Inhibition of MMPs by alcohols

Abstract

Objectives

While screening the activity of potential inhibitors of matrix metalloproteinases (MMPs), due to the limited water solubility of some of the compounds, they had to be solubilized in ethanol. When ethanol solvent controls were run, they were found to partially inhibit MMPs. Thus, the purpose of this study was to compare the MMP-inhibitory activity of a series of alcohols.

Methods

The possible inhibitory activity of a series of alcohols was measured against soluble rhMMP-9 and insoluble matrix-bound endogenous MMPs of dentin in completely demineralized dentin. Increasing concentrations (0.17, 0.86, 1.71 and 4.28 mol/L) of a homologous series of alcohols (i.e. methanol, ethanol, propanols, butanols, pentanols, hexanols, the ethanol ester of methacrylic acid, heptanols and octanol) were compared to ethanediol, and propanediol by regression analysis to calculate the molar concentration required to inhibit MMPs by 50% (i.e. the IC 50 ).

Results

Using two different MMP models, alcohols were shown to inhibit rhMMP-9 and the endogenous proteases of dentin matrix in a dose-dependent manner. The degree of MMP inhibition by alcohols increased with chain length up to 4 methylene groups. Based on the molar concentration required to inhibit rhMMP-9 fifty percent, 2-hydroxyethylmethacrylate (HEMA), 3-hexanol, 3-heptanol and 1-octanol gave the strongest inhibition.

Significance

The results indicate that alcohols with 4 methylene groups inhibit MMPs more effectively than methanol or ethanol. MMP inhibition was inversely related to the Hoy’s solubility parameter for hydrogen bonding forces of the alcohols (i.e. to their hydrophilicity).

Introduction

Dentin matrix metalloproteinases (MMPs) are host-derived proteolytic enzymes bound to the matrix. The MMPs have been detected in hard and soft tissues during tooth development . However, after development, the dentin matrix mineralizes, causing dentin collagen to become very stiff. All noncollagenous proteins bound to collagen, including MMPs, also become covered with apatitic nanocrystals making them immobile and nonfunctional. Mineralized dentin is stable structurally and functionally as long as it remains mineralized .

During adhesive bonding, dentin is acid-etched to remove the smear layer and to demineralize the relatively impermeable dentin matrix to a depth of 1–8 μm to expose the collagen fibrils of the matrix. These 100 nm diameter fibrils remain anchored to and continuous with the underlying mineralized dentin. During bonding, solvated liquid monomers flow around the demineralized collagen fibrils and envelop them with comonomers that are then polymerized. This mixture of resin and insoluble collagen fibrils is called the hybrid layer . It is a type of in situ tissue engineering in that it completely transforms the physicochemical nature of the surface from a wet hydrophilic crystalline surface to a more hydrophobic polymer surface that allows adhesion to resin composites. Unfortunately, acid-etching also uncovers and activates matrix-bound MMPs . These MMPs seem to remain active even after resin-infiltration . As hydrolases, these enzymes add water across specific collagen peptide bonds, splitting peptide chains into fragments. If there is no water in the hybrid layer, the MMPs are inactive. For instance, incubation of resin–dentin bonds in oil instead of water allows those bonds to remain stable over time . Resin–dentin bonds incubated in water show progressively lower bond strengths over 3–12 months . Transmission electron microscopy of hybrid layers in specimens exhibiting progressive decreases in bond strength, reveal loss of cross-banded collagen fibrils in the hybrid layer . We speculated that endogenous MMPs in hybrid layers are responsible for the degradation of bond strengths and that this might be prevented using chlorhexidine, an antimicrobial agent also known to inhibit MMP-2, 8 and 9 . The success of 2% chlorhexidine at preserving hybrid layers in vivo was shown by Hebling et al. , Carrilho et al. , Brackett et al. and Loguercio et al. . They used 2% chlorhexidine digluconate in water. To avoid incorporating water into the hybrid layer, we screened alcoholic solutions of chlorhexidine diacetate for their ability to inhibit the endogenous MMPs of dentin matrix. When alcohol controls were run, we were surprised to find that alcohols inhibited the endogenous collagenolytic activity of dentin matrices (see results in this study).

To evaluate this phenomenon more thoroughly, a series of alcohols were used (methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol and octanol) to inhibit soluble rhMMP-9 and insoluble matrix-bound MMPs. The purpose of this study was to test the null hypotheses that there are no differences among various alcohols in their ability to inhibit MMPs, and that there are no differences in alcohol inhibition of MMPs regardless of their hydrophilicity.

