Indomethacin hds drug-hybrid

MSPP35N

DISSERTATION

DEVELOPMENT OF A CONTROLLED-RELEASE DRUG

DELIVERY SYSTEM FOR INDOMETHACIN BASED ON A

HYDROXY DOUBLE SALT.

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Acknowledgement

I would like to first thank my supervisor Dr.Gareth Williams for

his guidance and support during the entire project. His

suggestions were very valuable and helped me a lot during the

project. I would like to convey my special thanks to Prof.Annie

Bligh for organising the course and project workshops which

helped me in the project. My thanks extend to my family &

friends who gave financial and psychological support to me.

Finally, I would to like to thank the department and the London

Metropolitan University for giving me this opportunity to pursue

the course during the academic year.

Mallika Piratla.

10036366.

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Abstract: The aim of this project is to incorporate a drug into a

Hydroxy Double Salt (HDS) to produce a HDS-drug hybrid for stable

and controlled release of the anions present in the drug. In this work,

Indomethacin, a non-steroidal anti-inflammatory drug, was chosen for

intercalation into a Zinc-HDS. Optimization reactions were performed

at varying conditions by changing the time, temperature and excess of

drug. Controlled release experiments were performed to assess the

release of the drug from the HDS. Various experimental techniques

were used to determine the success of the intercalation reactions

which are discussed in the report in detail.

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CONTENTS

Acknowledgement…………………………………………………………………………2

Abstract……………………………………………………………………..……….……….3

List of Figures……………………………………………………………………….…..…...6

List of tables……………………………………………………………………………..…...6

List of abbreviations…………………………………………………………………..….....7

1. Introduction………………………………………………………………….……….8

1.1 introduction……………………………………………………………………….9

1.2 aims of the project……………………………………………………..............10

2. Literature Review……………………………………………………………………11

2.1Properties of LDH’s and HDS’s………………………………………………………..12

2.2. Synthesis of LDH’s……………………………………………………………………..13

2.3. Pharmaceutical applications of LDH’s……………………………………………….13

2.4. Controlled drug delivery system………………………………………………………17

2.4.1: Factors to be considered for controlled release formulations…………………….17

2.4.2. Uses of LDH’s in drug delivery systems…………………………………………….18

2.4.3. Advantages & Disadvantages of controlled drug delivery systems……………….18

2.5: Applications of LDH’s in other fields…………………………………………………19

3. Materials and methods…………………………………………………………………...20

3.1. Experimental techniques used………………………………………...21

3.2. Preparation of Zinc hydroxy double salt ([Zn5 (OH) 8]. (NO3)2.2H2O)……………..22

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3.3. Optimization reactions of Indomethacin intercalated with [Zn5 (OH) 8].

(NO3)2.2H2O………………………………………………………………..22.

3.4. Preparation of large batch of optimized sample……………………..23

3.5. Preparation of Simulated Intestinal Fluid (SIF)……………………………………24

3.6. Controlled release experiments………………………………………………………..24

3.7. Drug recovery…………………………………………………………………………..24

3.8. Stability studies………………………………………………………………………....25

4. Results and discussion……………………………………………………...…………….26

4.1. ELEMENTAL ANALYSIS…………………………………………………………....27

4.2. FTIR ANALYSIS....................................................................................................…...27

4.3. X-RAY Diffraction……………………………………………………………………..29

4.4. Calibration curve…………………………………………………….......31

4.5.Controled release experiments…………………………………………..32

4.6. Stability studies…………………………………………………………..33

5. Conclusion………………………………………………………………..37

6. References………………………………………………………………….38

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List of Figures:

Figure.1: Chemical Structure of Indomethacin……………………………………….9

Figure.2: Schematic of controlled release of drug intercalated with HDS…………..16

Figure.3: FTIR spectra of Indomethacin, Zn-HDS and optimized sample…………..27

Figure.4: XRD pattern of Zn-HDS……………………………………………………..28

Figure.5: XRD pattern of optimized sample…………………………………………..29

Figure.6: Calibration curve of Indomethacin in SIF………………………………….30

Figure.7 : Controlled release profile for optimized sample…………………………....31

Figure.8: 1H NMR result of Indomethacin recovered from Zn-HDS in D2O………..32

Figure.9: 1H NMR result of Indomethacin in D2O……………………………………..33

Figure.10: 1H NMR result of degraded Indomethacin………………………………...34

Figure.11: 1H NMR result of degraded Indomethacin recovered from Zn-HDS stirred in

D2O…………………………………………………………………………………………35

List of Tables:

Table.1: Substances used for the preparation of [Zn5 (OH) 8]. (NO3)2.2H2O………….21

Table.2: Description of varying conditions for optimization reactions…………………22

Table.3: Quantity of substances used for optimization reactions………………………22

Table.4: Results from elemental analysis of Optimized sample…………………………26

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List of abbreviations:

Layered Double Hydroxide’s (LDH).

