et al, 1999;[START_REF] Grainger | TGF-β in blood: a complex problem[END_REF]. The promoter polymorphisms, although functional, have not been studied in posttransplant patients with gingival overgrowth yet. Furthermore, we evaluated haplotypes of TGFB1 gene as potential risk factors associated with gingival overgrowth in patients administered cyclosporine A.

Previous reports, similarly to our report included patients from Caucasian populations, but number of cases analysed was smaller than in the present study, study by [START_REF] Linden | The influence of transforming growth factor-β1 gene polymorphisms on the severity of gingival overgrowth associated with concomitant use of cyclosporine A and a calsium channel blocker[END_REF] for the studied SNPs: -800G>A, -509T>C, +869T>C, +915G>C were analysed. Similarly to genotypes, the analysed haplotypes did not discriminate the studied groups, i.e. kidney transplant patients medicated with cyclosporin A with and without gingival overgrowth.

Interaction between simultaneously administered drugs affecting enlargement have been also

reported. Cyclosporin A-treated patients are often comedicated with azathioprine and prednisolone, which can modify the severity of gingival overgrowth [START_REF] Wilson | Contribution of individual drugs to gingival overgrowth in adult and juvenile renal transplant patients treated with multiple therapy[END_REF]. In contrast patients on cyclosporin A who are also receiving calcium channel blockers present with a greater risk of the gingival lesions than patients treated with cyclosporine alone [START_REF] Thomasson | Nifedipine pharmacological variables as risk factors for gingival overgrowth in organ-transplant patients[END_REF]. However, in the present study, both groups, i.e. patients with and without gingival overgrowth were medicated similarly during observation period. So, the effect of treatment modality of gingival pathology could be neglected in the data analysis.

Another factor which could potentially influence the study conclusions is a number of evaluated cases. As in the present study analysed groups comprised of 84 and 140 cases the conclusions drawn should be considered as preliminary data, and the results of the study should be confirmed by observations from other populations involving larger groups of patients.

Based on the results from the present study it can be concluded that there is no significant association between the TGFB1 gene polymorphism and gingival overgrowth in kidney transplant patients administered cyclosporine A as a principal immunosuppressive agent. EH program (Jurg Ott, Rockefeller University, New York) was used to estimate haplotype frequencies based on results of typing for following SNPs: -800G>A, -509T>C, +869T>C, +915G>C. Among 16 potentially possible haplotypes derived from 4 SNPs, only four were observed in the studied sample of kidney transplant patients, with respective frequencies: G_C_T_G (0.545), G_T_C_G (0.324), G_C_C_C (0.071), and A_C_T_G (0.060).

  hospitalized and than followed-up at the Department of Nephrology, County Hospital, Szczecin, Poland were recruited into the study from 2002 to 2010 year, after giving informed consent. A total of 84 unrelated kidney transplant patients suffering from gingival overgrowth (61 males, 23 females) aged from 16 to 70 years (mean 42.2±13.1 years) were enrolled into the study. All patients were examined by two independent consultant periodontal specialists 6 months after kidney transplantation. Periodontists were blinded to SNP status. The patients were assessed using a clinical scoring method according to[START_REF] Pernu | Gingival overgrowth among renal transplant recipients related to immunosuppressive medication and possible local background factors[END_REF]. The patients were ascribed a general wholemouth score of between 0 and 3: 0=no overgrowth seen; 1=mild gingival overgrowth (thickening of the marginal gingiva and/or lobular granulation of the gingival pocket as well as overgrowth covering the gingival third of the crown or less); 2=moderate gingival overgrowth (overgrowth middle of the crown); 3=severe gingival overgrowth (overgrowth covering two thirds of the crown or affectation of the whole attached gingiva).

Figure 1 .

 1 Figure 1. Pairwise LD between the studied TGFB1 SNPs, estimated on base of analysis of all study subjects (n=224). Numbers represent r 2 (square correlation coefficient), and D' values (italics).

  [START_REF] Ellis | Plasma TGF-β1 as a risk factor for gingival overgrowth[END_REF]

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  The data were tested for Hardy-Weinberg equilibrium by calculating expected frequencies of genotypes and comparing them to the observed values using the χ2 test(Statistica 8.0, Statsoft Software, Warsaw, Poland). Associations between categorical variables were assessed by the Fisher exact test. The EH program (Jurg Ott, Rockefeller University, New York) was used to estimate

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  [START_REF] Edwards | Transforming growth factor beta modulates the expression of collagenase nad metalloproteinase inhibitor[END_REF][START_REF] Anderson | Effects of transforming growth factorbeta and platelet derived growth factor on human gingival fibroblasts grown in serum-containing and serum-free medium[END_REF][START_REF] James | Gingival fibroblast response to cyclosporine A and transforming factor beta 1[END_REF][START_REF] Overall | Independent regulation of collagenaze 72-kDa progelatinase and metalloproteinase inhibitor expression in human fibroblasts by transforming growth factor β[END_REF] Grainger et al, 1999;[START_REF] Grainger | TGF-β in blood: a complex problem[END_REF][START_REF] Buduneli | Evaluation of transforming growth factor-beta1 level in crevicular fluid of cyclosporin A-treated patients[END_REF][START_REF] Cotrim | Expression of matrix metalloproteinases in cyclosporine-related gingival fibroblasts is regulated by transforming growth factor (TGF)-β1 autocrine stimulation[END_REF][START_REF] Ellis | Plasma TGF-β1 as a risk factor for gingival overgrowth[END_REF] Yoshida et al, 2005;[START_REF] Gurkan | Gingival crevicular fluid transforming growth factor-β1 in cyclospotine and tacrolimus treated renal transplant patients without gingival overgrowth[END_REF]. However, available data on the

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	suggest potential role of the cytokine and polymorphism in the pathology of drug-induced including
	O cyclosporin A, gingival overgrowth (effects of TGFB1 gene polymorphism on gingival overgrowth in kidney transplant patients r medicated with cyclosporin A are controversial. Linden et al (2001) and Radwan-Oczko et al a l (2006) revealed that polymorphisms in TGFB1 gene located in coding regions of the gene, i.e.
	codon 10 -+869T>C Pro10Leu and codon 25 +915G>C Arg25Pro are genetic factors associated D i with risk of gingival overgrowth. Subsequent report from the latter group (Radwan-Oczko et al,
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  Nagata J, Yamane A (2005) Growth factors and proliferation of cultured rat gingival cells In response to cyclosporin A. J Periodontal Res 40: 11-19.

	Table 1 Patients' characteristics Yoshida T, Page 11 of 16	Oral Diseases -Manuscript Copy 9 Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy 11 Oral Diseases -Manuscript Copy 13
	Parameter		Patients with gingival	Patients without gingival	p
	O r a O r a O r a O r a Gender distribution (F/M) Age (years)* Place of residence: city/country 44/40 overgrowth (n=84) 23/61 42.2±13.1 Smoking 16 O r a	overgrowth (n=140) 49/91 39.7±11.4 83/57 31	0.301 0.233 0.332 0.615
	Diabetes	l l l l l	39	47	0.065
	D i D i D i D i Acute rejection All calculations performed by means of Fisher exact test, except *calculated by t-test 3 20 D i	0.011
		s e s e s e s e s e
		a s a s a s a s a s
			e e e e e
			s s s s s
			-----
			M a M a M a M a M a
			n u s n u s n u s n u s n u s
				c r c r c r c r c r
				i p i p i p i p i p
				t t t t t
				C C C C C
				o p y o p y o p y o p y o p y
			Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy	11 13

Table 2

 2 Characteristics of medication at monthly intervals after transplantation

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		1 month	2 months	3 months	4 months	5 months	6 months
			patients with gingival overgrowth (n=84)
	Cyclosporine
	concentration	420.0±234.2 380.1±206.7 308.3±170.2 306.3±198.2 276.5±193.3 280.8±179.4
	(ng/ml)
	Cyclosporine	300.0±84.1	280.9±78.8	260.7±79.3	251.2±76.9	245.2±68.6	235.4±65.4
	dose (mg/day)	(n=84)	(n=84)	(n=84)	(n=84)	(n=84)	(n=84)
	Diltiazem dose	173.8±24.5	173.8±24.5	173.8±24.5	173.8±24.5	173.8±24.5	173.8±24.5
	(mg/day)	(n=56)	(n=56)	(n=56)	(n=56)	(n=56)	(n=56)
	Verapamil dose	174.5±72.2	174.5±72.2	174.5±72.2	174.5±72.2	174.5±72.2	174.5±72.2
	(mg/day)	(n=28)	(n=28)	(n=28)	(n=28)	(n=28)	(n=28)
	Prednisone dose	18.8±7.4	16.3±5.2	15.1±4.3	12.4±3.7	10.6±3.0	9.5±3.1
	(mg/day)	(n=84)	(n=84)	(n=84)	(n=84)	(n=84)	(n=84)
			patients without gingival overgrowth (n=140)
	Cyclosporine
	concentration	413.2±258.1 370.8±288.2 326.8±263.5 276.6±159.8 296.9±195.6 249.8±172.2
	(ng/ml)
	Cyclosporine	282.4±99.1	274.4±88.2	256.5±85.4	254.9±83.6	246.2±77.2	238.4 ± 73.0
	dose (mg/day)	(n=140)	(n=140)	(n=140)	(n=140)	(n=140)	(n=140)
	Diltiazem dose	172.2±30.4	172.2±30.4	172.2±30.4	172.2±30.4	172.2±30.4	172.2±30.4
	(mg/day)	(n=104)	(n=104)	(n=104)	(n=104)	(n=104)	(n=104)
	Verapamil dose	184.3±60.5	184.3±60.5	184.3±60.5	184.3±60.5	184.3±60.5	184.3±60.5
	(mg/day)	(n=36)	(n=36)	(n=36)	(n=36)	(n=36)	(n=36)
	Prednisone dose	17.9±4.5	15.2±3.0	13.4±3.4	12.1±2.5	11.9±2.6	11.9±2.6
	(mg/day)	(n=140)	(n=140)	(n=140)	(n=140)	(n=140)	(n=140)

Table 3

 3 Distribution of tgfb1 alleles and genotypes among kidney transplant patients with and without gingival overgrowth All calculations performed by means of Fisher exact test, using homozygotes for a major (more frequent) allele or major allele frequency as reference.

