Controlled Release Of Chlorhexidine From Zinc Phosphate Cement Biology Essay

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This study was intended to prepare and characterize a controlled release system based on zinc phosphate cement loaded with chlorhexidine and their release profiles. Chlorhexidine is an antibacterial agent, on topical administration the drug will be washed out because of its flushing action of saliva. To overcome this problem, controlled release method was used to release the drug constantly for a long period. Chlorhexidine was used as active ingredient and incorporated at 1, 2 and 3% concentrations by mass. The release profiles were characterized by HPLC technique and it showed that the release was following diffusion mechanism. The release profiles have shown controlled release of chlorhexidine for a period of 4 weeks. Up to 0.8478% of the total drug released from zinc phosphate cement.

introduction:

The presence of decaying bacteria and other pathogenic organisms brings about the oral health hazards and is the main cause of teeth and implant loss in adult population. It causes vast primary leisions, gingival inflammations, chronic periodontitis, and enamel loss after orthodontic treatment, peri implantitis etc.

The use of mechanical procedures has technical difficulties to access the anatomical structures. Moreover the rough surface of implants favours the attachment and proliferation of micro organisms on the root and implant surfaces causing decontamination.

Hence the method for blocking the pathological process using topical application of antiseptic agents and sustained release of local drugs tremendously increase the benefits of conventional mechanical treatments. "1"

The eradication of microbial flora and it's infection in the oral cavity has been very difficult to be attained as a result of the dilution and rapid elimination of the applied drug, due to flushing of saliva. Therefore the drug delivery system in which drug is incorporated to sustain and prolong the effect, plays a major role.

In order to prolong the retention of drug in oral cavity, the controlled release of oral antiseptic like CHLORHEXIDINE has been extensively studied. Chlorhexidine widely used in clinical dental practise as an antiseptic. Chlorhexidine reduces the adhesion of microbes to oral mucosal cells.

The prolonged retention of drugs like chlorhexidine may be achieved by controlled release drug delivery. A study for by Palmer et al (2004) shows that the chlorhexidine released from experimental glass ionomer cement was very small. The study also stated that higher percentage release of chlorhexidine can be attained. Incorporation of antibacterial into dental cements brought about alterations in the physical properties of cements like restorative retension. This study aimed at understanding the controlled release profile of Chlorhexidine from luting cement- Zinc phosphate cement."2"

Chlorhexidine:

Chlorhexidine is a final irrigant prior to endodontic obturation. Chlorhexidine has the ability to restrict bacterial access and penetration into the dentinal tubules. It has immense range of substantivity (the ability of any oral antimicrobial agent to carry on its therapeutic activity for a prolonged time.), as it possess good anti microbial property and has the ability to adhere to root canal dentin. According to Lindblade R. M. (2010), it does not affect the immediate or the post cementation bonding. Certain irrigants like NaOCl used with EDTA resulted in reducing the immediate composite bond strength towards the root dentin or pulp chamber where as chlorhexidine did not show any of these negative effects rather it improved the immediate bond strength slightly with almost all posts/cements. It inhibits the collagen degrading enzymes, matrix metalloproteases etc and improves the durability of composite adhesive bonding to dentin.

Antiseptic of chlorhexidine usually contain 0.12% chlorhexidine, the chlorhexidine which gets retained in the oral cavity after rinsing are slowly released into the oral fluids. The drug is poorly absorbed from gastro intestinal tract. The excretion is mainly through faces and urine."4"

Structure of Chlorhexidine (5)

N', N''-Bis- (- 4-chlorophenyl)-3, 12 - diamino - 2, 4, 11, 13 tetraazatetradecanedimidamide

Synonyms:-

Chlorhexidine Diacetate, Chlorhexidine gluconate, Chlorhexidine Hydrochloride

Physical Properties:

Molecular formula

C22H30Cl2N10

Molecular weight

505.42

CAS Registry Number

55-56-1

Physical state

White to pale yellow powder

Odour

Odourless

Melting point

132-136oC

pH

5-8

Solubility in Water

Slightly soluble

Stability

Stable under ordinary conditions

Trade Names: Dyna-hex2, Hibatine, Hibaclens

MECHANISM OF ACTION:

Chlorhexidine has antibacterial effect. It is adsorbed on to the organism's cell wall thus, breaks up the integrity of the cell membrane causing the leakage of intracellular components.

USES:

Chlorhexidine is used as mouth wash.

Used as irrigation solution."7"

Used to relieve the pain in the treatment of mouth sorus.