Materials and methods

Experimental design

Two tiers of screening were used to evaluate the inhibitory activity of alcohols on MMPs. The first level of screening involved the use of a soluble recombinant human MMP-9 (rhMMP-9) in a colorimetric assay. The alcohols were diluted with distilled water to achieve concentrations of 0.17, 0.86, 1.71 and 4.28 mol/L if they were sufficiently soluble in water and did not cause precipitation of medium constituents. The second level of screening, used matrix-bound endogenous MMPs of completely demineralized dentin over a 30 day incubation. Both assays require the use of an incubation buffer containing calcium and zinc ions that are necessary to maintain enzyme structure and function. Preliminary experiments were done to determine the highest concentration of alcohols that could be achieved in the assay buffer. Some of the higher molecular weight alcohols were not completely soluble in the test buffers and could not be tested at all desired concentrations. Their highest soluble concentrations are listed in the tables that summarize the results. Because the second level of screening was labor-intensive and required 30 days, we only tested alcohols that reduced rhMMP-9 activity by over 50% and that were compatible with the calcium–zinc containing buffer. The alcohols that are listed are the highest concentrations that were completely soluble in the assay buffer along with the percent inhibition obtained at those concentrations. Lower concentrations of alcohols were then tested to permit calculation of the alcohol concentrations required to inhibit rhMMP-9 by 50%. This permitted comparison of the inhibitory potencies of each alcohol.

Use of soluble human recombinant MMP-9

This assay employed purified recombinant human MMP-9 (Cat#72009) and the Sensolyte Generic MMP colorimetric assay kit (Cat#72095) from AnaSpec, Inc. (San Jose, CA, USA) for screening anti-MMP activity of compounds of interest. The assay involves incubating a constant concentration of rhMMP-9 with a proprietary chromogenic substrate. The latter is a thiopeptolide that is cleaved by the MMPs and collagenases to release a sulfhydryl group. The sulfhydryl group reacts with 5,5′-dithiobis(2-nitrobenzoic acid) to produce the colored reaction product (2-nitro-5-thiobenzoic acid) which can be detected at 412 nm.

The thiopeptolide substrate solution was diluted to 0.2 mM with the supplied assay buffer in a 1:50 volume ratio. The 92-kDa rhMMP-9 was activated with 10 μg/mL of trypsin at 37 °C for 2 h immediately before the experiment to generate its 68-kDa active form. The trypsin was then inactivated with trypsin inhibitor. The assay was performed in a 96-well plate using five replicate wells for each experimental and control variable. Each well in the experimental groups (i.e. the various alcohol concentrations) contained 2 μL of rhMMP-9 (19.6 ng/well), X μL of the potential MMP inhibitor and 50 − (2 + x ) μL of the thiopeptolide substrate solution. All wells also received 50 μL of substrate to give a total volume in each well of 100 μL.

The control groups consisted of: (1) a positive control containing rhMMP-9 only without the potential anti-MMP agent, (2) an inhibitor control containing rhMMP-9 and 10 μL of 20 μM GM6001, a known MMP inhibitor supplied in the assay kit, (3) a test compound control containing assay buffer and the potential anti-MMP agent, and (4) a substrate control containing assay buffer. Additional assay buffer was added to bring the total volume of all control wells to 50 μL prior to adding 50 μL of the thiopeptolide substrate solution to obtain the 100 μL volume. The alcohol inhibitors were preincubated with the enzyme for 30 min to prevent a burst of uninhibited enzyme activity that occurred if all of the reagents were mixed together.

The reagents were mixed completely by vibrating the plate gently for 30 s and then read kinetically every 10 min for 60 min and absorbance was measured at 412 nm using a 96-well plate reader (Synergy HT, BioTek Instruments, Inc., Winooski, VT, USA).

Background absorbance was determined using the mean absorbance readings from the “substrate control” wells and subtracted from the readings of other wells containing the thiopeptolide substrate. The potencies of MMP-9 inhibition by the proprietary MMP inhibitor GM6001 (“inhibitor control”), and the alcohol inhibitors were expressed as percentages of the adjusted absorbance of the “positive control”, which was taken to be the maximum absorbance.