Hydroxy Double Salt’s (HDS’s).

Non-Steroidal Anti-Inflammatory Drugs (NSAID’s)

Zinc Hydroxy Double salt [Zn5 (OH) 8].(NO3)2.2H2O.

Metal cations (M+, M+2, M+3).

Metal Oxide (MeO-).

Attenuated Total Reflectance-Fourier Transformation Infra Red spectroscopy (ATR-

FTIR).

X-Ray Diffraction (XRD).

Nuclear Magnetic Resonance Spectroscopy (NMR).

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1. INTRODUCTION

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1.1. Introduction: Hydrotalcite like compounds such as Layered double hydroxides (LDH’s)

and Hydroxy Double Salts (HDS’s) are inorganic materials used as catalysts, adsorbents,

drug stabilizers and drug release modulators [15]. Hydrotalcite layers have positive charges

balanced by anions present in the interlayer spaces [15]. HDS’s have structural formula of

[(M+21-x, Me+2

1+x) (OH) 3(1-y)] +An

-(1+3y)/n.zH2O similar to LDH’s whose structural formula is 

[(M+21-x, M

+3x) (OH) 3]

x+Ax-. ZH2O

[8]. The layer structure depends on the divalent metal ions

and the anions can be monovalent such as Cl-, NO3-, or CH 3COO-, or divalent, such as SO4 -

2 or CO 3-2[8]. Structural principle of LDH’s includes substitution of metal ions (Me+2) ions by

divalent (M+2) and trivalent (M+3) in hydroxide layers which produces anion exchange

between metal ions and Anions present in the drug [16]. HDS’s are particularly very reactive

towards organic anions and anionic surfactants [16].

Non-Steroidal anti-inflammatory drugs (NSAIDS) are used in the treatment of Rheumatoid

and Osteo-arthritis [9]. Despite of the advantages of NSAIDS, their long term usage causes

gastro-intestinal toxicity such as ulceration, stricture formation in Oesophagus, Stomach and

Duodenum leading to severe bleeding, perforation and obstruction [9]. Indomethacin is an

ideal candidate for controlled release of the drug because of its short plasma half-life [9].

Controlled release formulation of Indomethacin minimizes initial drug release thereby

reducing the gastro-intestinal toxicity caused by the drug [9]. Indomethacin is a weak indole

acetic acid with a pka of 4.5 which is practically insoluble in simulated intestinal fluid [9] [18].

The structure of Indomethacin is shown in Figure.1.

N

O

Cl

OH

O

O

Figure.1: Chemical Structure of Indomethacin.

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1.2. Aims of the project:

To develop the use of Hydroxy Double Salts (HDS) for the controlled release of

Indomethacin.

To optimize the intercalation of Indomethacin into the HDS under varying

conditions given below:

Varying excesses the excess of drug used for intercalation (1.5, 3, 6 & 10

fold excess).

Varying reaction temperature (room temperature and 70°C).

Varying time periods of one day, three days and seven days.

To produce a large batch of the optimized Indomethacin intercalated HDS

sample.

To study controlled release of Indomethacin from Indomethacin intercalated

HDS sample in simulated intestinal fluid.

To observe the stability of both the Indomethacin and Indomethacin

intercalated HDS.

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2. LITERATURE REVIEW

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LDH’s are mineral and synthetic materials comprised of positively charged layers with

neutrality [3]. HDS’S are the layered materials which possess similar features to the LDH’s

with similar reactivity between the metal (cations) and anionic molecules [16]. LDH’s consists

of layers of metal cations (M+2 and M+3) with similar ionic radii which are coordinated by six

oxygen atoms forming M+2/ M+3(OH)6 octahedra [12]. These octahedra form into two-

dimensional sheets and stack together into many layers and are bonded together between

hydroxyl groups of adjacent layers by hydrogen bonding [12]. The first discovered material

was hydro-talcite which was discovered in 1842 which led to the development of large group

of naturally occurring minerals called as LDH’s in mid 19th century [12]. LDH’s are mineral

and synthetic materials comprising positively charged layers with anions located in the

interlayer region along with the water molecules to maintain electro neutrality [12].

2.1Properties of LDH’s and HDS’s:

HDS’s and LDH’s have similarity with each other in anion exchange reactivity, structural

parameters, crystallization, structural disorder and structure of the replacement anion [8]. Ion

exchange intercalation of drug anions was conducted by incorporation of the drug anions into

Zinc hydroxy double salt (Zn-HDS) whose formula is [Zn5 (OH) 8] (NO3)2. yH2O [3]. The

structure of Zn-HDS contains Zn (OH) 6 octahedral layers where the Zinc vacancies between

the layers are aligned with Zn (OH) 3[3]. The Zn atoms in the Zn (OH) 3 units show strong

interactions with water molecules present in the interlayer region and the anions present in

the drug molecule [3]. Zn-NO3 shows excellent bioavailability and hence commonly used for

intercalation reactions with NSAID’s like Ibuprofen, Indomethacin, Diclofenac etc [3].