		Oral Diseases -Manuscript Copy		Page 14 of 16
		Gingival Overgrowth	Healthy gingiva
			(n=84)		(n=140)	p value	OR (95%CI)
		n	(%)	n	(%)
	Genotype -800G>A
	GG	78	(92.9)	120	(85.7)	-	-
	GA	6	(7.1)	19	(13.6)	0.188	0.48 (0.18-1.27)
	AA	0	(0.0)	1	(0.7)	1.000	-
	minor allele carriers (GA+AA)	6	(7.1)	20	(14.3)	0.132	0.46 (0.17-1.20)
	minor allele frequency	6	(3.6)	21	(7.5)	0.103
	Genotype -509C>T
	CC	43	(51.2)	61	(43.6)	-	-
	CT	32	(38.1)	63	(45.0)	0.306	0.72 (0.40-1.28)
	TT	9	(10.7)	16	(11.4)	0.657	0.80 (0.32-1.97)
	minor allele carriers (CT+TT)	41	(48.8)	79	(56.4)	0.272	0.74 (0.43-1.27)
	minor allele frequency	50	(29.8)	95	(33.9)	0.404
	Genotype +869T>C
	TT	34	(40.5)	50	(35.7)	-	-
	TC	38	(45.2)	65	(46.4)	0.652	0.86 (0.47-1.55)
	CC	12	(14.3)	25	(17.9)	0.424	0.71 (0.31-1.55)
	minor allele carriers (TC+CC)	50	(59.5)	90	(64.3)	0.480	0.82 (0.47-1.42)
	minor allele frequency	62	(36.9)	115	(41.1)	0.404
	Genotype +915G>C
	GG	72	(85.7)	120	(85.7)	-	-
	GC	12	(14.3)	20	(14.3)	1.000	1.00 (0.46-2.16)
	CC	0	(0.0)	0	(0.0)	1.000	-
	minor allele carriers (GC+CC)	12	(14.3)	20	(14.3)	1.000	1.00 (0.46-2.16)
	minor allele frequency	12	(7.1)	20	(7.1)	1.000
		Oral Diseases -Manuscript Copy

Table 4

 4 Frequency of TGFB1 haplotypes among kidney transplant patients with and without gingival overgrowth.All calculations performed by means of Fisher exact test, using frequency of major haplotype as reference, except* -in relation to the sum of other haplotypes.

		Gingival Overgrowth	Healthy gingiva
			(2n=168)		(2n=280)	p value
		n	(%)	n	(%)
	G_C_T_G	100	(59.5)	144	(51.4)	*0.116
	G_T_C_G	50	(33.9)	95	(29.8)	0.236
	G_C_C_C	12	(7.2)	20	(7.1)	0.848
	A_C_T_G	6	(7.5)	21	(3.6)	0.063

Acknowledgements

The study was founded by the grant of Ministry of Science and Higher Education no. N N403 170637.

Conflict of interest

There is no conflict of interest.

Author contributions

Dr Kozak and Dr Dziewanowski were responsible for patient evaluation and data analysis, Dr Wajda, Dr Lapczuk and Dr Kurzawski were responsible for experiments, study design and manuscript preparation, Dr Lipski and Dr Drozdzik M were responsible for research design and manuscript revisions.


Benjamin R Disney

Robert Ds Watson

Andrew D Blann

Gregory Yh Lip

Dr Mark R Anderson
email: mark.anderson@swbh.nhs.uk

Review article: Proton pump inhibitors with clopidogrel; evidence for and against a clinically important interaction

Keywords: clopidogrel, PPI, proton pump inhibitor, drug interaction

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Introduction

The treatment of Acute Coronary Syndromes (ACS) with dual anti-platelet therapy (i.e. aspirin plus clopidogrel) reduces recurrent cardiovascular events [START_REF] Yusuf | for the Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation[END_REF]. However, with the advent of dual anti-platelet therapy comes an inevitable increased risk of bleeding as a side effect. With regards to gastrointestinal bleeding, this occurs in 1.3-2.7% of patients treated for ACS with aspirin, clopidogrel and enoxaparin [START_REF] Ng | Gastrointestinal bleeding in patients receiving a combination of aspirin, clopidogrel, and enoxaparin in acute coronary syndrome[END_REF][START_REF] Nikolsky | Gastrointestinal bleeding in patients with acute coronary syndromes: incidence, predictors, and clinical implications: analysis from the ACUITY (Acute Catheterization and Urgent Intervention Triage Strategy) Trial[END_REF]. Even patients on low dose aspirin have an incidence of gastrointestinal peptic ulcer bleeding of 0.5% per year (4).

In the context of ACS, gastrointestinal bleeding should be regarded as a serious condition that is independently associated with increases in both mortality and ischaemic complications [START_REF] Nikolsky | Gastrointestinal bleeding in patients with acute coronary syndromes: incidence, predictors, and clinical implications: analysis from the ACUITY (Acute Catheterization and Urgent Intervention Triage Strategy) Trial[END_REF].

Co-prescription with a proton pump inhibitor (PPI) could significantly reduce the risk of bleeding [START_REF] Ng | Gastrointestinal bleeding in patients receiving a combination of aspirin, clopidogrel, and enoxaparin in acute coronary syndrome[END_REF]. As a result, the near-universal use of a PPI is recommended in order to reduce the incidence of gastrointestinal bleeding in patients taking clopidogrel, most of whom are taking aspirin in addition [START_REF] Bhatt | ACCF/ACG/AHA 2008 expert consensus document on reducing the gastrointestinal risks of antiplatelet therapy and NSAID use: a report of the American College of Cardiology Foundation Task Force on Clinical Expert Consensus Documents[END_REF]. In addition to these guidelines, gastroenterologists frequently prescribe PPIs to prevent drug-induced upper gastrointestinal bleeding, supported by a Cochrane meta-analysis of PPI use to prevent NSAID induced peptic ulceration [START_REF] Rostom | Prevention of NSAID-induced gastroduodenal ulcers[END_REF].

The aim of this review article is to provide an overview of the evidence for the pharmacological interaction between proton pump inhibitors and clopidogrel and its impact on clinical outcomes.

A clinically relevant interaction between clopidogrel and PPI?

The possibility of a clinically relevant interaction between clopidogrel and PPIs was first noted in 2008 [START_REF] Gilard | Influence of omeprazole on the antiplatelet action of clopidogrel associated with aspirin: the randomized double-blind OCLA (Omeprazole CLopidogrel Aspirin) study[END_REF]. This came on the background of an observational study from 2006 which found clopidogrel activity on platelets to be diminished in patients receiving PPI treatment [START_REF] Gilard | Influence of omeprazol on the antiplatelet action of clopidogrel associated to aspirin[END_REF].

In response to studies suggesting that PPIs could diminish the effects of clopidogrel [START_REF] Juurlink | A population-based study of the drug interaction between proton pump inhibitors and clopidogrel[END_REF], the European Medicines Agency (EMEA) released a statement in May 2009 [START_REF]Public statement on possible interaction between clopidogrel and proton-pump inhibitors[END_REF], and the FDA also in 2009 [START_REF]Information for healthcare professionals: update to the labelling of clopidogrel bisulfate (marketed as Plavix) to alert healthcare professionals about a drug interaction with omeprazole (marketed as prilosec and prilosec OTC)[END_REF], warning of the potential interaction. Since then, clinicians have faced difficult and uncertain decisions regarding patient care. Various suggestions have been made

for prescribing, such as evening dosing of a PPI to ensure a 12 hour gap after a morning clopidogrel dose or the preferential use of pantoprazole [START_REF] Moayyedi | Proton Pump inhibitors and clopidogrel -hazardous drug interaction or hazardous interpretation of data?[END_REF]. However, there appears to be little evidence to back these approaches. Any potential interaction between PPIs and clopidogrel needs to be clarified due to the increased risk of poor cardiovascular outcomes in potentially large numbers of patients.

Pharmacological basis of a clopidogrel-PPI interaction

Clopidogrel, a thienopyridine, is a prodrug which is metabolised in the liver via the cytochrome p450 (mainly CYP2C19) pathway to its active metabolite [START_REF] Hulot | Cytochrome P450 2C19 loss-of function polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects[END_REF][START_REF] Richter | Potent mechanism-based inhibition of human CYP2B6 by clopidogrel and ticlopidine[END_REF]. This compound targets and irreversibly inhibits the ADP P2Y 12 receptor on circulating platelets which prevents platelet aggregation. CYP2C19, which has a major role in the metabolism of clopidogrel, also metabolises the PPIs. Reduced conversion of clopidogrel to its active metabolite by the CYP450 system could be in part responsible for clopidogrel resistance [START_REF] Erlinge | Patients with poor responsiveness to thienopyridine treatment or with diabetes have lower levels of circulating active metabolite, but their platelets respond normally to active metabolite ex vivo[END_REF] and clopidogrel resistance is associated with adverse cardiovascular events [START_REF] Miguel | Clinical implications of clopidogrel resistance[END_REF]. In addition to this, reduced intestinal absorption may play a role [START_REF] Erlinge | Patients with poor responsiveness to thienopyridine treatment or with diabetes have lower levels of circulating active metabolite, but their platelets respond normally to active metabolite ex vivo[END_REF][START_REF] Simon | French Registry of Acute ST-elevation and Non-ST-elevation myocardial infarction (FAST-MI) investigators. Genetic determinants of response to clopidogrel and cardiovascular events[END_REF].