Bacteriostatic spectrum of Chlorhexidine shows wide spectrum of activity against gram +ve bacteria."8"

SIDE EFFECTS9:

Some desquamation and soreness in oral mucosa is observed with the prolonged Chlorhexidine rinsing.

Use of Chlorhexidine has shown a side effect of plaque formation and gingival conditions.

Controlled release:

Controlled release dosage forms are designed to release drug in vivo according to determined rates that can be verified by in vitro measurements. Controlled release technology implies a quantitative understanding of physicochemical mechanism of drug availability to the extent that dosage form release rate can be specified. (lachman)

Recent approaches and potential development to controlled release drug delivery includes hydrodynamic pressure controlled systems, intragastric floatinc tablets, transmucossal tablets and microporus membrane coated tablets.

The basic principle involved in controlled drug delivery systems is to modify the biopharmaceutic, pharmacokinetic and pharmacodynamic properties of drug in such a way that its utility is maximized through reduction in side effects and cure or control of condition in the shortest possible time, by using smallest quantity of drug, administered by the most suitable route."10"

Mechanisms of controlled release:

Controlled release mechanisms can be classified as follows" 11":

1. Dissolution

Matrix

Encapsulation

2. Diffusion

Matrix

Reservoir

3. Combination of both dissolution & diffusion.

4. Osmotic pressure controlled system

Diffusion:

It is a process in which drug molecules diffuse from higher concentration to lower concentration until equilibrium is attained.

Matrix diffusion:

In this type of diffusion, drug is dispersed in an insoluble matrix or swellable hydrophobic substances. Materials for rigid matrix are insoluble plastics such as PVC and fatty materials like bees wax. This follows the Fick's first law of diffusion."10"

Reservoir diffusion:

Here, drug is surrounded by a water insoluble polymer. The release mechanism involves its partitioning into membrane with release into surrounding fluid by diffusion.

Fick's 2nd law of diffusion:

Fick's Second Law states that the rate of change of concentration in a volume element of a membrane, within the diffusional field, is proportional to the rate of change of concentration gradient at that point in the field."13"

Diffusion coefficient:

The rate at which a diffusing substance is transported between opposite faces of a unit cube of a system when there is unit concentration difference between them. Symbol D also called diffusivity

D = S2Ï€l2/4

Advantages"12":

Optimum drug therapeutic drug concentration is maintained in blood or cell.

Better patient acceptance and compliance.

Reduction in fluctuations at plasma drug levels.

Enhancement of activity duration for short half-life drugs.

Frequent dosing, wastage of drug and side effects are reduced.

Predictable and reproducible release rates for extended periods of time.

Disadvantages"12":

Poor in vitro-in vivo correlation.

Less systemic availability compared to immediate release conventional dosage forms.

Possibility of dose dumping.

High formulation cost.

Reduced potential for dose adjustment.

In case of toxicity, poisoning or hypersensitivity reactions recovery of drug is difficult.

Dental cement:

In the treatment of microbial flora of oral cavity by controlled release system use of dental cement as a medium is essential.

Dental cement is a material that produces mechanical effect on hardening and it is prepared by mixing a powder and liquid.

In zinc phosphate cement the powder consists of Zinc oxide and Magnesium oxide, liquid consists of phosphoric acid, water and a buffering agent.

Reasons for using zinc phosphate as cement:

In dentistry zinc phosphate is used as cement due to its various properties such as:

More retentiveness

Setting time

High compressive strength

High solubility

Disintegration.

ZINC PHOSPHATE:

Zinc phosphate is the widely used dental cement in dentistry for placing crowns, inlays, on lays, bridges and orthodontic bonds. It is prepared by mixing powders like zinc oxide and magnesium oxide with liquids like phosphoric acid, water and buffering agent. The chemical reaction that is occurring between the powder and liquid produces heat which depends upon the rate of reaction and size of the mix. The disintegration and solubility of the cement gets increased by increasing the powder-liquid ratio. Setting time and compressive strength of zinc phosphate cement also depends upon the powder liquid ratio. The setting time of cement is around 5-9 minutes. It produces mechanical interlocking effect on hardening and acts as protective, insulating and restorative material.

Chemical formula

Zn2.(PO3)4

Molecular weight

386.11

CAS number

7779-90-0

Physical state

White powder

Melting point

900oc

Density

3.99

Advantages:

Zinc phosphate has more retensive strength compared to other dental cements.

It has inconspicuous appearance.

It can be moulded easily as they form a thin layer for the cementing of crowns and inlays.

It provides easy handling with respect to setting.

It has low thermal conductivity when used beneath the metallic restoration.

It is 3 times stronger than polycarboxylate cements.