Preparation of demineralized dentin beams

Forty extracted human third molars were obtained with patient (18–22 year old) informed consent under a protocol approved by the Human Assurance Committee of the Medical College of Georgia. Ninety percent of the teeth had completely formed roots. None of the teeth were carious. Half were impacted and half were erupted. The teeth were stored at 4 °C in 0.9% NaCl supplemented with 0.02% sodium azide to prevent bacterial growth and used within three months after extraction. For each tooth, the enamel and superficial dentin were removed by horizontal sectioning 1 mm below the deepest central fissure using a diamond-coated copper disk (Isomet saw, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. A 1 mm thick dentin disk consisting of mid-coronal dentin was prepared from each tooth. Two 5 × 2 × 1 mm dentin beams were prepared from the middle of each disk under water cooling to obtain eighty beams.

Prior to demineralization, a dimple was made on the occlusal side of each beam at the end to allow for repeated measurements to be performed on the same surface. The beams were tumbled at 3 rpm in 10% phosphoric acid for 16 h at 25 °C to completely demineralize the dentin. Absence of residual minerals was confirmed using digital radiography. The initial modulus of elasticity of each demineralized dentin beam was determined using three-point flexure (see below). The beams were distributed to seven experimental groups and one control group ( N = 10) so that the mean initial elastic modulus of each group was similar in all groups.

Use of endogenous matrix-bound MMPs in beams of demineralized dentin

A recently developed assay uses beams of human coronal dentin that are completely demineralized. Using a miniature 3-point flexure fixture, the stiffness of the beams is measured before and after demineralization. Mineralized dentin has a stiffness of about 17–18,000 MPa, while completely demineralized dentin has a stiffness of 2–3 MPa. The control beams were separately incubated in a simplified medium containing 2.5 mM calcium chloride, 0.05 mM ZnCl 2 , 5 mM HEPES buffer pH 7.4, 0.3 mM NaN 3 , and 150 mM NaCl, pH 7.4. Many high molecular weight alcohols caused precipitations of more complex media and could not be tested.

Experimental beams were incubated in the same medium that contained twice the concentration of its constituents as the control group to permit dilution of the buffer by potential inhibitors to the media. Additional water was then added to dilute the final concentration of the medium to that of the control group. All beams were separately incubated at 37 °C in a shaking water bath (Precision Model 2873, Thermo Scientific, Marietta, OH, USA) in 1 mL of media in polypropylene tubes with a rubber “O” ring in the screw cap to prevent evaporation of water or inhibitors. The following variables were measured: decreases in dentin beam stiffness were measured by 3-point flexure at time 0 and 4 weeks using a Vitrodyne universal tester with a 1 N load cell on supports separated by 2.5 mm at 10% strain while the beams were under water.

Flexural modulus ( E ) was calculated as:

<SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='E=mL34bd3′>E=mL34bd3E=mL34bd3
E = m L 3 4 b d 3

m = slope of stress curve (N/mm); L = support span length (mm); b = width of beam (mm); d = thickness of beam (mm).

As the ASTM D790-03 standard for measuring the stiffness of polymeric beams is done at low strains and requires that the beam thickness be no more than one-sixteenth the length of the supported beam, our conditions violate those criteria and hence only provide an approximate stiffness value. However, the method is capable of accurately detecting differences in stiffness between groups.

Loss of beam stiffness over time was used as an indirect measure of the hydrolysis of matrix by endogenous MMPs. The loss of beam stiffness of experimental beams incubated with potential inhibitors was compared to that of controls. After measuring the initial modulus of elasticity, the beams were rinsed with water for 30 s to remove all media salts. They were then placed in sealed containers of anhydrous calcium sulfate (Drierite, W.A. Hammond Drierite Company, Ltd., Xenia, OH, USA) overnight. The next day, the dry mass of each beam was measured using a microanalytical balance to the nearest 0.01 mg. Pilot experiments indicated that these procedures yielded constant dry masses. These dry masses were measured on each beam at time 0 and 30 days. Each beam was rehydrated in water for 1 h before being placed in its incubating medium. Previous work showed that this was sufficient time for complete re-expansion of the dried beams . Loss of dry mass over time indicates solubilization of matrix by endogenous MMP activity because effective MMP inhibitors prevent the loss of dry mass over time.

The third index of matrix degradation over time was obtained by measuring the amount of collagen peptide fragments that were solubilized over 30 days of incubation. That is, each beam was incubated in 1 mL of medium. Any collagen peptide fragments that accumulated in the medium over 30 days could be quantitated by removing 400 μL of the 1 mL and mixing it with an equal volume of concentrated HCl to yield a final acid concentration of 6 N HCl in glass ampoules (Wheaton, Millville, NJ, USA). These vials were automatically sealed using an Ampulmatic Ampule Sealer (Bioscience, Inc., Allentown, PA, USA). The contents were hydrolyzed to amino acids in an oil bath at 118 °C for 18 h. After cooling, the glass vials were opened via prescored lines and placed in large glass dessicators containing NaOH pellets to trap HCl vapor and anhydrous calcium sulfate to trap water vapor. After 3 days in the vacuum dessicators, the dry contents of the vials were analyzed for hydroxyproline (HYP) using the colorimetric assay of Jamall et al. . After color development, the absorbance of all specimens and standards was measured at 558 nm in a 96-well plate reader.