As mentioned above, clopidogrel is metabolised to its active metabolite by various cytochrome p450 enzymes, the key enzyme being CYP2C19 [START_REF] Kurihara | In vitro metabolism of antiplatelet agent clopidogrel: cytochrome P450 isoforms responsible for two oxidation steps involved in the active metabolite formation[END_REF][START_REF] Rehmal | Interactions of two major metabolites of prasugrel, a thienopyridine antiplatelet agent with the cytochromes P450[END_REF]. Approximately 15% of the absorbed dose is converted to an active thiol metabolite, R-130964 [START_REF] Savi | The antiaggregating activity of clopidogrel is due to a metabolic activation by the hepatic cytochrome P450-1A[END_REF]. The extent of platelet inhibition by clopidogrel varies between patients and reduced platelet inhibition is associated with poor cardiovascular outcomes [START_REF] Siller-Matula | Thienopyridines in cardiovascular disease: focus on clopidogrel resistance[END_REF][START_REF] Snoep | Clopidogrel nonresponsiveness in patients undergoing percutaneous coronary intervention with stenting: a systematic review and meta-analysis[END_REF][START_REF] Angiolillo | Variability in individual responsiveness to clopidogrel: clinical implications, management, and future perspectives[END_REF][START_REF] Matetzky | Clopidogrel resistance is associated with increased risk of recurrent atherothrombotic events in patients with acute myocardial infarction[END_REF].

The activity of CYP2C19 can have a profound effect on the conversion of clopidogrel to its active metabolite. In healthy volunteers, those with CYP2C19 loss-of-function polymorphisms have impaired responsiveness to clopidogrel demonstrated by platelet aggregation and higher vasodilator-stimulated phosphoprotein (VASP) phosphorylation [START_REF] Hulot | Cytochrome P450 2C19 loss-of function polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects[END_REF].

It is thought that approximately 90% cases of poor metabolism is accounted for by the CYP2C19*2 allele [START_REF] Hulot | Cytochrome P450 2C19 loss-of function polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects[END_REF][START_REF] Fontana | Influence of CYP2C19 and CYP3A4 gene polymorphisms on clopidogrel responsiveness in healthy subjects[END_REF]. In patients suffering myocardial infarction who received clopidogrel, those with CYP2C19 loss-of-function alleles have lower levels of the active metabolite of clopidogrel and a higher rate of subsequent cardiovascular events than those [START_REF] Simon | French Registry of Acute ST-elevation and Non-ST-elevation myocardial infarction (FAST-MI) investigators. Genetic determinants of response to clopidogrel and cardiovascular events[END_REF][START_REF] Mega | Cytochrome P-450 polymorphisms and response to clopidogrel[END_REF][START_REF] Shuldiner | Association of cytochrome P450 2C19 genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy[END_REF][START_REF] Collet | Cytochrome P450 2C19 polymorphism in young patients treated with clopidogrel after myocardial infarction: a cohort study[END_REF]. The common polymorphisms of CYP2C19 are present in approximately 3-30% of whites, 3-40% of blacks and 12-100% of various Asian populations [START_REF] Desta | Clinical significance of the cytochrome P450 2C19 genetic polymorphism[END_REF][START_REF] Goldstein | Clinical relevance of genetic polymorphisms in the human CYP2C subfamily[END_REF]. In the study by Mega et al, 30% of healthy recruits had a loss-of-function allele for CYP2C19, which resulted in a relative reduction of 32.4% of the active metabolite of clopidogrel compared to non-carriers, and a 9% reduction in platelet aggregation [START_REF] Mega | Cytochrome P-450 polymorphisms and response to clopidogrel[END_REF]. This resulted in an increase in death from cardiovascular cause, myocardial infarction and stroke [START_REF] Mega | Cytochrome P-450 polymorphisms and response to clopidogrel[END_REF]. These effects are most notable in patients undergoing percutaneous coronary intervention with a HR of 3.58 (CI 1.71-7.51) for recurrent cardiovascular events [START_REF] Simon | French Registry of Acute ST-elevation and Non-ST-elevation myocardial infarction (FAST-MI) investigators. Genetic determinants of response to clopidogrel and cardiovascular events[END_REF]. The CYP2C19 loss-of-function polymorphism is also associated with stent thrombosis with rates as high as a factor of 3 as compared with noncarriers [START_REF] Mega | Cytochrome P-450 polymorphisms and response to clopidogrel[END_REF][START_REF] Sibbing | Cytochrome P450 2C19 loss-of-function polymorphism and stent thrombosis following percutaneous coronary intervention[END_REF]. Thus, any medication that reduces the conversion of clopidogrel to its active metabolite may reduce the platelet inhibition caused by clopidogrel. Indeed, clopidogrel has been at the centre of controversy previously, with a reported possible interaction with atorvastatin, which competes for CYP3A4 (see figure 1) [START_REF] Neubauer | Thienopyridines and statins: assessing a potential drug-drug interaction[END_REF]. Atorvastatin was found to attenuate the antiplatelet activity of clopidogrel in a dose-dependent manner in vitro [START_REF] Lau | Atorvastatin reduces the ability of clopidogrel to inhibit platelet aggregation: a new drug-drug interaction[END_REF]. However, this observation has since been refuted and was not found to be associated with worse clinical outcomes [START_REF] Farid | Effect of atorvastatin on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel in healthy subjects[END_REF][START_REF] Saw | Lack of adverse clopidogrel-atorvastatin clinical interaction from secondary analysis of a randomized, placebo-controlled clopidogrel trial[END_REF][START_REF] Wienbergen | Comparison of clinical benefits of clopidogrel therapy in patients with acute coronary syndromes taking atorvastatin versus other statin therapies[END_REF][START_REF] Saw | Lack of evidence of a clopidogrel-statin interaction in the CHARISMA trial[END_REF].

The potential mechanism of interaction lies in the fact that both clopidogrel and the PPIs, in varying degrees, are both metabolised by the same cytochrome p450 enzyme -CYP2C19 [START_REF] Hulot | Cytochrome P450 2C19 loss-of function polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects[END_REF][START_REF] Ishizaki | Review article: cytochrome P450 and the metabolism of proton pump inhibitors -emphasis on rabeprazole[END_REF]. PPIs have the potential to competitively inhibit the metabolism of clopidogrel to its active metabolite. This leads to reduced circulating concentrations of the active metabolite.

The earliest evidence of a pharmacological interaction between clopidogrel and PPIs is from platelet aggregation studies. One study of 105 patients found PPI users (omeprazole, n=24) had VASP phosphorylation levels than none PPI users and suggested an interaction [START_REF] Gilard | Influence of omeprazol on the antiplatelet action of clopidogrel associated to aspirin[END_REF]. In the OCLA study [START_REF] Gilard | Influence of omeprazole on the antiplatelet action of clopidogrel associated with aspirin: the randomized double-blind OCLA (Omeprazole CLopidogrel Aspirin) study[END_REF], which was a double-blind placebo-controlled trial, measurements of VASP expressed as platelet reactivity index (PRI) were measured in 124 patients undergoing coronary stent insertion. Patients receiving aspirin and clopidogrel were treated with omeprazole 20mg or placebo for 7 days. By day 7, 26.7% of placebo-treated patients and 60.9% of omeprazole-treated patients were classified as poor responders to clopidogrel (PRI>50%) indicating a significantly decreased effect of clopidogrel in the presence of a PPI.

As all PPIs are metabolised by CYP2C19 to varying degrees, and share the ability to inhibit CYP2C19, do all PPIs have negative effects on clopidogrel? Several studies have tried to answer this question.

One small study showed that lansoprazole 30mg given in addition to clopidogrel 300mg diminished the inhibition of platelet aggregation when compared to clopidogrel alone [START_REF] Small | Effects of the proton pump inhibitor lansoprazole on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel[END_REF]. However, no effect was seen with regards to PPIs interacting with prasugrel. Another study found that the inhibitory profiles for all PPIs were similar but with some heterogenicity; for example, lansoprazole is the most potent in vitro inhibitor of CYP2C19 with pantoprazole exhibiting more of an effect on CYP2C9 [START_REF] Li | Comparison of inhibitory effects of the proton pump-inhibiting drugs omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole on human cytochrome P450 activities[END_REF].

Although lansoprazole has been shown to be the most potent inhibitor of CYP2C19 [START_REF] Li | Comparison of inhibitory effects of the proton pump-inhibiting drugs omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole on human cytochrome P450 activities[END_REF], other evidence reports that in patients prescribed clopidogrel, concomitant treatment with lansoprazole does not alter the pharmacokinetics or pharmacodynamics of clopidogrel [START_REF] Small | Effects of the proton pump inhibitor lansoprazole on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel[END_REF].

Data from 1000 patients on clopidogrel undergoing angiography examined the effect the various PPIs had on platelet aggregation [START_REF] Sibbing | Impact of proton pump inhibitors on the antiplatelet effects of clopidogrel[END_REF]. Platelet aggregation was significantly greater in patients prescribed omeprazole (n=64) than those without PPI therapy. It was observed that Another study found that omeprazole (n=3132) was associated with adverse cardiovascular outcomes (e.g. death or rehospitalisation for ACS) [START_REF] Ho | Risk of adverse outcomes with concomitant use of clopidogrel and proton pump inhibitors following acute coronary syndrome[END_REF]. In the same study similar results were found with rabeprazole (n=151), however, small numbers weaken this data. Results from 300 patients looking at clopidogrel responsiveness by VASP phosphorylation and platelet aggregometry, in patients with coronary artery disease undergoing PCI found no difference between esomeprazole (n=74) and pantoprazole (n=152), and indeed no suggestion that either was associated with impaired response to clopidogrel [START_REF] Siller-Matula | Effects of pantoprazole and esomeprazole on platelet inhibition by clopidogrel[END_REF]. Unfortunately, omeprazole was not included in the analysis.

