CChhaapptteerr--II

1. INTRODUCTION

One of the most challenging aspects in formulation development

is the solubility behavior of drugs. With the advent of combinatorial

chemistry and high throughput screening, the number of poorly water

soluble compounds has dramatically increased. One of the most

frequent and greatest challenges to formulation scientists in the

pharmaceutical industry is the formulation of poorly soluble

compounds for oral delivery. Drug absorption from a solid dosage form

after oral administration depends on the release of the drug substance

from the drug product, the dissolution of the drug under physiological

conditions, and permeability across the gastrointestinal tract.

Current knowledge about the solubility, permeability, dissolution

and pharmacokinetics of a drug product should be considered in

defining dissolution test specifications for the drug approval process.

BCS is the scientific framework for classifying drug substances based

on their aqueous solubility and intestinal permeability. The

introduction of the Biopharmaceutics Classification System (BCS) in

1995 was the result of continuous efforts on mathematical analysis for

the elucidation of the kinetics and dynamics of the drug process in the

gastrointestinal (GI) tract. Since the BCS was introduced, it has been

used as a regulatory tool for the replacement of certain bioequivalence

(BE) studies with accurate in vitro dissolution tests. BCS recommends

method for classification according to dosage form dissolution along

with the solubility-permeability characteristics of the drug product.1,2

8

1.1. Biopharmaceutics Classification System (BCS)

BCS guidelines are provided by U.S. Food and Drug

administration (USFDA), World Health Organization (WHO) and the

European Medicines Evaluation Agency (EMEA).3

The objective of BCS is to predict the in vivo pharmacokinetic

performances of drugs from measurements of permeability and

solubility. It allows estimation of the contributions of three major

factors, viz. dissolution, solubility and intestinal permeability.

Dissolution and gastro intestinal permeability are the fundamental

parameters controlling rate and extension of drug absorption thus BCS

becomes fundamental tool in drug development.

BCS defines three dimensionless numbers, dose number (Do),

dissolution number (Dn) and absorption number (An), to characterize

drug substances. These numbers are combinations of physicochemical

and physiological parameters and represent the most fundamental view

of GI drug absorption.

The absorption number is the ratio of permeability (Peff) and the

gut radius (R) times the residence time (Tsi) in the small intestine which

can be written as the ratio of residence time and absorptive time (Tabs).

A� �P���

R� T� �

T�

T� -Eq.1.1

The dissolution number (Dn), is the ratio of the residence time to

the dissolution time (Tdiss), which includes solubility (Cs), diffusivity (D),

9

density (ρ), and the initial particle radius (r) of compound and the

intestinal transit time (Tsi ).

D� �3D

r��C

ρ� T� -Eq.1.2

The dose number, Do, is defined as ratio of dose concentration to

solubility.

D� �M V�⁄

C -Eq.1.3

Where Cs is the solubility, M is the dose and Vo is the volume of

water taken with the dose, which is generally set to be 250 ml.

Based on drug solubility and permeability, the following

Biopharmaceutics Classification System (BCS) is recommended in the

literature1:

Class 1: High solubility – High permeability Drugs

Class 2: Low solubility – High permeability Drugs

Class 3: High solubility – Low permeability Drugs

Class 4: Low solubility – Low permeability Drug.

The solubility of a drug is determined by dissolving the highest

unit dose of the drug in 250 ml of buffer adjusted between pH 1.0 and

8.0. A drug substance is considered highly soluble when the

dose/solubility volume of solution are less than or equal to 250 ml.

High permeability drugs are generally those with an extent of

absorption that is greater than 90% in the absence of documented

10

instability in the GI tract or those whose permeability has been

determined experimentally.

1.1.1. Class I drugs

The drugs of this class exhibit high absorption number and high

dissolution number. The rate limiting step is drug dissolution, and if

dissolution is very rapid, then the gastric-emptying rate becomes the

rate-determining step. These compounds are well absorbed, and their

absorption rate is usually higher than the excretion rate.4,5

1.1.2. Class II drugs

The drugs of this class have a high absorption number but a low

dissolution number. In vivo drug dissolution is then a rate-limiting step

for absorption except at a very high dose number. The absorption for

Class II drugs is usually slower than for Class I and occurs over a

longer period of time. In vitro–in vivo correlation (IVIVC) is usually

accepted for Class I and Class II drugs. The dissolution of a poorly

soluble compound is normally low (Dn < 1), while for many poorly

soluble compounds An and Do are usually high (Class II).

1.1.3. Class III drugs

Drug permeability is the rate limiting step for drug absorption,

but the drug is solvated very quickly. These drugs exhibit a high

variation in the rate and extent of drug absorption. Since the

dissolution is rapid, the variation is attributable to alteration of

physiology and membrane permeability rather than the dosage form

11

factors. If the formulation does not change the permeability or

gastrointestinal duration time, then Class I criteria can be applied.4,5,6

1.1.4. Class IV drugs

The drugs of this class are problematic for effective oral

administration. These compounds have poor bioavailability. They are

usually not well absorbed through the intestinal mucosa, and a high

variability is expected.

1.2. Approaches in improving drug solubility for oral delivery

Apart from the permeability, the solubility behavior of a drug is a

key determinant of its oral bioavailability. There have always been

certain drugs for which solubility has presented a challenge to the

development of a suitable formulation for oral administration.