Zinc phosphate has shown greater clinical success rate.

Disadvantages:

It shows slight solubility in saliva.

It has crushing strength of 12000 to19000 psi which considered being low in comparison to other cements.

High Performance Liquid Chromatography (HPLC):

Controlled release of chlorhexidine from zinc phosphate cement is analysed by using High Performance Liquid Chromatography (HPLC) by varying concentrations of chlorhexidine.

HPLC is the most versatile and universal type of analytical procedure. This technique is a powerful tool in analysis which uses highly improved form of column packing with smaller particle size, sample forced with high pressure using physically stable pumping system and highly sensitive detectors.

Principle:

A liquid mobile phase is pumped under pressure through a stainless steel column containing particles of stationery phase with a diameter of 3-10 µm. The analyte is loaded onto the head of the column via loop valve and separation of a mixture occurs according to the relative lengths of time spent by its components in the stationary phase. It should be noted that all components in the stationary phase spend more or less the same time in the mobile phase in order to exit the column. Monitoring of the column effluent is carried out with a variety of detectors.

HPLC Separation Modes:

Depending on the characteristics of chemical compounds such as polarity, molecular size, electrical charge, chirality etc different separation modes have been used. The techniques include:

Partition

Adsorption

Ion exchange and ion pair

Size exclusion

Chiral and affinity chromatography

Based on the polar nature of the analyte, two separation techniques such as Normal Phase and Reverse Phase Chromatography are used. There are certain atoms and molecules which attribute to the polar nature of the compounds. The location of these polar functional groups imparts its ability of chromatographic retention. A separation is created based on the relative attraction of each compound in the mixture towards stationary and mobile phase.

Normal Phase Chromatography9 consists of a polar stationary phase having pores large enough to accommodate and attracts the polar particles of the mixture. The mobile phase being non polar elutes away the less polar or non polar components.

Reverse Phase chromatography"9" is the most commonly used analysis strategy in which the stationary phase made of silica is derivatised with long hydrocarbon chains to make it non polar. The mobile phase is non polar and carries the mixture. The polar molecules move faster and longer with mobile phase. The non polar components will slow their way down the column because of attractive force like vanderwaals and dispersion force towards the hydrocarbon chain.

The Silica gets covalently bonded to the hydrocarbons containing C1 - C18 carbons, yielding a thermally and hydrolytically stable stationary phase. It imparts properties to the column as hydrophobic surface, small diameter, spherical particle size distribution etc. The larger the particle size, the broader the peaks become. The relative chromatographic retention of the components of a mixture due to the binding of some components to the stationary phase or the elution in the mobile phase depends on the polarity. The components get separated from each other by giving its characteristic retention time and the corresponding peak. The Chromatogram is obtained by plotting the retention time against the peak area.

Detector:

The commonly used method is UV spectrometer, as many organic molecules absorb UV light of various wave lengths. The amount of light absorbed depend on the amount of a particular analyte under investigation. The sensitivity of detectors is of significant importance, for the better resolution of peaks. For obtaining accurate chromatographic profile, the detector sampling volume should be small.

Pumps:

The solvent is mixed to attain the required gradient using the pumps and force the solvent through the column. Isocratic conditions where the solvent conditions are held constant, which is a very common in quality control analysis to confirm the identity of compounds.

Buffering Agents:

The variation in PH can affect the hydrophobic nature of the analyte. The buffering agents like sodium phosphate are used to control the ph variations. It also helps to neutralise any residual charge on the silica packing of stationary phase. These agents neutralise the charges on the analyte by acting as ion pairing agents.

Retention Time:

Retention time is the time taken by the analyte to elute down the column. It is measured as the time taken for the display to form the maximum peak height from the time of injection of sample. The different components show different retention time which depends on the pressure used, which internally affecting the flow rate of the solvent. The retention time also depends on the column.

Applications:

Accurate and precise quantitative analysis of pharmaceutical products is done by using the combination of HPLC and UV/Visible detectors.

Partition coefficients and pKa values of drugs can be determined.

Determination of drug protein binding.

Pure drug substances stability monitoring in formulations, with quantification of degradation products.

Strengths:

Readily automated.

Limited sample introduction ensures quantitative precision.

Easy handling.

HPLC has most intensive development in recent years, leading to development in columns, detectors and software control.

Availability of variety of columns and detectors helps in the selection of method which can be readily adjusted.

Less risk of sample degradation because heating is not required.

Limitations:

Organic solvents wastage is more, which more expensive to dispose off.

Prior to analysis drugs have to be extracted from the formulation.

Compounds without chromophore cannot be detected.