Other evidence suggests pantoprazole has less of a deleterious effect on clopidogrel than omeprazole [START_REF] Cuisset | Comparison of omeprazole and pantoprazole influence on a high 150-mg clopidogrel maintenance dose: the PACA (Proton Pump Inhibitors and Clopidogrel Association) prospective randomized study[END_REF]. PRI VASP was measured in 104 patients undergoing PCI who were randomised to either omeprazole (n=52) or pantoprazole (n=52) in addition to both aspirin and clopidogrel. After 1 month patients receiving pantoprazole had significantly better platelet response to clopidogrel as assessed with the PRI VASP (36% versus 48%).

These observations have led authors to put forward the case that pantoprazole is the preferred PPI in the context of concomitant clopidogrel use [START_REF] Norgard | Drug-drug interaction between clopidogrel and the proton pump inhibitors[END_REF][START_REF] Juhasz | Current standings of the proton pump inhibitor and clopidogrel co-therapy: review on an evolving field with the eyes of the Gastroenterologist[END_REF][START_REF] Juurlink | Proton pump inhibitors and clopidogrel: putting the interaction in perspective[END_REF]. Some data suggests that pantoprazole is less likely to inhibit CYP2C19 than omeprazole and does not attenuate the pharmacodynamic response to clopidogrel [START_REF] Li | Comparison of inhibitory effects of the proton pump-inhibiting drugs omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole on human cytochrome P450 activities[END_REF][START_REF] Siller-Matula | Effects of pantoprazole and esomeprazole on platelet inhibition by clopidogrel[END_REF][START_REF] Cuisset | Comparison of omeprazole and pantoprazole influence on a high 150-mg clopidogrel maintenance dose: the PACA (Proton Pump Inhibitors and Clopidogrel Association) prospective randomized study[END_REF][START_REF] Juurlink | Proton pump inhibitors and clopidogrel: putting the interaction in perspective[END_REF]. However, recently updated guidelines suggest that no one PPI should be used preferentially over another. This is due to the lack of head to head trials and that studies showing a difference between PPIs were in different populations using different methods [START_REF] Abraham | ACCF/ACG/AHA 2010 expert consensus document on the concomitant use of proton pump inhibitors and thienopyridines: a focused update of the ACCF/ACG/AHA 2008 expert consensus document on reducing the gastrointestinal risks of antiplatelet therapy and NSAID use[END_REF]. Although drug-drug interactions of omeprazole for CYP2C19 are thought to be one of competitive inhibition, the issue of its short plasma half-life (<1 h) has been brought up to refute this argument [START_REF] Norgard | Drug-drug interaction between clopidogrel and the proton pump inhibitors[END_REF]. However, omeprazole has a high affinity for CYP2C19 and its binding is extensive and it is unknown how long this inhibition persists. Omeprazole has a short half-life, however, separating the dose of omeprazole and clopidogrel is not a way of overcoming the interaction as some CYP2C19 may still be bound by omeprazole metabolism [START_REF] Norgard | Drug-drug interaction between clopidogrel and the proton pump inhibitors[END_REF]. In addition, there has been no obvious dose-response relationship found between PPI dose and adverse outcomes, which one would expect from competitive inhibition [START_REF] Ho | Risk of adverse outcomes with concomitant use of clopidogrel and proton pump inhibitors following acute coronary syndrome[END_REF][START_REF] Norgard | Drug-drug interaction between clopidogrel and the proton pump inhibitors[END_REF].

At present there is insufficient evidence to suggest that one particular PPI (e.g. pantoprazole) should be prescribed in preference to another to avoid an interaction. The ongoing SPICE (Evaluation of the Influence of Statins and Proton Pump Inhibitors on Clopidogrel Antiplatelet Effects) trial is directly comparing the effect of all PPIs on platelet aggregation and VASP and should help answer the question of which PPI is preferable. In addition the trial will examine clopidogrel resistance, CYP2C19 polymorphism and its effect on PPI on the antiplatelet activity of clopidogrel, mortality and the need to stop medication due to peptic ulcer disease or GI bleeding [START_REF]Evaluation of the Influence of Statins and Proton Pump Inhibitors on Clopidogrel Antiplatelet Effects (SPICE)[END_REF].

Clinical evidence -evidence of an interaction

From the evidence above there is no doubt that PPIs have an effect on the pharmacokinetics and pharmacodynamics of clopidogrel, as evidenced by laboratory assays, but these are surrogate markers of clinical effect. The question has to be asked, does a pharmacokinetic or pharmacodynamic effect have any impact on clinical outcomes?

Several studies suggest a link between co-prescription of a PPI and clopidogrel, and an increased risk of adverse clinical outcomes (see Table 1).

Data from 14383 patients in the Medco Integrated database was presented in abstract format at the AHA Scientific Sessions 2008 Symposium. The study population were patients who had undergone PCI and were prescribed clopidogrel alone or clopidogrel plus a PPI. Patients with no preceding cardiovascular disease taking PPIs had a 32.5% incidence of a major cardiovascular event within 1 year versus 21.2% of patients not taking a PPI (adjusted OR 1.79, 95% CI 1.62-1.97). Effects were more pronounced in patients with a preceding cardiovascular event (adjusted OR 1.86, 95% CI 1.63-2.12). However, confounding may be an issue and the study did not have information on valuable cardiovascular risk factors, such as smoking, blood pressure and serum cholesterol levels [START_REF] Aubert | Proton pump inhibitors effect on clopidogrel effectiveness: the Clopidogrel Medco Outcomes Study (abstract 3998)[END_REF].

Data retrospectively analysed from 4800 patients admitted with acute myocardial infarction and subsequently receiving clopidogrel classified patients as no PPI exposure, low PPI exposure and high PPI exposure based upon adherence rates to PPIs [START_REF] Pezalla | Initial assessment of clinical impact of a drug interaction between clopidogrel and proton pump inhibitors[END_REF]. One year event rates for acute myocardial infarction were 1.38% for no PPI, 3.08% in the low exposure and 5.03% in the high exposure group. However, patients prescribed PPIs may be older with greater co-morbidity, and indeed this is the case in this study which limits the applicability of [START_REF] Ho | Risk of adverse outcomes with concomitant use of clopidogrel and proton pump inhibitors following acute coronary syndrome[END_REF], the baseline characteristics varied considerably. Those on a PPI had greater comorbidity with higher rates of renal failure, diabetes and congestive cardiac failure.

The study also lacked data on smoking status and blood pressure. Again, because of these differences it is unclear if the adverse outcomes observed are related to PPI use or to confounding factors such as comorbidity [START_REF] Last | Review of recent evidence: potential interactions between clopidogrel and proton pump inhibitors[END_REF].

Thus, the results seen from these observational studies may simply reflect a greater tendency to prescribe prophylactic PPIs to patients at higher risk of cardiovascular events [START_REF] Moayyedi | Proton Pump inhibitors and clopidogrel -hazardous drug interaction or hazardous interpretation of data?[END_REF][START_REF] Rassen | Cardiovascular outcomes and mortality in patients using clopidogrel with proton pump inhibitors after percutaneous coronary intervention or acute coronary syndrome[END_REF].

Clinical evidence -evidence against a clinically relevant interaction

The first data against an interaction was presented in abstract format at the AHA Scientific Sessions 2008 Symposium was in direct conflict with Medco data presented at the same meeting [START_REF] Aubert | Proton pump inhibitors effect on clopidogrel effectiveness: the Clopidogrel Medco Outcomes Study (abstract 3998)[END_REF][START_REF] Dunn | Baseline proton pump inhibitor use is associated with increased cardiovascular events with and without the use of clopidogrel in the CREDO Trial (abstract 3999)[END_REF]. In this retrospective analysis of the Clopidogrel for the Reduction of Events During Observation (CREDO) Trial, no increase in cardiovascular or cerebrovascular events was seen in the clopidogrel-PPI group at 1 year. [START_REF] Rassen | Cardiovascular outcomes and mortality in patients using clopidogrel with proton pump inhibitors after percutaneous coronary intervention or acute coronary syndrome[END_REF]. Crude rates of MI hospitalisation or death were higher in the PPI group with a relative risk of 1.74 (CI 1.44-2.10). However, after multivariable adjustment the RR fell to 1.32 (CI 1.08-1.61). With high-dimensional propensity score (hd-PS) adjustment a RR of 1.22 (CI 0.99-1.51) for MI hospitalisation or death was seen. Thus, no statistically significant clopidogrel-PPI interaction is seen.

Zairis et al's study of 588 patients undergoing PCI showed no difference in cardiovascular outcomes in the PPI (omeprazole only n=340) group at 1 year (57). The primary endpoint was defined as cardiac death or rehospitalisation with for a non-fatal MI. This endpoint was reached by 10% in PPI group and 9.7% in the no PPI group -hazard ratio 1.1 (95% CI 0.6-1.8). The study groups were well matched in terms of baseline characteristics which is in contrast to other studies where patients on a PPI were older and with more comorbidity [START_REF] Juurlink | A population-based study of the drug interaction between proton pump inhibitors and clopidogrel[END_REF][START_REF] Ho | Risk of adverse outcomes with concomitant use of clopidogrel and proton pump inhibitors following acute coronary syndrome[END_REF].