Consideration of the modified Noyes – Whitney equation provides

some hints as to how the dissolution rate of even very poorly soluble

compounds might be improved to minimize the limitations to oral

bioavailability.7

dC

dt�AD�C � C�

h -Eq.1.4

Where dc/dt is the rate of dissolution, A is the surface area

available for dissolution, D is diffusion coefficient of the compound, Cs

is the solubility of the compound in the dissolution medium, C is the

concentration of the drug at time t and h is the thickness of the

diffusion boundary layer adjacent to the surface of the dissolving

compound.

12

To improve dissolution according to this analysis, the

possibilities are to increase the surface area available for dissolution by

decreasing the particle size of the compound and/or by optimizing the

wetting characteristics of the compound surface, to decrease the

boundary layer thickness, to ensure sink conditions for dissolution and

last but definitely not least, to improve the apparent solubility of the

drug under physiologically relevant conditions. Of these possibilities,

changes in the hydrodynamics are difficult to invoke in vivo and the

maintenance of sink conditions will depend on how permeable the

gastrointestinal mucosa is to the compound as well as on the

composition and volume of the luminal fluids. Although some research

efforts have been directed towards permeability enhancement using

appropriate excipients, results to date have not been particularly

encouraging. Administration of the drug in the fed state may be an

option to improve the dissolution rate and also to increase the time

available for dissolution. The magnitude of the food effect can be

predicted from dissolution tests in the relevant media. However, the

most attractive option for increasing the release rate is improvement of

the solubility through formulation approaches.

The various formulation and other approaches that can be taken

to improve the solubility or to increase the available surface area for

dissolution include i) physical modification such as particle size

reduction by micronization or formulating in the form of

nanosuspension, modification of crystal habit, polymorphism, pseudo

polymorphism, complexation, solid dispersion ii) chemical modification

13

such as preparing soluble prodrugs and salt formation iii) other

methods such as hydrotrophy, co crystallisation, solvent deposition.

Although salt formation, solubilization and particle size reduction

have commonly been used to increase dissolution rate and thereby oral

absorption and bioavailability of poorly water soluble drugs, there are

practical limitations of these techniques.8 Cyclodextrin complexation is

one of the widely used techniques in enhancing the solubility and

dissolution rate. The ability of cyclodextrins to form inclusion

compounds through molecular encapsulation has been known for

many years. Several pharmaceutical products on the market use this

formulation technology with different cyclodextrin derivatives.

1.3. Cyclodextrins9,10,11

Cyclodextrins are cyclic oligosaccharides which have received

increasing attention in the pharmaceutical field because of their ability

to form inclusion complexes with many lipophilic drugs.

Cyclodextrins (CD) comprise a family of cyclic oligosaccharides,

and several members of this family are used industrially in

pharmaceutical and allied applications. CDs are manufactured from

starch, one of the two glucose containing polymers produced by

photosynthesis (the other is cellulose). Starch consists of

D-glucopyranoside building blocks that have both E-1, 4- and E-1, 6-

glycosidic linkages. The degradation of starch (which is derived from

corn, potatoes and other sources) by the enzyme glucosyl transferase

generates primary products that are cyclic oligomers of

14

E-1, 4-D-glucopyranoside, or CDs by chain splitting and intramolecular

rearrangement. Cyclodextrins are cyclic (R-1, 4)-linked oligosaccharides

of R-D-glucopyranose containing a relatively hydrophobic central cavity

and hydrophilic outer surface. Due to lack of free rotation about the

bonds connecting the glucopyranose units, the cyclodextrins are not

perfectly cylindrical molecules but are toroidal or cone shaped. Based

on this architecture, the primary hydroxyl groups are located on the

narrow side of the torus while the secondary hydroxyl groups are

located on the wider edge.

Fig.1.1 shows chemical structure of βCD molecule. CDs derive

their system of nomenclature from the number of glucose residues in

their structure, such that the glucose hexamer is referred to as ECD,

the heptamer as βCD and the octamer as γCD (Fig. 1.2).

Characteristics of E, β, γ cyclodextrins are given in Table 1.1. There are

literally thousands of variations of CDs that have variable ring size and

random or site-specific chemical functionalization. Larger CDs,

containing more than eight glucopyranose units in the molecule, have

also been studied for their complexation phenomenon.13 The most

important property of CDs is their entrapping of hydrophobic guest

molecules into their cavity in the aqueous phase as shown in Fig.1.3.

This complexation ability of CD is due to their chemical structure and

the glucopyranose units conformation. In cyclodextrin molecules, the

glucopyranose units are present in the chair conformation. Therefore,

the hydroxyl functional groups are orientated to the cone exterior with

the primary hydroxyl groups of the sugar residues at the narrow and

15

wider edges, which gives it a hydrophilic outer surface. The central

cavity is formed by the skeletal carbons and ethereal oxygens of glucose

residues, which gives the CD molecule a comparatively hydrophobic

inner cavity. The polarity of this cavity has been estimated to be similar

to that of an aqueous ethanolic or methanolic solution.13,14

Fig. 1.1: The chemical structure of β cyclodextrin molecule

Fig. 1.2: Structure of (A) 3 cyclodextrin (B) γ cyclodextrin

(A) (B)

16

Fig. 1.3: Schematic presentation of drug-cyclodextrin

complex formation

In Fig.1.3, small circles represent water molecules, ellipse

represent drug molecules. Water molecules are repulsed both by the

hydrophobic drug molecules and the hydrophobic cavity of the

truncated CD cylinder. The main driving force for inclusion is mainly

the substitution of the polar–apolar interactions (between the apolar

CD cavity and polar water) for apolar–apolar interactions (between the

drug and the CD cavity).