Requirement of inexpensive and reliable detectors.

Experimental

Chemicals used:

Chlorhexidine

Zinc phosphate

Acetonitrile

Sodium phosphate monobasic

Trimethylamine

Ortho-Phosphoric acid.

Equipments used:

pH meter:

Instrument

HannapH meter

Electrode

H1 1110B pH electrode

HPLC

HPLC Instrument

Agilent 1200

Mode of Separation

Reverse Phase Chromatography

Column

Phenomenex

Serial No

84668

Stationary phase

Hypersil C18

Pore size

5 µm

Column dimensions

150*4.6 mm

Mobile phase

Acetonitrile: Phosphate Buffer (45:55)

Detector

UV-Visible Spectrometer

(239nm)

Pump

Isocratic

Methodology:

Preparation of sample:

1.8g of Zinc phosphate cement and Chlorhexidine were weighed and placed on a ceramic tile. The mixture was mixed for uniform distribution of Chlorhexidine in zinc phosphate. 0.5 ml of liquid was added to the powder mixture and mixed to form a smooth paste.

Then freshly prepared paste was placed in the silicone rubber moulds to form discs. Three sets of specimens were prepared containing 1, 2 and 5% Chlorhexidine by mass. The moulds were placed in between two glass slides and clamped. They were allowed to dry in an incubator for 10 minutes at 370C. Dried discs were carefully removed from rubber moulds and placed in an individual centrifuge tubes of capacity 50 ml and 5ml deionised water was added to each tube. Centrifuge tubes were kept in incubator for curing at 370C for 1 hour.

Chlorhexidine release was then determined from each sample at an intervals of 1, 2, 3, 4, 24hrs, 1, 2, 3 and 4 weeks respectively by using HPLC (Agilant 1200 series).

S.NO

Percentage of Chlorhexidine

Zinc oxide powder (gm)

Phosphoric acid (ml)

Chlorhexidine required (gm)

Deionised water (ml)

1

1%

1.8

0.5

0.023

5

2

2%

1.8

0.5

0.046

5

3

5%

1.8

0.5

0.115

5

Table 1

Preparation of buffer solution:

Accurately weighed 9.5984g of sodium phosphate monobasic was dissolved in 1000ml of deionised water with 5ml of Triethylamine in it. After complete dissolution of sodium phosphate monobasic, pH of the solution was adjusted to 3.0 using pH meter by adding diluted 85% Ortho-Phosphoric acid.

Preparation of mobile phase:

Mobile phase was prepared by mixing Acetonitrile and Buffer solution in the ratio of 45:55v/v. Degassing of mobile phase is done every time to avoid air bubbles.

Preparation of solvent:

Solvent consists of Acetonitrile and Formic acid (1%) in the ratio of 20:80 v/v.

Preparation of standard stock solution:

50mg of Chlorhexidine was weighed and transferred to a 50ml volumetric flask. Small amount of solvent was added to dissolve Chlorhexidine. Then volume was made up to 50ml with solvent.

From the standard stock solutions different dilutions were prepared, by using the formula:

C1V1=C2V2

Where,

C1= Concentration of stock solution

C2= Concentration of required dilution

V1= Volume of unknown concentration to be calculated.

V2= Volume of volumetric flask in which dilution is made.

Volume of stock solution (ml)

Volume of volumetric flask for makeup (ml)

Concentration (µg/ml)

0.1

100

1

1

100

10

2.5

100

25

5

100

50

7.5

100

75

10

100

100

Table 2

Experimental procedure:

The mobile phase was degassed and connected to pump. A bottle for collecting waste was arranged. The HPLC equipment (Agilent 1200 series) was turned on by switching on the detector and isocratic pump. Then instrument online icon on screen was clicked to get the connection with the instrument.

All the experimental conditions were setup and a directory was created. The pump was turned on, flow rate was set to 0.5 ml and gradually increased to 1.5 ml till the constant base line was observed.

Experimental conditions:

Flow rate

2.5 ml/min

Detector wave length

239 nm

Retention time

1.6 - 1.8 min

Injecting volume of sample

20 µl

Stop time

4 min

Table

Solvent was injected to check the presence of impurities. After a straight base line without any noise observed, samples were injected to analyse. Before running the sample, sample information was entered.

First the dilution samples were run to analyse followed by specimens. Before injecting sample syringe should be washed cleanly to avoid impurities. Every sample was run for every concentration. The release profiles were recorded and mean release was calculated. Then graphs of peak area against time were plotted.