LDH’s possess the following features:

High specific surface area of the particles [7].

Homogenous dispersion of the metal ions for maintenance of thermal stability to

form stable crystallites [7].

Interaction between the elements causes close dispersions between molecules

which result in development of hydrogenating properties [7].

Reconstruction of the original structure under mild conditions when reacted with

solutions containing various anions [7].

Good capacity for anion-exchange [7].

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Intercalation reactions with LDH’s utilise the chemical, electronic, optical and magnetic

properties and can also work at low temperatures to prepare novel materials [12]. The factors

which are to be considered for intercalation reactions of LDH’s are number, size and

orientation of the guest molecules and the interactions of the negatively and positively

charged hosts [12]. The interlayer region between the LDH contains water which can be

determined by factors such as nature of interlayer anions, vapour pressure and the

temperature [12]. The metal hydroxide layers connect the water molecules and the anions

between the layers by hydrogen bonding [12].

2.2. Synthesis of LDH’s:

LDH’s are synthesised when a metal oxide (MeO-) reacts with a solution containing Mono-

(M+), di-(M+2) and trivalent (M+3) metal cations[16]. HDS’s are synthesised when a metal

oxide (MeO-) reacts with divalent M+2 metal cations [16].

Bohm.et.al first described the conversion of an oxide into layered double hydroxide [12]. The

first prepared layered hydroxide was Zinc chromium hydroxide [Zn2 Cr (OH) 6] NO3. 2H2O

which was prepared by reaction of Zinc Oxide with solutions of Cr (NO3) [12]. 6H2O resulted

in violet colour of ZnO saturated by ethanolic chromium nitrate solution that led to initiation

of this procedure [12]. Sabatier, Recoura and Mailhe described the formation HDS’s by

reacting solutions containing Ni+2, Co+2, Zn+2 and Cd+2 ions with Cupric Oxide (CuO) and

their structural studies were described by Feitknecht and Maget [12].

2.3. Pharmaceutical applications of LDH’s:

LDH’s are widely applicable in pharmaceutical applications due to their acid buffering

capacity and anion exchange capacity which acts as anti-acidic and anti-peptic in the

formulation [4]. LDH’s have wide pharmaceutical applications with Diclofenac, Gemfibrozil,

Ibuprofen and Naproxen etc could be intercalated into LDH’s for their storage and controlled

release [4]. Hybridization of LDH’s with anti-inflammatory drugs attributes to high

inflammatory property and controlled release property of the drug which enhances water

solubility [4]. Ambrogi.et.al further concentred on the hybridization of LDH’s with NSAID’s

such as Indomethacin, Tiaprofenic acid and Ketoprofen [4]. The work of Ambrogi.et.al on

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intercalation of LDH’s with NSAID’s showed rapid dissolution rates of LDH’s in acidic

medium that could release intercalated drugs in ionic form which lacks crystallinity resulting

in the enhanced solubility of the drug [4]. Hydrotalcite like materials are studied as carriers for

controlled drug delivery of NSAID’s because of the following reasons:

To assess the effect of the intercalated drug in an acidic medium [15].

To evaluate the gastric permeability of hydrotalcite materials that can modify

the anti-inflammatory absorption of the intercalated drug and the invitro release

profile of the intercalated drug in intestinal environment [15].

Properties like low toxicity and good biocompatibility made nano-hybrids efficient carriers

in developing controlled release formulations for drug delivery [4]. Solubility of water also

plays a crucial role in drug bioavailability especially in the case of poorly soluble drugs; the

water solubility depends on the dose delivery and also the undesirable side effects of the drug [4]. Hybridization offers excellent features such as protected delivery, controlled release,

enhanced water solubility, increase in the dispersion ability and utilization of targeted drug

delivery [4]. Layered double hydroxides show strong electro static attractions between the

layers when they are intercalated with multivalent anions [4]. Monovalent anions such as

nitrates and chloride ions are good carriers for exchange reactions with layered double

hydroxides [4]. The solubility of layered double hydroxides is pH dependent and

heterogeneous hybrids of LDH’s stabilize and protect the bio molecules [4].

Li.et.al also developed hybridization of LDH’s with anti-inflammatory drugs such as

Fenbufen-LDH hybrids and demonstrated that the inorganic-drug hybrid materials showed

controlled release capacity which was proved to be an effective drug delivery system [4].

Desigaux.et.al recently proposed an approach to synthesize new labile molecular complexes

which resulted with the association of DNA molecules with LDH’s [4]. Intercalation of bio

molecules such as DNA, polymers and anions with LDH’s has captivating advantages such

as simplicity of preparation of LDH-bio molecule hybrid, high dispersion capacity, cost

effectiveness of the drug, safety, efficient delivery of the drug[4].