The main driving force for complex formation is thought to be the

release of enthalpy rich water from the cavity due to the entrapping of

guest molecules of CD.15,16,17 Weak van der Waals forces, hydrogen

bonds, and hydrophobic interactions keep the complex together. No

covalent bonds are formed or broken during drug-CD complex

formation. Therefore, the complexation process can be considered as a

replacement of water molecules with drug molecules.

Generally, in an aqueous solution, the cyclodextrin cavity

(slightly apolar) is occupied by water molecules, which is

CD

17

thermodynamically unfavorable (polar-apolar interaction). Therefore,

the water molecules inside the cavity have fewer tendencies to form

hydrogen bonds in the same way as in solution and result in a higher

enthalpy and higher energy. When hydrophobic guest molecules are

incorporated into this system, the energy of the system is lowered by

substituting these enthalpy rich water molecules with those

hydrophobic guest molecules to form the complex of CDs and guest

molecules.

In aqueous solution, equilibrium is reached with the formation of

a complex of the drug and CD and with the dissociation of the

complexes. Therefore, the complexation can be studied with methods

such as chemical reactions. Most frequently, the complexation happens

between one cyclodextrin and one guest (1:1 ratio) molecule. However,

2:1, 1:2, 2:2, and higher order complex equilibria always exist

simultaneously in the system. Phase solubility diagrams are normally

used to analyze the complexation stoichiometry.

In addition, the complexation is determined both by the CD’s

inner cavity size and by the appropriate size of those organic

compounds or guest molecules.18 Only those guest molecules with

suitable shape and size can be incorporated into the CDs inner cavity

to form inclusion complexes. The cavity size of CDs is dependent on the

number of glucose in the molecule as shown in Table 1.1. The cavity

size of ECD is the smallest of the three CDs and insufficient for many

drugs. γCD has the largest cavity size of all three CDs. However, it is

18

much more expensive than the other CDs. Therefore, βCD is most

widely used in research and manufacturing due to its cost and suitable

cavity size for most drug molecules.

Table 1.1: Characteristics of cyclodextrins

Property 3 β γ

Number of glucose units 6 7 8

Molecular weight 972 1135 1297

Water solubility (g/100 mL) 14.5 1.85 23.2

pKa 12.33 12.2 12.08

Inner diameter (nm) 0.45-0.57 0.62-0.78 0.79-0.95

Outer diameter (nm) 1.37 1.53 1.69

Depth/Height (nm) 0.79 0.79 0.79

Cavity volume (nm3) 0.174 0.262 0.472

1.4. Cyclodextrin derivatives

The aqueous solubility of E, β and γCD is much lower than that

of comparable linear dextrins, most probably due to relatively strong

binding of the cyclodextrin molecules in the crystal state (i.e. relatively

high crystal energy). In addition, βCD molecules form intramolecular

hydrogen bonds that diminish their ability to form hydrogen bonds with

the surrounding water molecules. It was discovered that substitution of

any of the hydroxyl groups, even by hydrophobic moieties such as

methoxy functions, resulted in dramatic increase in their aqueous

solubility. With increasing degree of methylation the solubility of βCD

(in cold water) increases until about 2/3 of all the hydroxyl groups have

been methylated, and then it decreases again upon further

methylation.19 Later several new derivatives came available including

the 2-hydroxypropyl derivatives of both β and γCD, the sulfo butyl

19

ether derivative of β cyclodextrin, and the branched (glucosyl- and

maltosyl-) βCD.20 The main reason for the solubility enhancement in

the alkyl derivatives is that chemical manipulation transforms the

crystalline E, β and γ cyclodextrins into amorphous mixtures of

isomeric derivatives.16,21 Statistically there are about 130,000 possible

heptakis (2-O-(hydroxylpropyl)) βCD derivatives, and given that

introduction of the 2-hydroxypropyl function also introduces an optical

center, the total number of isomers, i.e. geometrical and optical, is even

much greater.16 Since the reactivity of the three hydroxyl groups on the

cyclodextrin forming glucose units have been shown to be slightly

different the substitution is usually not totally random and found to

depend on, for instance, the basicity of the aqueous reaction media.21,22

This could explain the slight differences found in the complexing

abilities of identical cyclodextrin derivatives from different suppliers

and sometimes from one batch to another from the same supplier.

Fully substituted derivatives were shown to have lower aqueous

solubility than partly substituted derivatives which could be related to

the fact that the number of possible isomers decreases as the

cyclodextrin molecule becomes close to fully substituted. The ability of

the cyclodextrin derivatives to form water soluble complexes is also

dependent on the degree of substitution (i.e. the solubility of the

cyclodextrin molecule and the access of the guest molecule to the

cyclodextrin cavity). Thus, the degree of substitution is in general

optimized with regard to the solubilizing abilities of the cyclodextrins.