The genetic determinants of response to clopidogrel have been evaluated and the use of omeprazole, or any other PPI, had no effect on the clinical response to clopidogrel [START_REF] Simon | French Registry of Acute ST-elevation and Non-ST-elevation myocardial infarction (FAST-MI) investigators. Genetic determinants of response to clopidogrel and cardiovascular events[END_REF]. A large retrospective analysis of 20596 Medicare patients again drew negative results with regards to the effect any drug interaction had on clinical events. Those patients receiving both clopidogrel and a PPI (pantoprazole n=4707, omeprazole n=683) had a HR 0.99 (CI 0.82-1.19) for serious cardiovascular disease (ACS or revascularisation). The study also raises the 15 importance of PPIs in this group of patients with regards to reducing the incidence of gastrointestinal bleeding. In those patients receiving concurrent PPI the incidence of hospitalisation with gastrointestinal bleeding was 50% lower than those not prescribed a PPI [START_REF] Ray | Outcomes with concurrent use of clopidogrel and protonpump inhibitors: a cohort study[END_REF].

The recently published Clopidogrel and the Optimisation of Gastrointestinal Events (COGENT) trial is the first, and only, prospective study of the potential interaction [START_REF] Bhatt | Clopidogrel with or without omeprazole in coronary artery disease[END_REF]. The study was well designed specifically to address the issue of a clinically relevant interaction.

Unfortunately the trial was halted early due to bankruptcy of its sponsor with 3873 of 5000 patients enrolled. The study groups were well matched. With respect to cardiovascular outcomes 109 patients had an event (MI, stroke, coronary artery bypass graft, PCI, CV death), 4.9% and 5.7% with omeprazole and placebo respectively (HR 0.99, 95% CI 0.68-1.44). In addition, overt upper gastrointestinal bleeding was reduced in those patients prescribed a PPI (HR 0.13, 95% CI 0.03-0.56). The authors err on the side of caution, however, stating "that the results do not rule out a clinically meaningful difference in cardiovascular events due to use of a PPI." This is due to a low number of clinical events during follow up and, as a result, wide confidence intervals.

It should be highlighted that confounding can have a significant effect on results and their

Does bleeding have an impact?

An alternative hypothesis is that bleeding per se in the ACS setting is bad. Indeed, bleeding in the context of ACS is an independent risk for poor short and long term clinical outcomes [START_REF] Spencer | Does comorbidity account for the excess mortality in patients with major bleeding in acute myocardial infarction?[END_REF][START_REF] Mehran | Associations of major bleeding and myocardial infarction with the incidence and timing of mortality in patients presenting with non-ST-elevation acute coronary syndromes: a risk model from the ACUITY trial[END_REF][START_REF] Manoukian | Impact of major bleeding on 30-day mortality and clinical outcomes in patients with acute coronary syndromes: an analysis from the ACUITY trial[END_REF][START_REF] Eikelboom | Adverse impact of bleeding on prognosis in patients with acute coronary syndromes[END_REF]. Therefore, it may not be that PPIs are associated with adverse clinical outcomes but that patients taking them may have had a history of bleeding resulting in PPI prescription or may be deemed at high risk of bleeding. A bleeding episode could lead to a prolonged prothrombotic state which in turn leads to increased adverse clinical events. This is an area which requires further studies to clarify.

Other possibilities include that PPIs do have a direct effect on cardiovascular risk, or that PPIs interact with clopidogrel but it is individuals with genetic polymorphism of the gene encoding CYP2C19 leading to loss-of-function that may be more susceptible to this interaction. The TRITON-TIMI 38 study examined those patients with a loss-of function CYP2C19 polymorphism and current PPI use in patients prescribed clopidogrel. The study was underpowered to address this specific point, however, no association of increased adverse events was observed (HR 0.76, CI 0.39-1.48) [START_REF] O'donoghue | Pharmacodynamic effect and clinical efficacy of clopidogrel and prasugrel with or without a proton-pump inhibitor: an analysis of two randomised trials[END_REF].

Ultimately though, the association between clopidogrel and PPIs is likely to suffer from channelling bias -that is that physicians tend to prescribe PPIs to patients with more severe underlying illness [START_REF] Rude | Proton-pump inhibitors, clopidogrel, and cardiovascular adverse events: fact, fiction, or something in between?[END_REF]. In addition to the concerns regarding clopidogrel there are also conflicting reports on the use of PPIs with aspirin. Studies have shown that omeprazole has the ability to reduce the absorption and bioavailability of aspirin in animals [START_REF] Giraud | Effect of omeprazole on the bioavailability of unmodified and phospholipid-complexed aspirin in rats[END_REF] and by doing so has the ability to reduce the analgesic and antipyretic effects [START_REF] Lichtenberger | Nonsteroidal anti-inflammatory drug and phospholipid prodrugs: combination therapy with antisecretory agents in rats[END_REF].

However, with respect to aspirin the interaction is thought to lie in the PPIs ability to increase intragastric pH and thereby reduce the absorption and thus bioavailability of aspirin, rather than inhibit the enzymes that metabolise it, as is the mechanism with clopidogrel. Aspirin is normally absorbed in a lipid-soluble state by diffusion. By increasing intragastric pH above the pKa of aspirin absorption is reduced [START_REF] Giraud | Effect of omeprazole on the bioavailability of unmodified and phospholipid-complexed aspirin in rats[END_REF][START_REF] Hollander | Intestinal absorption of aspirin[END_REF] Unfortunately, as with the clopidogrel interaction, there is ongoing debate as to the importance and accuracy of the aspirin-PPI interaction in humans. Two studies on small numbers of patients report that PPIs do not interfere with the antiplatelet effects of aspirin; one studied omeprazole [START_REF] Iñarrea | Omeprazole does not interfere with the antiplatelet effect of low-dose aspirin in man[END_REF] and the other lansoprazole (70) in 14 and 24 patients respectively. One study, however, reported enhanced antiplatelet activity of aspirin when coadministered with pantoprazole [START_REF] Kasprzak | Pantoprazole may enhance antiplatelet effect of enteric-coated aspirin in patients with acute coronary syndrome[END_REF]. The largest study examining this interaction which enrolled 418 patients with stable coronary disease found that the antiplatelet effect of aspirin was reduced by PPIs [START_REF] Würtz | The antiplatelet effect of aspirin is reduced by proton pump inhibitors in patients with coronary artery disease[END_REF]. Patients who have a GI bleed have increased mortality, and the protection PPIs offer against such events is not to be underestimated [START_REF] Marshall | Peptic ulcer bleeding: an expedition of 20 years from 1989-2009[END_REF]. Based on the available evidence, the decision to prescribe a PPI to a patient on clopidogrel should be made after an individual risk assessment balancing gastrointestinal bleeding risk against thrombotic risk. Further prospective randomised clinical trials are needed, but in the meantime, we must be careful not to jump to the wrong conclusions, and deny our patients the protection that PPIs clearly offer, compared with a concern about cardiovascular outcomes that has not yet been proven. However, a clinically important interaction cannot be excluded in certain groups such as those patients with loss-of-function polymorphisms and further work should be aimed at this area. Following intestinal absorption, clopidogrel undergoes a two step process to form its active metabolite. These processes are dependent on various cytochrome P450 (CYP450) enzymes.

F

  similar in patients taking clopidogrel and pantoprazole (n=162), esomeprazole (n=42) or no PPI.

  after adjustment, the high exposure group still had a relative risk of 337% compared to the no exposure group.Further evidence suggesting a clinically relevant interaction comes from the retrospective analysis of 8205 patients from Veteran Affairs hospitals between 2003-2006 who had been admitted with ACS[START_REF] Ho | Risk of adverse outcomes with concomitant use of clopidogrel and proton pump inhibitors following acute coronary syndrome[END_REF]. Of these patients 63.9% were prescribed PPI at discharge, during follow up or both: omeprazole n=3132, rabeprazole n=151, lansoprazole n=22, pantoprazole n=15, more than one PPI n=1924. Death or rehospitalisation for ACS occurred in 20.8% of patients prescribed clopidogrel without PPI and 29.8% of patients prescribed clopidogrel plus a PPI. The use of a PPI during any point was associated with an increased risk of death or rehospitalisation with ACS -Odds Ratio (OR) 1.25 (95% CI 1.11-1.41). Adjusted OR for all cause mortality revealed no difference between the two groups. The authors, however, conclude that concomitant use of clopidogrel and a PPI may be associated with an attenuation of the benefits of clopidogrel but stop short of confirming a causal relationship. Again, one limitation of this study is the difference in baseline characteristics -those prescribed a PPI tended to be older with more comorbidity (e.g. diabetes, prior MI, heart failure, CVA, COPD, renal and liver disease) and may therefore represent a population with a higher risk of adverse outcomes, rather than PPIs being the problem.One Canadian population based study examined patients admitted with acute myocardial infarction between 2002-2007[START_REF] Juurlink | A population-based study of the drug interaction between proton pump inhibitors and clopidogrel[END_REF]. PPI prescribing was common -31% of patients receiving a PPI within 90 days of discharge. The authors report a significant association between readmission due to MI and current use of a PPI (adjusted OR 1.27, 95%CI 1.03-1.57) and a 40% increase in recurrent MI within 90 days (OR 1.4, 95% CI 1.10-1.77). Pantoprazole and histamine receptor antagonists were not associated with adverse outcomes. Unfortunately the number of prescriptions and breakdown of which PPI was prescribed is not given. As with