Effective inhibitors lowered the concentration of hydroxyproline (HYP) in the medium hydrolysate relative to that of controls which contained no inhibitors.

MMP inhibitors

All alcohols were purchased from Sigma Chemicals (St. Louis, MO, USA) and were used as received. They included methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, tert-butanol, 2-hydroxyethylmethacrylate (HEMA), 1-pentanol, hexanols, heptanols, octanols, 1,2-ethanediol, and 1,3-propanediol.

Statistics

Regression analyses were made of the degree of inhibition of rhMMP-9 by the highest soluble alcohol concentrations to permit calculation of percent inhibition. Those alcohols that inhibited rhMMP-9 at their highest solubility were then reanalyzed at lower concentrations (e.g. 1.71, 0.86 and 0.17 mol/L) to determine the dose–response relationship of that alcohol. Using those regression equations, we calculated the highest soluble molar concentration of alcohols required to inhibit MMPs by 50% and designated this as Inhibitory Concentration 50 (IC 50 ). This mathematical construct allows one to compare the same inhibitory activities of inhibitors with variable solubilities. The measured changes in beam stiffness, loss of dry mass and solubilization of collagen peptides were not normally distributed. As the normality and homoscedasticity assumptions of the data appeared to be violated, the data were expressed as least square means and the common standard error of the least square means. Least square means are the expected values of group or subgroup means for a balanced design involving the group variables with all covariates at their common mean values. Pair-wise multiple comparisons were performed using the Tukey test. Statistical significance was set at α = 0.05.

Materials and methods

Experimental design

Two tiers of screening were used to evaluate the inhibitory activity of alcohols on MMPs. The first level of screening involved the use of a soluble recombinant human MMP-9 (rhMMP-9) in a colorimetric assay. The alcohols were diluted with distilled water to achieve concentrations of 0.17, 0.86, 1.71 and 4.28 mol/L if they were sufficiently soluble in water and did not cause precipitation of medium constituents. The second level of screening, used matrix-bound endogenous MMPs of completely demineralized dentin over a 30 day incubation. Both assays require the use of an incubation buffer containing calcium and zinc ions that are necessary to maintain enzyme structure and function. Preliminary experiments were done to determine the highest concentration of alcohols that could be achieved in the assay buffer. Some of the higher molecular weight alcohols were not completely soluble in the test buffers and could not be tested at all desired concentrations. Their highest soluble concentrations are listed in the tables that summarize the results. Because the second level of screening was labor-intensive and required 30 days, we only tested alcohols that reduced rhMMP-9 activity by over 50% and that were compatible with the calcium–zinc containing buffer. The alcohols that are listed are the highest concentrations that were completely soluble in the assay buffer along with the percent inhibition obtained at those concentrations. Lower concentrations of alcohols were then tested to permit calculation of the alcohol concentrations required to inhibit rhMMP-9 by 50%. This permitted comparison of the inhibitory potencies of each alcohol.

Use of soluble human recombinant MMP-9

This assay employed purified recombinant human MMP-9 (Cat#72009) and the Sensolyte Generic MMP colorimetric assay kit (Cat#72095) from AnaSpec, Inc. (San Jose, CA, USA) for screening anti-MMP activity of compounds of interest. The assay involves incubating a constant concentration of rhMMP-9 with a proprietary chromogenic substrate. The latter is a thiopeptolide that is cleaved by the MMPs and collagenases to release a sulfhydryl group. The sulfhydryl group reacts with 5,5′-dithiobis(2-nitrobenzoic acid) to produce the colored reaction product (2-nitro-5-thiobenzoic acid) which can be detected at 412 nm.

The thiopeptolide substrate solution was diluted to 0.2 mM with the supplied assay buffer in a 1:50 volume ratio. The 92-kDa rhMMP-9 was activated with 10 μg/mL of trypsin at 37 °C for 2 h immediately before the experiment to generate its 68-kDa active form. The trypsin was then inactivated with trypsin inhibitor. The assay was performed in a 96-well plate using five replicate wells for each experimental and control variable. Each well in the experimental groups (i.e. the various alcohol concentrations) contained 2 μL of rhMMP-9 (19.6 ng/well), X μL of the potential MMP inhibitor and 50 − (2 + x ) μL of the thiopeptolide substrate solution. All wells also received 50 μL of substrate to give a total volume in each well of 100 μL.