The degree of substitution of the pharmaceutical grades is about 0.65

20

for 2-hydroxypropyl β cyclodextrin (i.e. on the average 0.65

hydroxypropyl moieties are on each glucose unit) and about 1.8 for

randomly methylated β cyclodextrin (i.e. on the average 1.8 methoxy

moiety on each glucose unit). Some of the pharmaceutically important

derivatives of cyclodextrins are enumerated in Table 1.2.23

Table 1.2: Pharmaceutical derivatives of β cyclodextrin

Cyclodextrin R=H or

β cyclodextrin -H

2 hydroxy propyl β cyclodextrin -CH2CHOHCH3

Sulfobutyl ether β cyclodextrin sodium salt

-(CH2)4SO3+Na-

Randomly methylated β cyclodextrin

-CH3

Branched β cyclodextrin Glucosyl or maltosyl group

1.5. Applications of Cyclodextrins9,10,24-28

Since each guest molecule is individually surrounded by a

cyclodextrin (derivative) the molecule is micro-encapsulated from a

microscopical point of view. This can lead to advantageous changes in

the chemical and physical properties of the guest molecules.

• Stabilization of light or oxygen sensitive substances.

• Modification of the chemical reactivity of guest molecules.

• Fixation of very volatile substances.

• Improvement of solubility of substances.

• Modification of liquid substances to powders.

• Protection against degradation of substances by microorganisms.

21

• Masking of ill smell and taste.

• Masking pigments or the colour of substances.

• Catalytic activity of cyclodextrins with guest molecules.

These characteristics of cyclodextrins or their derivatives make

them suitable for applications in analytical chemistry, agriculture, the

pharmaceutical field, in food and toilet articles.29 Until the late 1960s

almost all cyclodextrin related chemistry was carried out in Europe but

the obtained technological advances did not lead to notable industrial

explorations of these oligosaccharides. However, in the early 1970s a

number of industrial applications were investigated, such as within the

food and cosmetic industry.30 In the food industry, cyclodextrins were

investigated as stabilizers for flavoring agents and to reduce unpleasant

odor and taste. In the cosmetic industry cyclodextrins were being tested

as stabilizers of chemically labile compounds, to obtain prolonged

action, to decrease local irritation and to reduce unpleasant odors. In

Japan, there is a tradition for industrial usage of natural products and

the Japanese regarded the parent cyclodextrins as natural materials

originating from starch and thus as “non-toxic” natural products. By

1970, the Japanese were already actively studying the chemistry of

cyclodextrins as well as their production and in the early 1980s

cyclodextrins were introduced as industrial raw materials, mainly for

the food and cosmetic industries.31 Some of the marketed products

prepared with cyclodextrins are given in Table 1.3.

22

Table 1.3: Marketed products prepared with cyclodextrins

Drug Formulation Trade name Company

3CD

Alprostadil (PGE1)

IV solution Prostavasin Ono (Japan)

Cefotiam hexetil HCl

Oral tablet Pansporin T Takeda (Japan)

βCD

Benexate HCl Oral capsule Ulgut

Teikoku Kagaku Sangyou

(Japan)

Dexamethasone Dermal ointment

Glymesason Fujinaga (Japan)

Nicotine Sublingual tablet

Nicorette Pharmacia (Sweden)

Nitroglycerin Sublingual tablet

Nitropen Nihon Kayaku (Japan)

Piroxicam Oral tablet Brexin Chiesi (Italy)

Tiaprofenic acid Oral tablet Surgamyl

Roussel-Maestrelli (Italy)

HPβCD

Cisapride Suppository Propulsid Janssen (Belgium)

Indomethacin Eye drop solution

Indocid Chauvin (France)

Itraconazole Oral and IV

solutions Sporanox Janssen (Belgium)

Mitomycin IV solution

Mitozytrex

MitoExtra

SuperGen (USA)

Novartis(Switzerland)

RMβCD

17β-Oestradiol Nasal spray Aerodiol Servier (France)

Chloramphenicol Eye drop solution

Clorocil Oftalder (Portugal)

SBE7βCD

Voriconazole IV solution Vfend Pfizer (USA)

Ziprasidone maleate

IM solution Geodon, Zeldox

Pfizer (USA)

23

HPγCD

Diclofenac sodium

Eye drop solution

Voltaren ophtha

Novartis (Switzerland)

1.6. Study of CD inclusion complexation & dilution effect

The most widely used approach to study inclusion complexation

(Fig. 1.4) is the phase solubility method described by Higuchi and

Connors,27 which examines the effect of a solubilizer, i.e. CD or ligand,

on the drug being solubilized, i.e. the substrate. Phase solubility

diagrams are categorized into A and B types; A-type curves indicate the

formation of soluble inclusion complexes while B-type suggest the

formation of inclusion complexes with poor solubility. BS type response

denotes complexes of limited solubility and a BI curve indicates

insoluble complexes. A-type curves are subdivided into AL (linear

increases of drug solubility as a function of CD concentration), AP

(positively deviating isotherms), and AN (negatively deviating isotherms)

subtypes. βCD often gives rise to B-type curves due to their poor water

solubility whereas the chemically modified CDs like HPβCD and

SBE7βCD usually produce soluble complexes and thus give A-type

systems.32

24

Fig. 1.4: Phase solubility diagram

In the case of a 1:1 complex, the equilibrium binding or

association constant, K, is determined from the slope of the linear

portion of the curve (Eq.1.5).

-Eq.1.5

Where So is the intrinsic solubility of the drug studied under the

conditions.

For many drug/CD complexes, binding constant values are in the

range of 100 to 20000 M-1. The ratio of free to complexed drug upon

dilution of a sparingly water-soluble drug/CD complex depends on the

phase solubility behavior of the system. Dilution will not result in drug

precipitation when the relationship between drug solubility and CD

concentration is linear, e.g. in a 1:1 interaction of CD and drug.