LDH’s are widely used in cosmetic preparations especially in ultraviolet screening agents,

anti-wrinkling and skin regulating agents [4]. Intercalation of cosmetics with LDH’s provides

high adsorption capacity, excellent anion exchange capacity, stabilizing potential and also

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improves rheological properties of the formulation [4]. The research on cosmetic preparations

based on LDH’s has led to high potentiality of intercalated cosmetic formulations which have

a great future in the field of cosmetics [4].

An LDH-drug nano hybrid can be obtained by reacting LDH with counter anion [14]. In drug

and gene delivery, cellular uptake is the main step [14]. LDH nano particles act as efficient

delivery agents to provide maximum efficacy of the cellular uptake by adjusting the LDH

particle size, conjugating the ligands to enhance the endocytosis mediated by the receptors [14]. The mechanism of cellular drug delivery is partial dissolution of LDH layers which

buffers the excess protons and hence improves the capability of drug to penetrate into

cytoplasm thereby enhancing the efficient drug delivery [14]. Intercalation of LDH’s with

anionic drug molecules determines the quality of the intercalated molecules as materials for

transport, storage and release the drug under controlled conditions [12]. NSAIDS cause

formation of gastric-duodenal ulcers which are aromatic compounds with ionisable

carboxylic groups which can be embedded in the hydrotalcite layers to produce an effective

and sustained release formulation [1]. Hwang.et.al and Yang.et.al described the intercalation

of Vitamins A (vitamin retinol), E (tocopherol) & C (ascorbic acid) into LDH’s which led to

the development of intercalation of multi-vitamin drugs with LDH’s [4].

LDH’s can intercalate with negative charge anions such as oligomers, single or double

stranded DNA which have wide applications in gene therapy [4]. Bio molecules such as

Cytosine Mono-phosphate (CMP), Adenosine Mono-phosphate (AMP), Guanosine Mono-

phosphate (GMP) and DNA form potential nano-hybrids with LDH’s by anion exchange

reactions which have extensive applications in medicinal field [4]. The ingenious works of

Choy.et.al have led to the development of hybrid systems of LDH’s with polymers, edible

dyes and anions which gave a successful outcome in the research of hybrid systems with

LDH’s [4]. Choy.et.al described the intercalation of bio molecules such as DNA, ATP and

nucleosides with LDH’s to develop new DNA carriers for delivery of genetic materials into

cells [12]. The LDH-drug hybrid develops a neutral complex in the body if it is not degraded in

the body [12]. The neutral complex does not interact with the negatively charged cell

membrane and anionic bio molecules during transfer to mammalian cells via endocytosis [12].

The unstable LDH will be dissolved by the lysosymes and releases the intercalated drug

molecule in acidic conditions [12].

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The controlled release effect of LDH-drug is effective as the delivery of drug is slow, stable

and controlled which provides non-toxic concentration of the drug in the body for a long

period of time [19]. The intercalation reactions of LDH are possible only if the drug is anion or

zwitter ion [20]. In anion exchange method the precursor anionic drug will be used for

intercalation reactions with LDH’s to form LDH-nano hybrids [20]. The anion exchange

method was proved to be very efficient method for the exchange of anions in the drugs [20].

There are two ways of release patterns of interlayer anionic drugs where the anionic drugs

can be intercalated into the interlayer via anion exchange method and the second one is the

anions will be de-intercalated and surrounded by the ions such as chloride ions or

phosphonates [20].

2.4. Controlled drug delivery system: Desired pharmacological response at the target site

without adverse effects and interactions of drug in the body is the main step for an effective

drug therapy [11]. Controlled release drug delivery systems have many advantages and

disadvantages as well [11]. Currently marketed controlled release systems are used to develop

novel dosage forms which are biocompatible with polymers and machineries used in the

preparation of drug [6]. The approaches for the survival of controlled are as follows:

Controlled release technology uses various polymers for coating the tablets,

sugar coated beads etc [6].

Matrix systems developed by swellable and non-swellable polymers [6].

Devices used to release osmotically controlled systems [6].

Controlled release formulation technology was introduced in 1952 by Smith Kline & French [6]. The goal behind the controlled release formulation was to release the drug constantly at

definite time intervals [6]. The main drawback of controlled release systems is lack of invitro-

invivo correlation [6]. Drugs which show invitro-invivo correlation have high permeability

across gastro-intestinal epithelium such that the controlled release of the drug is achieved [6].

In vitro dissolution rates of drugs could be useful for the prognostic studies of in vivo

absorption rates which will help in development of controlled release rate of the drug [6].Controlled release of the drug (anionic guest) intercalates with HDS or LDH (host) to form

a hybrid molecule[6]. The biological conditions in vitro will initiate the controlled release of

anions from the HDS or LDH intercalated drug. The schematic of controlled release process

of drug intercalated with HDS or LDH is shown in the Figure.2 below.

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Figure.2: Schematic of controlled release of drug intercalated with HDS.