  Perhaps the most definitive data on whether mechanistic changes translate into clinical data come from over 13000 patients participating in the Prasugrel In Comparison to Clopidogrel for Inhibition of Platelet Activation and Aggregation-Thrombolysis in Myocardial Infarction (PRINCIPLE-TIMI) 44 and Trial to Assess Improvement in Therapeutic Outcomes by Optimising Platelet Inhibition with Prasugrel-TIMI (TRITON-TIMI) 38 studies. Both studies were subanalysed to look at individual PPI use and showed no association between PPI and adverse cardiovascular outcomes. The authors found no independent association between any of the PPIs and the risk of MI or cardiovascular death (55). These two randomised trials studied over 13000 patients. There was no difference among the PPIs used: omeprazole (n= 1675), lansoprazole (n=441), esomeprazole (n=613), pantoprazole (n=1844). As with previous studies patients taking PPIs were older and sicker, with more frequent history of peptic ulcer disease and lower baseline haemoglobin. After adjustment for confounders, no significant association between use of a PPI and risk of primary endpoint (composite of cardiovascular death, non-fatal-MI, or non-fatal stroke) with HR 0.94 (95% CI 0.80-1.11, p=0.46). Consistent PPI use (i.e. use of PPI for the whole duration of follow up) was not associated with an increased risk of cardiovascular death, MI or stroke in patients treated with clopidogrel (HR 1.05, 95% CI 0.85-1.30). An accompanying editorial commended the work the patients included in the TRITON-TIMI trial were much younger, with less diabetes or renal failure than those in the previously highlighted observational studies (56). Rassen et al analysed data of 64561 patients who underwent PCI or hospitalisation for ACS between 2001 and 2005

  subsequent interpretation. Work by Valkhoff et al has highlighted the problems with confounding by indication when analysing data retrospectively collected regarding the use of PPIs with clopidogrel (60). This analysis examined 23655 patients admitted with MI, with 1247 readmitted with recurrent MI. All PPIs were included in the analysis. PPI users were noted to be older, with more co-morbidity and co-medication. With current users of clopidogrel, current PPI use was associated with recurrent MI (OR 1.62, 95% CI 1.15-2.27).

  applying past PPI use as the reference category, rather than PPI non-use, no association between recurrent MI and current PPI use was noted (OR 0.95, 95% CI 0.38-2.41). The authors suggest that the observed association between current PPI use and recurrent MI when PPI non-use was the reference may be the result of residual confounding.

19 Conclusion

 19 Although, the current data are conflicting, much concern has been raised in the medical press regarding the potential negative effects of PPIs on the efficacy of clopidogrel. There is extensive evidence demonstrating that PPIs do have an effect on the pharmacokinetics and pharmacodynamics of clopidogrel, but it is important to highlight that abnormal laboratory indices do not always translate into clinically relevant interactions.

44 )

 44 No link. P = pantoprazole, O = omeprazole, E = esomeprazole, L = lansoprazole, R = rabeprazole, OR = odds ratio, HR = hazard ratio, AHR = adjusted hazard ratio, MACE = major adverse cardiac event, ARR = adjusted rate ratio, NA = not available, MI = myocardial infarction a = composite of cardiovascular death, non-fatal MI or non-fatal stroke.

• 22 Figure 1 .

 221 Figure 1. Metabolism of clopidogrel.

44 )

 44 No link. P = pantoprazole, O = omeprazole, E = esomeprazole, L = lansoprazole, R = rabeprazole, OR = odds ratio, HR = hazard ratio, AHR = adjusted hazard ratio, MACE = major adverse cardiac event, ARR = adjusted rate ratio, NA = not available, MI = myocardial infarction a = composite of cardiovascular death, non-fatal MI or non-fatal stroke.

•Figure 1 .

 1 Figure 1. Metabolism of clopidogrel.

Table 1 . Summary of studies examining the interaction between clopidogrel and PPIs

 1

	Author	Ref. PPI	No. of	Study design	Results & Conclusion
				patients on
				PPI &
				clopidogrel
	Ho	42	NA	5244	Retrospective	OR 1.25 for death or
						rehospitalisation for
						PPI group.
						Positive link.
	Juurlink	9	POLR	734	Retrospective	OR1.4 (CI 1.1-1.77)
						for OLR for
						reinfarction.
						OR 1.02 (CI 0.7-1.47)
						for P for reinfarction.
						Positive link.
	Aubert	50	NA	4521 o r	Retrospective	OR 1.86 (CI 1.63-2.12) for PPI group. Positive link.
	Pezalla Dunn	51 54	NA NA	712 P e e Retrospective 176 Retrospective r	5% rate of MI at 1 year in high PPI exposure group (1.38% in controls) Positive link. OR 1.63 (CI 1.01-2.63) for PPI group.
	O'Donoghue 55	POEL	4529	Retrospective R e	No link. AHR 0.94 for primary endpoint. a No link.
	Rassen Zairis	53 57	POELR O	3996 340	Retrospective v i ARR for MI/death 1.22 (CI 0.99-1.51). No link. e Retrospective HR for MI/death 1.1
	Simon	17	POELR	NA	Retrospective	(CI 0.6-1.8). No link. w PPI had no effect on
						clinical response to
						clopidogrel.
						No link.
	Ray	58	POELR	7593	Retrospective	HR for CVS events
						0.99 (CI 0.82-1.19)
	Bhatt	59	O	1876	Prospective	HR 0.99 (CI 0.68-1.
						20

Table 1 .

 1 Summary of studies examining the interaction between clopidogrel and PPIs

	Author	Ref. PPI	No. of	Study design	Results & Conclusion
				patients on
				PPI &
				clopidogrel
	Ho	42	NA	5244	Retrospective	OR 1.25 for death or
						rehospitalisation for
						PPI group.
						Positive link.
	Juurlink	9	POLR	734	Retrospective	OR1.4 (CI 1.1-1.77)
						for OLR for
						reinfarction.
						OR 1.02 (CI 0.7-1.47)
						for P for reinfarction.
						Positive link.
	Aubert	50	NA	4521	Retrospective	OR 1.86 (CI 1.63-2.12)
						for PPI group.
						Positive link.
	Pezalla	51	NA	712	Retrospective	5% rate of MI at 1 year
						in high PPI exposure
						group (1.38% in
						controls)
						Positive link.
	Dunn	54	NA	176	Retrospective	OR 1.63 (CI 1.01-2.63)
						for PPI group.
						No link.
	O'Donoghue 55	POEL	4529	Retrospective	AHR 0.94 for primary
						endpoint. a
						No link.
	Rassen	53	POELR	3996	Retrospective	ARR for MI/death 1.22
						(CI 0.99-1.51).
						No link.
	Zairis	57	O	340	Retrospective	HR for MI/death 1.1
						(CI 0.6-1.8).
						No link.
	Simon	17	POELR	NA	Retrospective	PPI had no effect on
						clinical response to
						clopidogrel.
						No link.
	Ray	58	POELR	7593	Retrospective	HR for CVS events
						0.99 (CI 0.82-1.19)
	Bhatt	59	O	1876	Prospective	HR 0.99 (CI 0.68-1.

Alimentary Pharmacology & Therapeutic


Dr Pierpaolo Cortellini
email: studiocortellini@cortellini.191.it

Maurizio Tonetti

Maurizio S Tonetti #$

Without Regenerative Materials. An Initial Randomized Controlled Trial in Intrabony Defects

Keywords: clinical trial, periodontal regeneration, microsurgery, minimally invasive surgery, osseous defects clinical trial, periodontal regeneration, microsurgery, minimally invasive surgery, osseous defects

Clinical Relevance

Scientific rationale for study. To compare the efficacy of M-MIST to M-MIST with the additional use of regenerative materials in the treatment of intrabony defects.

Principal findings. M-MIST alone resulted in excellent clinical and radiographic improvements. Addition of regenerative materials did not provide further improvements. The procedure produced very limited patient discomfort and wound failure was limited to only 1 case.

Practical implications. Clinical and radiographic healing following use of M-MIST needs to be confirmed in large multicenter trials.

Introduction

Regeneration is a healing outcome that can occur when the systemic and local conditions are favourable. In periodontal regeneration, the local conditions include the presence of space for the formation of the blood clot at the interface between flap and root surface [START_REF] Haney | Periodontal repair in dogs: expanded polytetrafluorethylene barrier membrane support wound stabilisation and enhance bone regeneration[END_REF], Sigurdsson et al 1994, Cortellini et al 1995, Tonetti et al 1996[START_REF] Wikesjo | Periodontal repair in dogs: gingival tissue occlusion, a critical requirement for guided tissue regeneration[END_REF][START_REF] Kim | Periodontal repair in surgically created intrabony defects in dogs. Influence of the number on bone walls on healing response[END_REF], the stability of the blood clot to maintain a continuity with the root surface avoiding formation of a long junctional epithelium [START_REF] Linghorne | Studies in the regeneration and reattachment of supporting structures of teeth. I. Soft tissue reattachment[END_REF][START_REF] Hiatt | Repair following mucoperiosteal flap surgery with full gingival retention[END_REF][START_REF] Wikesjo | Periodontal repair in dogs: effect of wound stabilisation on healing[END_REF][START_REF] Haney | Periodontal repair in dogs: expanded polytetrafluorethylene barrier membrane support wound stabilisation and enhance bone regeneration[END_REF], and the soft tissue protection to avoid bacterial contamination [START_REF] Selvig | Regenerative surgery of intrabony periodontal defects using e-PTFE barrier membranes. Scanning electron microscopic evaluation of retrieved membranes vs. clinical healing[END_REF], Tonetti et al 1993[START_REF] Nowzari | Periodontal pathogens on polytetrafluoroethylene membrane for guided tissue regeneration inhibit healing[END_REF], De Sanctis et al. 1996[START_REF] Sanz | Treatment of intrabony defects with enamel matrix proteins or barrier membranes: results from a multicenter practice-based clinical trial[END_REF].

Development of periodontal regenerative medicine in the last 25 years has followed two distinctive, though totally interlaced paths. The interest of researchers has so far focused on regenerative materials and products on one side and on novel surgical approaches on the other side.

In the area of materials and products, three different regenerative concepts have been mainly explored: barrier membranes, grafts, and wound healing modifiers, plus many combinations of the aforementioned.