However, dilution may cause drug precipitation when the relationship

between drug solubility and CD concentration is nonlinear.32,33

AP

Concentration of the drug

AL

AN

S0

Bi

BS

25

Equilibrium binding of drug and CD to form a 1:1 complex can

be represented as

Drug + CD → Drug: CD Complex -Eq.1.6

Since equilibrium binding usually establishes with half lives of

much less than 1 second, the kinetics of dissociation of drug CD

complexes are generally expected to be much faster than many

physiological processes.34,35

1.7. Mechanism of drug release from cyclodextrin complexes

Different mechanisms play an important role in drug release from

the drug CD complex. Complexation of the drug to CD occurs through

a non covalent interaction between the molecule and the CD cavity.

This is a dynamic process whereby the drug molecule continuously

associates and dissociates from the host CD. Assuming a 1:1

complexation, the interaction will be as mentioned in Eq.1.6.

Two parameters, the complexation constant (K) and the lifetime

of the complex, are very important for the drug release mechanism.

1.7.1. Dilution

Dissociation due to dilution appears to be a major release

mechanism. Dilution is effective in releasing freed drug from weak drug

and CD complexes and is significant contributor even for strongly

bound drugs. The recent example reported by Piel et al.36 for

miconazole, a more strongly bound drug compared to prednisolone,37

supports the probable role of dilution. Dilution is minimal when a

26

drug-CD complex is administered ophthalmically. Efficient corneal

absorption is further exacerbated by contact time.

1.7.2. Competitive displacement

Competitive displacement of drugs from their CD complexes

probably plays a significant role in vivo. Addition of parabens to

parenterals not only leads to decreased antimicrobial activities of the

parabens, due to complexation, but also decreases the drug solubility

due to its displacement from complexes.38 Van Stam et al.39 showed

that alcohol displaces 2-napthol from βCD complexes. Tokumura et

al.40,41 reported that the βCD complex of a poorly water soluble drug,

cinnarizine, was more soluble in vitro than cinnarizine alone. Oral

administration of the complex showed less bioavailability than

expected, based on the in vitro dissolution experiments. It was

suggested that cinnarizine was too strongly bound to the CD so that

complex dissociation was limiting oral bioavailability.

Co-administration of phenylalanine, a displacing agent, improved the

bioavailability of cinnarizine from the complex but not from

conventional cinnarizine tablets.

1.7.3. Protein binding

Drug binding to plasma proteins may be an important

mechanism by which the drug may be released from a drug-CD

complex. It is evident that proteins may effectively compete with CDs

for drug binding and thus facilitate the in vivo release of drugs from

drug-CD complexes. Frijlink et al.42 studied the effect of HPβCD on the

27

displacement of both naproxen and flurbiprofen from plasma binding

sites in vitro. They found that tissue distribution of flurbiprofen and

naproxen was higher when HPβCD drug solution was administered

compared to drug solution in plasma, 10 minutes after parenteral dose,

meaning that more drug was free from CD solution to distribute to the

tissues than from the plasma solution.

1.7.4. Drug uptake by tissue

A potential contributing mechanism for drug release from CD is

preferential drug uptake by tissues. When the drug is lipophilic and

has access to tissue, and is not available to the CD or the complex, the

tissue then acts as a sink, causing dissociation of the complex based

on simple mass action principles. This mechanism is more relevant for

strongly bound drugs or when the complex is administered at a site

where dilution is minimal, e.g., ocular, nasal, sublingual, pulmonary,

dermal or rectal sites. For example, CD has been used in ophthalmic

delivery of poorly water soluble drugs to increase their solubility and/or

stability in the tear fluid, and in some cases to decrease irritation.42,43

The relative contribution of these mechanisms will however

depend on the route of administration (dilution effects), volume of

distribution of drug and cyclodextrin (dilution effects), binding strength

and concentration of drug and CD (dilution effects), binding constant

and concentration of competing agent (competitive displacement), and

association constant and protein concentration (protein binding).

28

1.8. Methods of preparation of inclusion complexes

Different methods are available for the preparation of CD

inclusion complexes. Brief principles of these methods are given below.