The above Figure.2 is a representation of controlled release process of anionic drug

2.4.1: Factors to be considered for controlled release formulations:

If the guest species contains drugs in controlled release formulations, there are some

Modification of the pharmacokinetic features of the drug [5].

r long periods to

icle in the desired target [5].

increase [5].

intercalated with HDS which acts as a host. The incorporation of the anionic drug into the

HDS molecule forms an intercalated drug-HDS hybrid molecule. Once the hybrid reaches the

SIF, the anions from the drug are released in a controlled system.

considerations applicable to the pharmaceutical technologies which are as follows:

Maintenance of the pharmacological activity of the drug fo

avoid repeated administration [5].

The drug should act as a veh

Solubilization rate and bioavailability of the drug should

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2.4.2. Uses of LDH’s in drug delivery systems:

Intercalation of the drugs into LDH’s produces a novel drug delivery system that helps in the

following reasons:

They enhance the bioavailability of the drug’s chemical and biological

properties [13].

Modification of the pharmacokinetic properties can be achieved through

controlled release of the drug [13].

Increase in the solubilisation and bioavailability of the drug can be achieved [13].

Cellular uptake efficiency of the drug can be maximized [13].

2.4.3. Advantages & Disadvantages of controlled drug delivery systems:

Controlled drug delivery systems have many advantages which are as follows:

Therapeutic advantage by reducing the drug plasma level concentrations and by

maintaining steady plasma concentration of the drug avoiding fluctuations of the drug

concentration in the body [6] [11].

Reduction in adverse side effects is achieved by multiple dosing of the drug which

improves the tolerability of the drug by reduction in the cost of the drug as well [6].

Oral drug delivery is the most common and convenient method for patients because of

the reduction in the dosing frequency which causes patient compliance and comfort [6].

Disadvantages of controlled drug delivery are they require more time to achieve therapeutic

blood concentrations, dose dumping and also increase in the cost of the drug [11]. Drugs which

are suitable for controlled release systems should possess very short and long half lives, poor

absorption of the drug through gastro-intestinal tract and has low solubility [11]. Controlled

release systems are more suitable for long term therapies such as treatment for cardiovascular

diseases and rheumatoid arthritis etc [11].

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2.5: Other applications of LDH’s:

LDH’s have industrial applications in the field of separation chemistry, polymer preparation,

pharmaceutical preparations etc as they are easy to prepare and are environmentally friendly

compounds [19]. Bio active molecules such as carboxylic acids are suitable for the

intercalation process of drugs into LDH’s [19]. LDH’s are the solid materials which have the

ability to exchange the interlayer anions which are both organic (aliphatic carboxylates,

sulphonates, phosphonates, porphyrins, pthalocyanins etc) and inorganic anions (carbonates,

nitrates, chlorides, anionic coordination compounds, oxomethalates etc) [1]. Agricultural

applications of LDH’s help to achieve controlled release of active agro-substances with high

buffering capacity, high water retention ability and acid neutralizing capacity [4]. LDH’s are

used as potential flame retardants for polymers because of their clay like crystalline structure [17]. The typical metal hydroxide properties of LDH’s help them in the direct participation of

the Mg-Al-LDH in flame inhibition by endothermic decomposition and char formation [17].

LDH’s have been explored more widely than compared to HDS’s, although

they both possess the same structural features and act similarly as hosts for the storage and

controlled drug delivery process[3]. They are very few materials available on the research of

HDS’s when compared to LDH’s which shows the gap in the research on HDS’s. In this

project, HDS’s are used as hosts for the controlled release of Indomethacin

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3. MATERIALS AND METHODS

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3.1. Experimental techniques used:

1. Elemental analysis: Elemental analysis results were recorded on an EA1108 CHN

Fison instrument by Mr. Stephen Boyer in the School of Human Sciences, London

Metropolitan University (LMU).

2. Attenuated Total Reflectance- Fourier Transform Spectroscopy (ATR-FTIR):

ATR-FTIR spectra were recorded on a Bruker Vector 22 instrument. Spectra were

recorded over the range of 400-4000 cm-1 range with 16 scans per sample at resolution of

2cm-1.

3. X-Ray Diffraction (XRD): XRD patterns were recorded using Philips PW1830

powder diffraction. The instrument was operated at 40.kv and 25 m.A with CuKα

radiation (λ=1.5418 A°) by Dr.Gareth Williams, LMU.

4. Ultra Violet-visible Spectroscopy (UV spectroscopy): UV absorption spectra were

recorded on Shimadzu-1800 spectrometer using wavelength scan mode and quartz

mode.

5. Visible spectroscopy: A Jenway 7315 spectrometer was used to record the

absorbance values at λmax.