The control groups consisted of: (1) a positive control containing rhMMP-9 only without the potential anti-MMP agent, (2) an inhibitor control containing rhMMP-9 and 10 μL of 20 μM GM6001, a known MMP inhibitor supplied in the assay kit, (3) a test compound control containing assay buffer and the potential anti-MMP agent, and (4) a substrate control containing assay buffer. Additional assay buffer was added to bring the total volume of all control wells to 50 μL prior to adding 50 μL of the thiopeptolide substrate solution to obtain the 100 μL volume. The alcohol inhibitors were preincubated with the enzyme for 30 min to prevent a burst of uninhibited enzyme activity that occurred if all of the reagents were mixed together.

The reagents were mixed completely by vibrating the plate gently for 30 s and then read kinetically every 10 min for 60 min and absorbance was measured at 412 nm using a 96-well plate reader (Synergy HT, BioTek Instruments, Inc., Winooski, VT, USA).

Background absorbance was determined using the mean absorbance readings from the “substrate control” wells and subtracted from the readings of other wells containing the thiopeptolide substrate. The potencies of MMP-9 inhibition by the proprietary MMP inhibitor GM6001 (“inhibitor control”), and the alcohol inhibitors were expressed as percentages of the adjusted absorbance of the “positive control”, which was taken to be the maximum absorbance.

Preparation of demineralized dentin beams

Forty extracted human third molars were obtained with patient (18–22 year old) informed consent under a protocol approved by the Human Assurance Committee of the Medical College of Georgia. Ninety percent of the teeth had completely formed roots. None of the teeth were carious. Half were impacted and half were erupted. The teeth were stored at 4 °C in 0.9% NaCl supplemented with 0.02% sodium azide to prevent bacterial growth and used within three months after extraction. For each tooth, the enamel and superficial dentin were removed by horizontal sectioning 1 mm below the deepest central fissure using a diamond-coated copper disk (Isomet saw, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. A 1 mm thick dentin disk consisting of mid-coronal dentin was prepared from each tooth. Two 5 × 2 × 1 mm dentin beams were prepared from the middle of each disk under water cooling to obtain eighty beams.

Prior to demineralization, a dimple was made on the occlusal side of each beam at the end to allow for repeated measurements to be performed on the same surface. The beams were tumbled at 3 rpm in 10% phosphoric acid for 16 h at 25 °C to completely demineralize the dentin. Absence of residual minerals was confirmed using digital radiography. The initial modulus of elasticity of each demineralized dentin beam was determined using three-point flexure (see below). The beams were distributed to seven experimental groups and one control group ( N = 10) so that the mean initial elastic modulus of each group was similar in all groups.

Use of endogenous matrix-bound MMPs in beams of demineralized dentin

A recently developed assay uses beams of human coronal dentin that are completely demineralized. Using a miniature 3-point flexure fixture, the stiffness of the beams is measured before and after demineralization. Mineralized dentin has a stiffness of about 17–18,000 MPa, while completely demineralized dentin has a stiffness of 2–3 MPa. The control beams were separately incubated in a simplified medium containing 2.5 mM calcium chloride, 0.05 mM ZnCl 2 , 5 mM HEPES buffer pH 7.4, 0.3 mM NaN 3 , and 150 mM NaCl, pH 7.4. Many high molecular weight alcohols caused precipitations of more complex media and could not be tested.

Experimental beams were incubated in the same medium that contained twice the concentration of its constituents as the control group to permit dilution of the buffer by potential inhibitors to the media. Additional water was then added to dilute the final concentration of the medium to that of the control group. All beams were separately incubated at 37 °C in a shaking water bath (Precision Model 2873, Thermo Scientific, Marietta, OH, USA) in 1 mL of media in polypropylene tubes with a rubber “O” ring in the screw cap to prevent evaporation of water or inhibitors. The following variables were measured: decreases in dentin beam stiffness were measured by 3-point flexure at time 0 and 4 weeks using a Vitrodyne universal tester with a 1 N load cell on supports separated by 2.5 mm at 10% strain while the beams were under water.

Flexural modulus ( E ) was calculated as:

E=mL34bd3
E = m L 3 4 b d 3
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Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Inhibition of MMPs by alcohols
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