1.8.1. Physical blending method

Physical mixture of drug and CDs are prepared simply by

mechanical trituration. In laboratory scale CDs and drug are mixed

together thoroughly by trituration in a mortar and passed through

appropriate sieve to get the desired particle size in the final product.44

1.8.2. Kneading method

This method is based on impregnating the CDs with little amount

of water or hydroalcoholic solutions to make a paste. The drug is then

added to the above paste and kneaded for a specified time. The

kneaded mixture is then dried and passed through appropriate sieve.45

1.8.3. Co precipitation technique

This method involves the co-precipitation of drug and CDs in a

complex. In this method, required amount of drug is added to the

solution of CDs. The system is kept under magnetic agitation with

controlled process parameters and the formed precipitate is separated

by vacuum filtration and dried at room temperature in order to avoid

the loss of the structure water from the inclusion complex.46,47,48

1.8.4. Solution/solvent evaporation method

This method involves dissolving of the drug and CDs separately

in two mutually miscible solvents, mixing of both solutions to get

29

molecular dispersion of drug and complexing agents and finally

evaporating the solvent under vacuum to obtain solid inclusion

compound. This method is quite simple and economic both on

laboratory and large scale production and is considered as an

alternative to the spray drying technique.44

1.8.5. Neutralization precipitation method

This method is based on the precipitation of inclusion

compounds by neutralization technique and consists of dissolving the

drug in alkaline solutions like sodium/ammonium hydroxide and

mixing with an aqueous solution of CDs. The resultant clear solution is

then neutralized under agitation using hydrochloric acid solution till

reaching the equivalence point. A white precipitate is being formed at

this moment, corresponding to the formation of the inclusion

compound. This precipitate is filtered and dried. Limitation associated

with this method is acid and alkaline susceptible drugs can undergo

degradation during this process.49

1.8.6. Microwave irradiation method

This technique involves the microwave irradiation reaction

between drug and complexing agent using a microwave oven. The drug

and CD in definite molar ratio are dissolved in a mixture of water and

organic solvent in a specified proportion into a round bottom flask. The

mixture is reacted for short time of about one to two minutes at 60°C in

the microwave oven. After the completion of reaction, adequate amount

of solvent mixture is added to the above reaction mixture to remove the

30

residual, uncomplexed free drug and CD. The precipitate so obtained is

separated using Whatman filter paper, and dried in vaccum oven at

40°C for 48 hrs. Microwave irradiation method is a novel method for

industrial scale preparation due to its major advantage of shorter

reaction time and higher yield of the product.49-52

1.8.7. Atomization/Spray drying method

Spray drying is a common technique used in pharmaceuticals to

produce a dry powder from a liquid phase. Another application is its

use as a preservation method, increasing the storage stability due to

the water elimination.53 This method represents one of the most widely

employed methods to produce the inclusion complex starting from a

solution. The product obtained by this method yield the particles in the

controlled manner which in turn improves the dissolution rate of drug

in complex form. Thermal stress and low yield of the final product are

the limitations associated with this technique.54

1.8.8. Milling/Co-grinding technique

Solid binary inclusion compounds can be prepared by grinding

and milling of the drug and CDs with the help of mechanical devices.

Drug and CDs are mixed intimately and the physical mixture is

introduced in an oscillatory mill and grinded for suitable time.

Alternatively, the ball milling process can also be utilized for

preparation of the drug-CD binary system. This technique is superior to

other approaches from economic as well as environmental stand point

31

in that unlike similar methods it does not require any toxic organic

solvents.55

1.8.9. Lyophilization/ Freeze drying technique

Lyophilization/freeze drying technique is considered as a suitable

technique in getting a porous, amorphous powder with high degree of

interaction between drug and CD.56,57 In this technique, the solvent

system from the solution is eliminated through a primary freezing and

subsequent drying of the solution containing both drug and CD at

reduced pressure. Thermolabile substances can be successfully made

into complex form by this method. The limitations of this technique are

time consuming process and yield poor flowing product.

1.9. Techniques for characterization of inclusion complexation58-60

The complexation depends largely on the dimensions of the

cyclodextrins and the particular sterical arrangement of the functional

groups of the molecules, which leads to a relatively hydrophilic outside

and a hydrophobic inside cavity of the molecule. Inclusion complexes

formed between the guest and cyclodextrin molecules can be

characterized both in the solid and solution state by the following

techniques.

1.9.1. Solid state characterization

Inclusion complexation in solid state is characterized by the

following techniques.

32

1.9.1.1. Thermo analytical methods

These are used to determine whether the guest substance

undergoes some change before the thermal degradation of CDs.

1.9.1.2. Scanning Electron Microscopy (SEM)

SEM is used to study the microscopic aspects of the raw material

(CD and the guest substances, respectively). The difference in

crystallization state of the raw material and the product seen under

electron microscope indicates the formation of the inclusion complexes.

1.9.1.3. X-ray diffractometry

Powder X-ray diffractometry may be used to detect inclusion

complexation in the solid state. The difference in diffractograms of

newly formed substance than the uncomplexed guest and CD molecule

indicates complex formation.

1.9.1.4. Wettability and dissolution tests

The wetting of the solid phase by a solvent is always the first step

of any dissolution process. Cyclodextrin complexation of the lipophilic

drug often not only improves the wettability in water considerably, but

also simple addition of βCD to non wettable solid enhances their

wettability.

1.9.1.5. Infra Red (IR) spectroscopy

IR is used to estimate the interaction between CD and the guest

molecules in the solid state.

33

1.9.1.6. Thin Layer Chromatography (TLC)

In thin layer chromatography, the retention factor (Rf) values of a

guest molecule diminishes to considerable extent and this helps in

identifying the complex formation between guest and host molecule.

1.9.2. Solution state characterization

Inclusion complexation in solid state is characterized by the

following techniques.

1.9.2.1. Electrochemistry

In electrochemistry, 3 different methods polarography,

conductivity and polarimetry are used to determine CD complexation.

Polarography is a suitable method to study inclusion complexation if

the electron distribution of a complexed electro active guest molecule in

aqueous solution is different from that in the non-complexes state in

aqueous solution. Conductivity measurement may be used to

characterize inclusion complexation. Anionic surfactants having

different polar heads, different tail configurations, the same Na+

counter ion and their solution conductivities are dramatically affected

by inclusion complex formation with CDs. A polarimetric study was

conducted as a supporting tool for the complex formation because βCD

is optically active in nature.

34

1.9.2.2. Solubility studies

Changes in solubility of the guest are plotted as a function of the

CD concentration to check whether the solubility of potential guest

increases with increase in CD concentration.

1.9.2.3. Spectroscopic studies

Spectroscopic methods include Nuclear Magnetic Resonance

(NMR) spectroscopy, Electron Spin Resonance (ESR), Ultraviolet/Visible

(UV/VIS) spectroscopy, Fluorescence spectroscopy, Circular dichroism

spectroscopy.