6. NMR spectroscopy: 1H NMR spectra were collected by Mr Jon Crowder on a Bruker

500 MHz spectrometer; D2O was used as a solvent.

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3.2. Preparation of Zinc hydroxy double salt ([Zn5 (OH) 8]. (NO3)2.2H2O):

Zinc hydroxy double salt was prepared by the method reported by Stählin and Oswald [3].The

materials used in the preparation of [Zn5 (OH) 8]. (NO3)2.2H2O are presented in Table.1

below.

Table.1: Substances used for the preparation of [Zn5 (OH) 8]. (NO3)2.2H2O.

Substance Molar mass No. of moles Mass needed in

grams

Zinc oxide (ZnO) 81.39 g/mol 0.0125 1.0173 g

Zinc nitrate hexa

hydrate

([Zn(NO3)2(H2O)6]

297.49 g/mol 0.019 5.6523 g

Deionised water. 18.01528 g/mol 0.67 12.070 ml

To prepare [Zn5 (OH) 8]. (NO3)2.2H2O, a reaction mixture of 0.0125 moles of ZnO, 0.019

moles of [Zn (NO3)2(H2O) 6] and 0.67 moles of deionised water were stirred vigorously at

room temperature (RT) for seven days on a magnetic stirrer. After seven days, the product

was collected by vacuum filtration and the filtered product was washed thoroughly with

deionised water and then dried in an oven at 80°C for two hours.

3.3. Optimization reactions of Indomethacin intercalated with [Zn5 (OH) 8].

(NO3)2.2H2O:

A variety of reactions were performed to optimize Indomethacin intercalated [Zn5 (OH) 8].

(NO3)2.2H2O. The conditions used for optimization reactions are shown below in Table.2.

22  

Table.2: Description of varying conditions for optimization reactions.

Reaction time Excess of Indomethacin Reaction temperature

1 Day 1.5 3 6 10 RT 70°C in oil

bath

3 days 1.5 3 6 10 RT 70°C in oil

bath

7 days 1.5 3 6 10 RT 70°C in oil

bath

To deprotonate the anions present in the drug, one mole equivalent of sodium hydroxide

(NaOH) was added to all the optimization reaction mixtures. Indomethacin has one ionisable

H+ ion and it was not available as a sodium salt and hence, one mole equivalent of NaOH was

added to the reaction mixture. For each reaction, the quantity of HDS, Indomethacin and

NaOH solution were placed in a boiling tube and water added to a total volume of 10 mL.

The boiling tube was placed over a magnetic stirrer and continuously stirred. The details of

amount of HDS, Indomethacin and NaOH are shown below in Table.3.

Table.3: Quantity of substances used for optimization reactions.

Excess of Drug Moles HDS Moles drug

needed

Mass HDS(g) Amount of

NaOH added in

ml.

Mass drug (g)

1.5 2 X 10-4 3 X 10-4 0.1231 g/mol 0.6 0.1073g

3 2 X 10-4 6 X 10-4 0.1231 g/mol 1.2 0.2146g

6 2 X 10-4 12 X 10-4 0.1231 g/mol 2.4 0.4293g

10 2 X 10-4 20 X 10-4 0.1231 g/mol 4 0.7155g

The collected product was recovered by vacuum filtration and washed thoroughly with

deionised water and dried at 80°C for two hours.

23  

3.4. Preparation of large batch of optimized sample: Among the optimization

reactions for all the excess folds of drug shown in tables.2 & 3, excess 3 fold of Indomethacin

intercalated with HDS at RT was found to be the optimized sample by elemental analysis

data. Large batch of the optimised sample was prepared as calculated above in table.3, by

taking 5 folds excess of the optimised sample for further use in controlled release

experiments.

3.5. Preparation of Simulated Intestinal Fluid (SIF): SIF was prepared by mixture of

2.1020 g of potassium hydroxide (KOH) and 13.6105 g of potassium dihydrogen phosphate

(KH2PO4) added in a 1000ml volumetric flask. Deionised water was carefully added to make

the volume up to 1000ml. SIF was chosen for the controlled release experiments as it acts as

a phosphate buffer with pH of 7.3, the same as that of intestinal fluid in the Gastro-intestinal

system. The concentration of Indomethacin in SIF was determined by plotting a calibration

curve between concentration and absorbance at wavelength of 308 nm (λmax) which is

discussed in the results and discussion part of this report.

3.6. Controlled release experiments: Controlled release experiments were carried for the

optimized HDS-Indomethacin nanocomposite. For the controlled release experiments, the

temperature should be maintained at 37°C (biological condition in intestinal fluid) in oil bath

constantly by checking with thermometer on a hot plate. 0.25g of the Indomethacin

intercalated HDS was taken in a 250 ml Round Bottomed Flask (RBF) and 250ml of SIF was

added to the 250ml RBF. A magnetic stirrer bead was added to the RBF, which was then

placed in the oil bath with magnetic stirrer. 3.5 ml aliquots were collected at regular time

intervals. All the collected aliquots were analysed by spectrophotometer to calculate their

concentration at 308nm (λmax) obtain a controlled release profile as shown in the results part

in this report.