In the area of the surgical approaches, clinical innovation in flap design and handling, has radically changed surgery and has allowed a drastic limitation of interdental wound failure from 100% with conventional flap approaches to less than 10% with the more modern approaches. The modified minimally invasive surgical technique (M-MIST, Cortellini and Tonetti 2009) has been recently proposed and tested. The M-MIST was designed to: i) improve flap stability; ii) maintain space for regeneration; and iii) preserve an increased portion of the blood supply at the level of the crest and the papilla. The surgical approach consists of a limited interdental incision in which only a buccal triangular flap is elevated, while the papilla is left in place, connected to the root of the crest-associated tooth with its supracrestal fibres. The palatal/lingual tissues are not involved in the surgery. A similar approach used in combination with a bioresorbable barrier, has been recently proposed by [START_REF] Trombelli | Single-flap approach with buccal access in periodontal reconstructive procedures[END_REF]. Clinical and radiographic outcomes were evaluated at 1 year.

Study population

Patients with advanced periodontal disease, in general good health, presenting with at least one isolated deep, predominantly interdental intrabony defect were considered eligible for this study. Patients were included after completion of cause related therapy consisting of scaling and root planing, motivation and oral hygiene instructions. Flap surgery for pocket elimination in sites different from the experimental ones was performed, when indicated, before the regenerative treatment. All subjects gave written informed consent. Inclusion/exclusion criteria were as previously reported (Cortellini and Tonetti 2009). Patients were enrolled three months after completion of periodontal therapy, when baseline clinical measurements were recorded.

Sample Size and Randomization

The primary outcome of the study was clinical attachment level gain at 12 months. Changes in probing pocket depths and percentage fill of the radiographic intrabony component of the defect were secondary outcomes. Sample size was set at 15 subjects/treatment arm based on logistic considerations, the results of a previously reported case series (Cortellini and Tonetti 2009) and the pilot nature of this trial.

After verification of the entry criteria, 45 subjects gave informed consent and were enrolled into the study. Subjects were assigned a patient number and were randomly assigned to one of the three treatment regimens. Assignment was performed by a clinical research support infrastructure (ERGOPerio, Genova, Italy) using a custom made program based on balanced random permuted blocks. Furthermore, to reduce the chance of unfavourable splits among groups in terms of key prognostic factors, the randomization process balanced average clinical attachment levels and probing pocket depths. To conceal assignment, opaque envelopes were assigned to the specific subject and were opened during

Clinical measurements at baseline and at 1-year follow-up visit

The following clinical parameters were evaluated at baseline before regenerative therapy and at the 1-year follow up visit by a single calibrated clinician (MT). Full mouth plaque scores (FMPS) were recorded as the percentage of total surfaces (4 aspects per tooth) with the presence of plaque (O Leary 1972). Bleeding on probing (BOP) was assessed dichotomously and full mouth bleeding scores (FMBS) were then calculated (Cortellini et al 1993a) Probing pocket depth (PPD) and recession of the gingival margin (REC) were recorded to the nearest millimeter at the deepest location of the selected interproximal site. All measurements and BOP were taken with a pressure sensitive manual periodontal probe at 0.3 N (Brodontic probe equipped with a PCP-UNC 15 tip, Hu-Friedy, Chicago, IL). Clinical attachment levels (CAL) were calculated as the sum of PPD and REC.

Periapical radiographs were taken at baseline and 1-year as previously described (Tonetti et al 1993b). The baseline radiographic defect angle; the distance between the cemento-enamel junction and the bottom of the defect (X-ray CEJ-BD); the distance between the cemento-enamel junction and the interdental bone crest (X-ray CEJ-BC) were performed on high resolution scanned radiographs (8 bit, 1200 dpi) using the program ImageJ (NIH, Bethesda, USA). The radiographic Primary closure of the flaps and early healing events were evaluated at completion of surgery and at weekly recalls for a period of 6 weeks.

Clinical characterization of the intrabony defects

Defect morphology was characterized after flap elevation and debridement in terms of distance between the cemento-enamel junction and the bottom of the defect (CEJ-BD) and total depth of the intrabony component of the defect (INFRA), essentially as previously described (Cortellini et al 1993b). The defects were described as 1, 2, 3-wall or combination defects.

Surgical and patient outcomes

Surgical-time was measured with a chronograph, starting at delivery of local anesthesia through the completion of sutures. Primary closure of the flap was checked with mgnification at the end of surgery and weekly for 6 weeks. The presence of a discontinuity in the soft tissues was registered as wound failure.

Patients were asked at the end of surgery to report about intra-operative pain and personal feeling of the hardship of the procedure. A visual-analog scale (VAS) 10 cm long was used to indicate the intensity (0=no pain/hardship; 10=unbearable pain/hardship). Patients were asked at week one for their experience with postoperative pain and discomfort using a standard questionnaire; pain intensity was quantified with a VAS essentially as described (Cortellini et

Surgical approach (M-MIST)

All the surgical procedures were performed as previously described (Cortellini and Tonetti 2009, Video Clip 1) with the aid of an operating microscope (Global Protege, St Louis, Mo) at a magnification of 4X to 16X (Cortellini & Tonetti 2001, 2005). The defect-associated interdental papilla was surgically approached either with a diagonal incision following the pattern of the simplified papilla preservation flap when the width of the interdental space was 2mm or narrower (SPPF, [START_REF] Cortellini | The simplified papilla preservation flap. A novel surgical approach for the management of soft tissues in regenerative procedures[END_REF] or with a horizontal incision according to the modified papilla preservation technique at interdental sites wider than 2mm (MPPT, Cortellini et al 1995). Flap elevation was limited to the buccal flap. No interdental and/or lingual intrasulcular incisions were performed. The supra-crestal interdental tissues, therefore, i) remained attached to the root cement of the crest-associated tooth with its supracrestal fibers, ii) maintained continuity with the palatal tissue and iii)

were not elevated or displaced. After removal of the granulation tissue by careful dissection and root debridement, the root surface was chemically conditioned with a 2-minute application of an EDTA gel (Preph-gel, Straumann AG, Basel, Switzerland). The randomization envelope was opened and treatment continued based on the assignment.

A single modified internal mattress suture was positioned at the defectassociated interdental area (6-0 or 7-0 e-PTFE Gore-tex, WL Gore & Associates, Flagstaff AZ, USA). In the control group, that did not receive any regenerative materials, the suture was tightened to reach primary closure of the defectassociated papilla (Cortellini & Tonetti 2001, 2005, 2007).

In the two test groups that received additional regenerative materials the suture was left loose. In the EMD test group, EMD was applied on the rinsed and airdried root surface. In the EMD BMDX test group the xenograph was mixed with EMD on a sterile plate. EMD was applied on the rinsed and air-dried root surface and the mixture of EMD and BMDX was positioned into the defect with a sterile instrument with no attempt to tightly pack the material into the defect or to overfill it. When the regenerative materials were properly in place the suture was tightened to reach primary closure of the defect-associated papilla.

The post-operative regimen was as previously described (Tonetti et al 2002, Cortellini andTonetti 2009). Patients were requested to avoid brushing, flossing and chewing in the treated area. At week 1, sutures were removed and patients resumed careful tooth-brushing with a soft tooth-brush (Vitis Surgical, Barcelona, Spain). Patients resumed interdental cleaning after 3 to 4 weeks. At week 4, patients performed full oral hygiene and resumed mastication in the treated area.

Weekly prophylaxis was delivered for 6 weeks. At the end of the "early healing phase", patients were placed on a 3-months recall system for 1 year. (College Station, Texas, USA). Post-hoc power analysis was calculated using the program G*Power 3.1 [START_REF] Faul | G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences[END_REF].

Comparisons between baseline and 1 year measurements within each group were performed applying the paired Student t-test (α=0.05). Comparisons among the experimental groups at baseline and at 1 year were performed applying the ANOVA. Radiographic percent fill of the baseline intrabony component of the defect was calculated as: Bone fill% = (X-ray Bone Gain)/ X-ray INFRA * 100.

Results

Experimental population and surgical approach

Forty-five subjects were enrolled in this 3-arm randomized controlled clinical trial.

The M-MIST alone was applied in 15 subjects (mean age 48.9±7.9, range 34 to 59 years, 6 females, 1 smoker). The M-MIST plus EMD was applied in 15 subjects (mean age 47.2±8.5, range 34 to 64 years, 8 females, 2 smokers). The M-MIST plus EMD-BMDX was applied in 15 subjects (mean age 53.5±11.9, range 28 to 71 years, 7 females, 2 smokers). No subject discontinued participation in the study and no data points were missing for analysis (Figure 1).

Baseline subject and defect characteristics are described in Table 1. The defect morphology of the 3 groups was well matched in terms of width of the defect and number of residual bony walls. The defects were mainly combinations of 3, 2 and 1-wall components. A 1-wall component was present in the majority of sites ( 9sites in the M-MIST group and 10 sites in the other two). No differences were detected in any of the baseline clinical measurements among the 3 groups.

Postoperative course and early healing phase

The surgical time was rather short for all the 3 procedures. The shortest was recorded by M-MIST alone (average time 52.9±5.6 minutes, min 45', max 63'), followed by the M-MIST EMD (average time 54.2±7.4 minutes, min. 42', max. None of the patients reported intra-operative pain or personal feeling of hardship of the procedure at the end of surgery. At 1 week none of the patients reported having experienced post-operative pain. Slight discomfort was reported by 3 patients of the M-MIST group (average VAS value 10.7±2.1), by 2 patients of the M-MIST EMD (average VAS value 11.5±0.7) group and by 4 patients of the M-MIST EMD+BMDX one (average VAS value 12.3±3.1). Few patients needed pain control medications (ibuprofen): 3 patients from the control group (average number of 600 mg pills 0.4±0.7, max 2), 4 patients from the EMD group (average 0.3±0.6, max 2), 4 patients from the EMD+BMDX group (average 0.5±1, max 3).