The most direct evidence for the inclusion of a guest into CD

cavity is obtained by 1H-NMR spectroscopy. It is also used to determine

the direction of penetration of guest molecule into the CD cavity. ESR is

a useful method to investigate inclusion complexation with radicals in

aqueous solutions. The complexation causes a change in the

absorption spectrum of a guest molecule. During the spectral changes,

the chromophore of the guest is transferred from an aqueous medium

to the non polar CD. When fluorescent molecules in aqueous solution

are included in cyclodextrins, fluorescence spectra may be influenced

which indicates the formation of inclusion complexes. Circular

dichroism is a useful method to detect cyclodextrin inclusion complexes

in aqueous solution. When an achiral guest molecule is included within

the asymmetric locus of the cyclodextrin cavity which consists of chiral

glucose units, new circular dichroism bands can be induced in the

absorption bands of the optically inactive guest.

35

1.9.2.4. pH-Potentiometric Titration

If the guest compound has a prototropic function, the

potentiometric titration method can be used to detect inclusion

complex formation. Due to the fact that cyclodextrin usually favour the

unionized guest molecules having a higher hydrophobicity, rather than

the ionized ones, the pKa value of an acidic guest molecule is usually

increased, while those of basic ones is usually decreased by binding to

CDs.

1.9.2.5. Microcalorimetry

Changes in thermodynamic properties due to inclusion

complexation can be measured by microcalorimetry. These changes in

enthalpy and entropy are associated with the change in the behaviour

of water structure within the cavity, removal of the water from the

cavity, restructuring of water around the guest molecule and release of

water into the bulk. Other contributions to the overall energies of

reaction are due to the restriction in rotation around the glycosidal

linkages of the cyclodextrin when the guest molecule enters the cavity.

1.10. Factors influencing inclusion complex formation

There are several factors effecting complex formation which are

briefed as below.

1.10.1. Type of cyclodextrin

Type of cyclodextrin can influence the formation as well as the

performance of drug/CD complexes. For complexation, the cavity size

36

of cyclodextrin should be suitable to accommodate a drug molecule of

particular size.33,61-65 Compared with neutral CDs, complexation can be

better when the CD and the drug carry opposite charge but may

decrease when they carry the same charge.66,67 For many acidic drugs

forming anions, the cationic (2- hydroxy-3-[trimethyl ammonio] propyl)

βCD acted as an excellent solubilizer.16 In the case of ionisable drugs,

the presence of charge may play a significant role in drug/CD

complexation and hence a change in the solution pH can vary the

complex constant.

1.10.2. Temperature

Temperature changes can affect drug/cyclodextrin complexation.

In most cases, increasing the temperature decreased the magnitude of

the apparent stability constant of the drug/CD complex and the effect

was reported to be a result of possible reduction of drug/CD interaction

forces, such as van der Waals and hydrophobic forces with rise of

temperature. However, temperature changes may have negligible effect

when the drug/CD interaction is predominantly entropy driven (i.e.,

resulting from the liberation of water molecules hydrated around the

charges of guest and host molecules through inclusion complexation.68

1.10.3. Method of preparation

Method of preparation viz co grinding, kneading, solid dispersion,

solvent evaporation, co precipitation, spray drying, or freeze drying can

affect drug/CD complexation. In many cases, spray drying66,69,70 and

37

freeze drying71,72 were found to be most effective for drug complexation

and leads to more drug solubility and bioavailability.

1.11. Past work carried out on cyclodextrin complexation

Loftsson T et al. in a review discussed the importance of

cyclodextrins as functional excipients that are used in over

40 marketed products in various global regions. Increasing the ability

of cyclodextrins to complex with drug through a manipulation of their

complexation efficiency (CE) may expand the use of these materials to

the increasing list of drug candidates and marketed drugs that may

benefit from this technology. The review assessed tools and materials

that have been suggested for increasing the CE for pharmaceutically

useful cyclodextrins and drugs. They stated that expanding the use of

cyclodextrins in oral dosage forms will require mechanisms to limit

their amounts as otherwise formulation bulk becomes limiting.

Techniques that may be interesting in this regard include those that

impact both apparent drug solubility as well as the efficiency by which

the drug interacts with the cyclodextrin molecule. The use of drug

salts, polymers, and co solvents may be useful to varying degrees in

this regard. In addition, processing approaches that may make

cyclodextrins function as better solubilizers should be considered and

include the use of heat during processing as well as volatile bases,

acids and processing solvents. Finally, they concluded that considering

overarching formulation concepts such as super saturation may further

38

help in the optimal use and placement of cyclodextrin in solid dosage

forms.73

A survey of the literature related to use of cyclodextrins for

enhancing oral bioavailability was conducted by Rebecca L Carrier et

al. The focus of the literature reviewed was on the solubilising

capabilities of cyclodextrins and their contribution to bioavailability

enhancement and on the use of cyclodextrins in oral pharmaceutical

formulations. Twenty eight studies were examined in which the focus

was on the use of cyclodextrins as solubilizers to enhance

bioavailability. Commonly observed factors included: utilization of

pre-formed complex rather than physical mixtures, drug

hydrophobicity (log P > 2.5), low drug solubility (typically <1 mg/ml),

moderate binding constant (< 5000 M−1), low dose (< 100 mg), and low

CD: drug ratio (< 2:1). Most studies reported successful improvement in

bioavailability. Of these studies, the majority reported an increase in

AUC between 0 and 100% (1 to 2 times). They concluded that the

physical and chemical properties of a given drug, cyclodextrin, and

dosage form interact to influence kinetics of key processes of oral drug

delivery, including dissolution and absorption. These interactions

create difficulty in precisely specifying one range of values for one

parameter (e.g., binding constant, solubility) that will result in

successful bioavailability enhancement using cyclodextrins.74

A brief literature review on cyclodextrin complexation is given in

Table 1.4 to 1.7.