3.7. Drug recovery: 0.05 g of Zn-HDS intercalated with Indomethacin, 0.1g of sodium

carbonate (Na2CO3) and 3ml of heavy water (D2O) were placed in a boiling tube with a

magnetic stirrer bead. The whole mixture was stirred continuously at 80°C overnight. The

product was carefully recovered by filtering the solution in a side arm boiling tube. The

filtrate and 0.05g of powdered Indomethacin were separately analysed by NMR

spectroscopy. The drug recovery process was performed to check whether the anions from

the drug could be recovered from the HDS.

24  

3.8. Stability studies: 0.05g of Indomethacin and 0.05g of optimized sample were taken in

two separate vials and placed in an oven at 100°C for 48hrs. After this time, 0.1 g of Na2CO3

and 3ml of D2O were added to the optimized sample and placed in a boiling tube. The boiling

tube was stirred at 80°C overnight. The samples were analysed by NMR spectroscopy to

check the stability of the drug after the heat treatment.

25  

4. RESULTS AND DISCUSSION

26  

4.1. ELEMENTAL ANALYSIS: All the samples prepared by optimization reactions as

shown in the Table.3. From the elemental micro analysis data of all the samples prepared,

excess 3 fold of Indomethacin intercalated with HDS was chosen as optimized sample.

Results of elemental analysis of optimized sample are given in the Table.4 below.

Table.4: Results from elemental analysis of Optimized sample.

Element Expected percentage Observed percentage

Carbon 12 28.746

Hydrogen 3 3.092

Nitrogen 1 2.942

From the above values, the formula of optimized sample was calculated to be Zn5 (OH) 8

(C19H16ClNO4)1.2(NO3)0.8 (H2O). It is clear that the nitrate ions are replaced by the drug,

which shows the success of intercalation, although it did not prove possible to achieve

complete replacement of nitrate from the drug.

4.2. FTIR ANALYSIS:

ATR-FTIR spectra of Indomethacin, Zn-HDS and the optimized sample are shown in

Figure.3. IR spectra were recorded to determine the success of all the intercalation reactions.

The broad peak centred at 3400 cm-1 shows the vibrations of the hydrogen bonded O-H

groups of the HDS layers. The peaks between 1410 cm-1 and 1340 cm-1 show the stressed

peaks due to intermolecular vibrations in the drug molecule. The sharp peak of Zn-HDS at

1360 cm-1 can be ascribed to stretching vibrations of nitrate ions present.

27  

Figure.3: FTIR spectra of Indomethacin, Zn-HDS and optimized sample.

From Figure.3, the small broad peak (2-3 peaks) at 2800 cm-1 in both Indomethacin and

optimized sample ascribed to C-H bond stretching (C-H bonds are present in Indomethacin).

The peaks at 794 cm-1 in Indomethacin and optimized sample show the presence of C-Cl

bonds in Indomethacin. The sharp peak at 1692 cm-1 in Indomethacin and optimized sample

is the bending vibrations of N-H bonds. The peaks of Indomethacin and optimized sample are

similar except the peak at 3400 cm-1 which shows the absence of O-H bonding in

Indomethacin. From the above FTIR spectra shown in Figure.3, it is clear that the IR spectra

of the Indomethacin intercalated Zn-HDS nanocomposites and indomethacin have significant

similarities. The FTIR results support with the elemental analysis data, suggests that the drug

has been successfully incorporated into the HDS.

28  

4.3. X-RAY Diffraction: The XRD technique was used to determine the distance of

interlayer spacing between the layers. XRD patterns obtained for Zn-HDS and optimized

sample are shown in the Figures.4 and 5 below respectively.

Figure.4: XRD pattern of Zn-HDS

From Figure.4, the graph clearly show reflections at 9A�. The interlayer spacing is

calculated to be 9.85 Å.

29  

Figure.5: XRD pattern of optimized sample.

The value of 2sin2θ is 3A� and by using Bragg’s law (D=λ/ 2 sinθ) as the reaction is first

order reaction, the distance between the layers is 29.65 A�. By comparing both the patterns

as shown in Figures 4 & 5, The interlayer spacing of optimized sample(29.65 A�) is more

than that of Zn-HDS(9.85A�) which shows the distance between the layers in Indomethacin

intercalated HDS.

30  

4.4. Calibration curve: The concentration of Indomethacin in SIF was determined by

plotting a calibration curve between concentration and absorbance at wavelength of 308 nm

(λmax). Calibration curve plotted between wavelength Vs absorbance of Indomethacin in SIF

is shown in the Figure.6 below.

Figure.6: Calibration curve of Indomethacin in SIF.

From the above Figure.6, the Pearson correlation value of R2=0.999 which is very close to

value “1” shows that the method obeys Beer-Lambert’s law where concentration and

absorbance are in linear relation with each other.