Results:

Measurable amounts of release were found for all the three concentrations of chlorhexidine. No steep or sudden release of chlorhexidine were found, release was steady. Release profiles of all concentrations of chlorhexidine (1, 2 and 5%) were shown in figures 1, 2 and 3 respectively.

Standard calibration graph:

This graph is plotted for concentration against mean peak area for serially diluted samples of standard stock solution of Chlorhexidine. A straight line was observed passing through origin. Slope of the graph and regression were also observed. By using this graph amount of chlorhexidine released was calculated.

Serial Number

Concentration (µg/ml)

Mean peak area (mAU*s)

1

1

33.52

2

10

370.85

3

25

944.87

4

50

1711.33

5

75

2721.15

6

100

3584.66

Table

Graph 1

The slope of graph is 35.775 and the regression coefficient was found to be 0.9991

Chlorhexidine release profile graphs:

Graphs 1, 2 and 3 [time interval (hours) against mean peak area (mAU*s)] shows the release profiles of the sample solutions for 4 weeks. Mean peak area is obtained as mean of 3 consecutive results for each sample.

1% Chlorhexidine release profile:

S.No

Time interval (Hours)

Mean peak area

(mAU*s)

1

1

93.6

2

2

198.6

3

3

227.57

4

4

269.12

5

24

275.28

6

168

509.79

7

336

664.35

8

504

705.8

9

672

708.57

Table

Graph 2

2% Chlorhexidine release profile:

S.No

Time interval (Hours)

Mean peak area

(mAU*s)

1

1

136.7

2

2

191.12

3

3

235.01

4

4

382.92

5

24

539.43

6

168

677.59

7

336

969.32

8

504

1068.09

9

672

1100.74

Table

Graph 3

5% Chlorhexidine release profile:

S.No

Time interval (Hours)

Mean peak area

(mAU*s)

1

1

197.21

2

2

414.24

3

3

493.14

4

4

500.56

5

24

734.83

6

168

950.86

7

336

1016.54

8

504

1211.03

9

672

1303.17

Table

Graph 4

Comparison of release profile graphs:

S.No

Time interval (Hours)

Mean peak area (mAU*s)

1% chlorhexidine

2% chlorhexidine

1

1

93.6

136.7

2

2

198.6

191.12

3

3

227.57

235.01

4

4

269.12

382.92

5

24

275.28

539.43

6

168

509.79

677.59

7

336

664.35

969.32

8

504

705.8

1068.09

9

672

708.57

1100.74

Table

Graph 5

Diffusion graphs:

After 4 weeks release levels were almost levelled. For calculating diffusion coefficients 4th week values were used as M∞ while substituting in equation,

D = S2Ï€l2/4

Where,

D = diffusion coefficient

S = slope of the graph Mt/M∞ against √ t

l = specimen thickness (1 mm for the samples we used in experiments).

The Mt/M∞ against √ t graphs for all the 3 concentrations of first 3 hours were plotted and they were shown in graphs 6, 7 and 8 respectively. Best fit equations are shown in table () and regression coefficients were shown in table ().

Diffusion graph for 1% Chlorhexidine:

√ t

Mt/M∞

60

0.133

84.85

0.283

103.92

0.3251

120

0.3844

Table

Graph 6

Diffusion graph for 2% Chlorhexidine:

√ t

Mt/M∞

60

0.124

84.85

0.173

103.92

0.213

Table

Graph 7

Diffusion graph for 5% Chlorhexidine

√ t

Mt/M∞

60

0.1517

84.85

0.3186

103.92

0.379

Table

Graph 8

Diffusion equations and regression coefficients:

Initial concentration (%)

Equation

Regression coefficient (r)

1

y=0.0041x - 0.0938

0.9591

2

y=0.002x + 0.0022

0.9997

5

y= 0.0053x - 0.1524

0.9649

Diffusion coefficients:

Initial concentration (%)

Diffusion coefficient

1

1.3*10-7

2

0.3*10-7

5

2.2*10-7

Discussion:

The release profile graphs have shown 3 distinct phases of release rates: rapid increase, slow increase rate and a plateau. For the first 4 hours there was a rapid increase in the release, after that release was slow for next 2 weeks. In 3rd and 4th week the release has reached a constant level.

The release of chlorhexidine follows Fick's second law of diffusion. As the release follows the diffusion, diffusion coefficients were calculated which are in the range of 0.3*10-7 (for 2% concentration) to 2.2*10-7 (for 5 % concentration).

The percentage of release from each concentration was calculated which are in range from 0.32% (for 5% concentration) to 0.8478% (for 1% concentration). As the concentration of Chlorhexidine increased the release of it from zinc phosphate cement also slightly increased.

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