1-year clinical outcomes

At 1 year, the 3 groups presented with low levels of FMPS and FMBS, shallow residual pockets, significant amounts of CAL gains and limited increase in gingival recession (table 2). Differences between baseline and 1 year were statistically significant in the 3 groups in terms of PPD reduction (P<0.0001 for all groups, t-test) as well as in terms of CAL gain (P<0.0001 for all groups, t-test).

Minor changes in the position of the gingival margin occurred between baseline and 1 year in the 3 groups (average recession increase of 0.3mm): the difference between baseline and 1 year was not statistically significant in the M-MIST and in the M-MIST EMD+BMDX groups, while reached statistical significance in the M-MIST EMD one (P=0.02).

Comparisons among the 3 groups showed no statistically significant difference in any of the measured clinical outcomes (analysis of variance, table 2). In particular, CAL gains of 4,1±1,4mm were observed in the M-MIST control group, 4,1±1,2mm in the EMD group, and 3,7±1,3mm in the EMD+BMDX one.

The frequency distribution of gains in CAL is reported in table 3. None of the sites gained less than 2mm of attachment, while 73.3% of the M-MIST group, 60% of the EMD group, and 46.6% of the EMD+BMDX group gained 4mm or more.

Bone changes were measured on scanned radiographs (table 4). The 3 groups gained a substantial amount of bone at 1 year as compared to baseline. The percent bone fill of the intrabony component was 77±19% in the M-MIST control group, 71±18% in the EMD group and 78±27% in the EMD+BMDX group. Differences among groups were not statistically significant.

  The aim of the present 3-arm randomised controlled pilot clinical trial was to initially compare the clinical efficacy of the "modified minimally invasive surgical technique" alone versus the clinical efficacy of the M-MIST combined with two well-recognised regenerative materials, enamel matrix derivative (EMD) alone and in combination with Bone Mineral Derived Xenograph (BMDX), in the treatment of isolated, interdental intrabony defects. parallel group, randomized, controlled clinical trial comparing the efficacy of 3 treatment modalities in 45 intrabony defects. A single defect was treated in each patient. All the experimental sites were accessed with the modified minimally invasive surgical technique (M-MIST, Cortellini and Tonetti 2009) and carefully debrided. EDTA was applied on the instrumented and dried root surfaces. Enamel matrix derivative (EMD: Emdogain, Institute Straumann AG, Basel, Switzerland) was applied to the debrided root surface in one group (15 defects); EMD plus Bone Mineral Derived Xenograph (BMDX, BioOss, Gestlich, Switzerland) in another group (15 defects). The third group (15 defects) did not receive the application of any regenerative material / device. Flaps were sutured with modified internal mattress sutures. Patients were enrolled in a stringent postoperative supportive care program with weekly recalls for 6 weeks, and then included in a 3-months periodontal supportive care program for 1 year.

  debridement and EDTA application. Every defect, therefore, received EDTA at the end of defect / root instrumentation.

  componet (X-ray INFRA) was calculated as (CEJ BD) -(CEJ BC).

  al 2001, Tonetti et al

  as means ± standard deviation of 15 defects in 15 patients. All calculations were performed using the software Stata version 11.1

  67'). Slightly more time was required for M-MIST EMD+BMDX that accounted for 58.9±6.2 minutes, on average (min 45', max 63'). The difference among groups reached statistical significance (P=0.036, analysis of variance).

  Primary closure was obtained in all treated sites at completion of surgery. All the treated sites remained closed during the 6 weeks of early healing period with the exception of one M-MIST EMD+BMDX site that presented at suture removal (week 1) with a slight discontinuity of the interdental wound on the side of the defect-associated tooth. Few BMDX granules were surfacing the soft tissues and were carefully removed. At week 2 the gap appeared closed. No oedema or haematoma was noted in any of the treated sites.

  When a defect wraps around the lingual aspect of a tooth, elevation of the interdental soft tissues becomes necessary and a Minimally Invasive Surgical Technique (MIST) becomes the preferred approach.Independent full-scale clinical trials are needed to confirm the results of the present study. It is also important to determine the exact nature of the healing observed following application of M-MIST as the clinical healing observed in this study does not necessarily equate to periodontal regeneration. If such research will confirm these results and provide histological evidence of periodontal regeneration, M-MIST may become the procedure of choice for the treatment of interdental intrabony defects.

Figure 2a .

 2a Figure 2a. Site treated with M-MIST alone. Lower right lateral incisor presenting

Figure 2d .

 2d Figure 2d. Primary closure of the defect associated papilla was obtained with an

Figure 2e .

 2e Figure 2e. The 1 year photograph shows a 3mm residual PD and a CAL of 8mm.

Figure 2f .

 2f Figure 2f. Complete radiographic resolution of the intrabony component at 1 year.

Figure 3a .

 3a Figure 3a. Site treated with M-MIST and EMD. Lower left second bycuspid

  Figure 3d. EMD has been applied on the air dried root surface and primary

Figure 3e .

 3e Figure 3e. The 1 year photograph shows a 2mm residual PD and a CAL of 5mm.

Figure 3f .

 3f Figure 3f. The 1 year radiograph shows the almost complete resolution of the

Figure 4a .

 4a Figure 4a. Site treated with M-MIST and EMD plus BMDX. Upper left central

Figure 4d .

 4d Figure 4d. EMD plus BMDX has been applied on the air dried root surface and

Figure 4e .

 4e Figure 4e. Postoperative radiograph showing the BMDX grafted to fill the

Figure 4f .

 4f Figure 4f. The 1 year photograph shows the nice preservation of the interdental

Figure 4g .

 4g Figure 4g. The 1 year radiograph shows the complete resolution of the intrabony

  Figure 1. Consort diagram

Table 1 .

 1 Baseline patient and defect characteristics of the 3 experimental groups (N=45).

	M-MIST (N=15)	M-MIST+EMD (N=15)	M-MIST + EMD BMDX	Significance
			(N=15)

Table 2 .

 2 Clinical outcomes at 1 year (N=45).

				M-MIST +
		M-MIST	M-MIST+EMD	EMD BMDX	Significance
		(N=15)	(N=15)	(N=15)
	FMPS (%)	10,2±4,4	9,9±4,0	10,6±4,8	P=0,925
	FMBS (%)	7,0±5,2	5,7±3,0	7,0±3,6	P=0,605
	PD (mm)	3,1±0,6	3,4±0,6	3,3±0,6	P=0,327
	REC (mm)	2,4±1,4	2,3±1,4	3,1±2,1	P=0,354
	CAL (mm)	5,5±1,6	5,7±1,7	6,4±2,4	P=0,397
	Delta PD (mm)	4,4±1,6	4,4±1,2	4,0±1,3	P=0,657
	Delta REC (mm) -0,3±0,6	-0,3±0,5	-0,3±0,7	P=1
	CAL Gain (mm)	4,1±1,4	4,1±1,2	3,7±1,3	P=0,639

Table 3 .

 3 Frequency distribution of CAL gains at 1 year (N=45).

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				M-MIST +
		M-MIST	M-MIST+EMD	EMD BMDX
		(15)	(15)	(15)
	Cal Changes (mm) N of sites	N of sites	N of sites
	0-1 mm	0	0	0
	2-3 mm	4	6	8
	4-5 mm	9	7	6
	≥6 mm	2	2	1

Table 4 .

 4 Baseline radiographic measurements and 1-year radiographic outcomes (N=45).

		M-MIST (N=15)	M-MIST+EMD (N=15)	M-MIST + (N=15) EMD BMDX	Significance
	CEJ-BD 0 (mm) 9,0±1,7	9,3±2,0	10,5±2,1	P=0,102
	INFRA (mm)	4,7±1,0	4,7±1,3	4,5±1,3	P=0,128
	CEJ-BD 1 (mm) 5,5±1,3	6,0±1,5	7,2±2,1	P=0,025
	Bone gain (mm) 3,5±1,0	3,3±1,2	3,3±1,1	P=0,815
	Bone fill (%)	77±19	71±18	78±27	P=0,603

Journal of Clinical Periodontology -PROOFJournal of Clinical Periodontology -PROOF

Acknowledgements

This study was partly supported by the Accademia Toscana di Ricerca Odontostomatologica, Firenze Italy and the European Research Group on Periodontology (ERGOPerio), Berne, Switzerland.

this study has been self-supported by the authors, the European Research Group on Periodontology and the Accademia Toscana di Ricerca Odontostomatologica.

Data analysis

Discussion

In this trial the Modified Minimally Invasive Technique alone resulted in pocket depth reductions, clinical attachment level gains and radiographic bone fill better than the reported ranges with access flap surgery in intrabony defects (Tu et al 2009). In particular, these cases expressed a very high clinical healing potential that resulted in the almost complete resolution of the intrabony defects (77±19% radiographic bone fill), good amounts of CAL gains (4.1±1.4mm) and stability of the gingival margin. Wound failure was observed in only one instance (2.2%) treated with EMD + BMDX, and complications and morbidity were minimal.

The additional application of regenerative materials (EMD or EMD plus Bone Mineral Derived Xenograph) did not improve the results as expected from the current literature utilizing more conventional papilla-preservation surgery (Trombelli and Farina 2008). While the power of this initial study to detect a true difference was limited to a difference of 0.96 mm, the fact that the outcomes among the 3 groups could not be discriminated is interesting and raises a series of hypotheses that focus on the intrinsic healing potential of the surgical approach. The M-MIST was designed to optimize wound and blood clot stability, flap margin blood perfusion and provide a stable space for regeneration (Cortellini and Tonetti 2009). The importance of these factors has been systematically assessed in pre-clinical studies and specific solutions have been introduced in periodontal regenerative surgery by progressively evolving flap designs (Cortellini et al 1994[START_REF] Cortellini | The simplified papilla preservation flap. A novel surgical approach for the management of soft tissues in regenerative procedures[END_REF], Cortellini and Tonetti 2001, 2007, 2009).

As previously indicated, the M-MIST is not always applicable (Cortellini et al