31  

4.5.Controled release experiments: Controlled release experiments were performed to

study the release of drug anions from the Zn-HDS intercalates. Samples were stirred at 37°C

in SIF (pH-7.3); the results are given in Figure.7.

Figure.7 : Controlled release profile for optimized sample.

From the above controlled release profile shown in Figure.7, the controlled release

experiments are repeated thrice to check the constancy in the release profile of Indomethacin.

Controlled release of the drug took place at 35 minutes and it is almost similar in series 2 and

series 3. Although the three controlled release experiments were performed under similar

conditions, the differences in the controlled release profile might be due to the fluctuations in

the temperature.

32  

4.6. Stability studies: Studies were performed to determine the recovery of the drug

anions from the Zn-HDS host. 1H NMR results of stability studies are shown below in

Figures.8 and 9 for Indomethacin and optimized sample respectively

.

Figure.8: 1H NMR result of Indomethacin recovered from Zn-HDS in D2O.

From the above Figure.8, the chemical shifts from 6.80-7.83 at 2.36, 1.05, 1.04 and 1.02

ppm shows the presence of heterocyclic ring structures which are present Indomethacin.

The chemical shift recorded at 4.88 ascribes to the H environments of the D2O with the

drug intercalates the chemical shifts recorded at 3.87 and 3.55 shows the presence of C-Cl

bonding present in the drug.

33  

Figure.9: 1H NMR result of Indomethacin in D2O.

From the above Figure.9, the chemical shifts at 6.76-8.46 shows the presence of heterocyclic

aromatic molecules present in Indomethacin. Chemical shift present at 4.85 might be H

environment present in Indomethacin. Chemical shifts at 3.60 and 3.88 shows the presence of

C-Cl bonds and 2.25 & 1.92 shows the presence of H bonded with the alkyl groups.

From the Figure.8, presence of heterocyclic rings and Cl-bonds present in Indomethacin

suggest that the drug was recovered from the Zn-HDS when compared to Figure.9.

34  

Figure.10: 1H NMR result of degraded Indomethacin.

From the above Figure.10, the chemical shifts recorded from 6.76-8.46 denotes the presence

of heterocyclic aromatic compounds present in Indomethacin. Chemical shifts at 3.60 and

3.88 shows the presence of C-Cl bonding present in the drug. Chemical shifts at 1.92 and

2.25 shows the presence of H environment present between the drug and D2O. Presence of

heterocyclic aromatic molecules present in Indomethacin shows the stability of the drug even

after degradation at 100°C. Degradation of the drug took place as per the chemical shifts and

also extra peaks found in the degraded drug when compared with Figure.10.

35  

Figure.11: 1H NMR result of degraded Indomethacin recovered from Zn-HDS stirred in

D2O.

From the above Figure.11, the chemical shifts recorded from 6.77-7.40 denotes the presence

of heterocyclic aromatic compounds present in Indomethacin. Chemical shifts at 3.84 and

3.52 shows the presence of C-Cl bonding present in the drug. Chemical shifts at 2.32 shows

the presence of H environment present between the drug and D2O. Presence of heterocyclic

aromatic molecules present in Indomethacin shows the stability of the drug even after

degradation at 100°C. The presence of heterocyclic rings in the degraded Indomethacin

recovered from Zn-HDS suggests that drug was not degraded which shows stability of

Indomethacin even after degradation. By comparing Figures.8 & 11, the chemical shifts show

that degradation of the drug was not observed in the Indomethacin an intercalated HDS

sample which shows that the intercalation reactions of Indomethacin with HDS are stable and

successful.From the NMR results of Indomethacin in D2O, it is clear that the Indomethacin

showed degradation after heating the drug. The results of Indomethacin intercalated HDS

showed the stability of the drug even after heating the drug.

36  

5. Conclusion:

The optimisation process of drug was achieved with the optimized sample of excess 3 folds

of Indomethacin with HDS. The results of FTIR suggested that the optimized sample showed

similarity with Indomethacin which shows the success of the intercalation reactions. The

controlled release experiments presented in the report showed success in the intercalation

reactions of the Indomethacin with HDS. Hence, intercalation into HDS’s could be suitable

and most efficient delivery matrices for controlled release systems.

Future work could include preparation of enteric coated beads of the optimal HDS-

indomethacin sample. Controlled release experiments of enteric coated beads would be

conducted under the same biological conditions (stirring at 37°C in SIF) as that of the

uncoated beads. The controlled release profiles of both uncoated beads and enteric coated

beads could be compared to begin to develop an improved drug delivery system.

Recent developments of nano composites of bio active molecules in particularly with drugs

by intercalation reactions with LDH or HDS provide efficient controlled release of the drug.

The HDS nanocomposites are very easy to prepare and are suitable for controlled release

purpose as they are also friendly. In future, the developments on the intercalation chemistry

between anionic drugs and LDH’s and HDS’s might be useful in pharmaceutical field both as

low-cost and efficient carriers of anions to desired target in drug therapy

37  

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