39

Table 1.4: Summary of literature on cyclodextrin complexation with different drugs

Drug Type of CD Method used for complexation

Purpose of the work Reference

Celecoxib βCD Kneading, evaporation and

freeze drying

Improvement of aqueous solubility and dissolution rate

75

Celecoxib HPβCD Physical mixing, co grinding, kneading and evaporation

Fast dissolution 76

Rofecoxib SBE7βCD Kneading Better solubility enhancement with SBE7βCD than βCD

77

Valdecoxib HPβCD and SBE7βCD

Kneading and co

evaporation

Enhanced solubility, dissolution rate and similar in vivo absorption rate with both CDs

78

Captopril HPβCD and perbutanoyl

βCD(TBβCD)

Kneading Binary HPβCD release rate was faster than binary TBβCD. Ternary captopril, TBβCD and

HPβCD system gave better plasma profile

79

Flurbiprofen βCD, MβCD and hydroxyl

ethyl βCD

Physical mixing, kneading, sealed

heating, co evaporation and

co lyophilization

Solubility enhancement obtained by different binary systems varied from a minimum of 2.5

times up to a maximum of 120 times, depending on both the cyclodextrin type and

the preparation method

80

Eflucimibe γCD Kneading Enhancement of solubility 81

40

Glimepiride βCD, HPβCD and SBE7βCD with or without water

soluble polymers

Kneading Highest dissolution rate was observed with SBE7βCD than with HPβCD and βCD. Ternary systems showed an increase in dissolution

rate compared to binary systems and duration of action was longer for ternary systems than

for innovator product.

82

Clofibrate βCD Co precipitation, kneading and

sealed heating

Complex prepared by sealed heating method showed greatest improvement in dissolution

rate and suitable for large scale industrial use and does not require the use and costly elimination of large amounts of water while the complex prepared by co precipitation showed

lowest rate of dissolution.

83

Tolbutamide βCD and HPβCD Kneading, co precipitation and

freeze drying

The extent of dissolution rate enhancing effect was found to be dependent on the method

used for the preparation of the mixture.

84

Amlodipine, Felodipine

MβCD Kneading and lyophilization

For amlodipine, 3 times enhancement in solubility with both methods of preparation

and for felodipine, 16 times increase in solubility was observed

85

Tolbutamide βCD in demineralized water

and in aqueous solutions of

different surfactants

Physical mixing and kneading

The presence of surfactants with proper shape and structure in dissolution media or in the

formulation containing tolbutamide-βCD inclusion complex gave rise to unexpected

dissolution

86

41

Warfarin βCD Physical mixing, kneading, co evaporation and

freeze drying

Freeze drying was the best method to obtain the highest dissolution rate of the drug and higher solubility and also suggested possible improvement of

oral warfarin base bioavailability

87

Lycopene HPβCD Kneading Enhancement in solubility 88

Ursodeoxycholic acid

βCD and co precipitation with choline dichloride

Kneading, freeze drying, sealed heating and spray drying

Only spray drying provided complete complexation and ursodeoxycholic acid- βCD-CDC complex obtained by spray drying showed highest dissolution rate

26

Bicalutamide βCD Kneading, solvent evaporation and spray drying

Increase in dissolution rate order with different techniques was found to be kneading>spray drying>solvent evaporation>physical mixture>pure drug

89

Beclomethasone diproprionate

γCD Spray drying to obtain powder for inhalation/lung

delivery

In vitro tests indicated that the preparation of particles with right characteristics for lung deposition, by spray drying, is favoured by high Tin and low solution flows,

which result in higher Tout values.

90

Phenytoin HPβCD, HPγCD and

MβCD

Freeze drying Phenytoin complex with MβCD showed enhancement in solubility and dissolution rate and pharmacological

evaluation in mice indicated the possibility for developing new parenteral formulation similar to that

of phenytoin sodium

91

Ketoprofen βCD and SBE7βCD

Hot-melt extrusion, physical mixing, co grinding and freeze drying

Hot-melt extrusion was found to be superior in the extent of intimacy of mixing and dissolution compared to other methods.

92

42

Table 1.5: Summary of literature on cyclodextrins based on factors effecting complexation

Factor Drug CDs studied Observation Reference

Type of CD Albendazole,

Mebendazole, Ricobendazole Fenoprofen

Ketoprofen

Cocaine

βCD, HPβCD,

MβCDs E, β, γ, HPβCDs

MβCD, βCDs

E, β, γ CDs

More effective enhancement of

solubility with substituted CDs Better stability constant values of

pharmaceutical interest with only βCD and HPβCD complexes.

Better dissolution performance of MβCD complex.

Drug binding with reasonable affinity only to βCD in aqueous solution

71

93

94

95

Cavity size Gliclazide

Digitoxin

β, ECDs

δCD

Cavity size of βCD was suitable for

complexation while that of αCD was insufficient to include GL rings Enhanced solubility due to partial

inclusion of the drug in CD Cavity

58

96

pH and ionization

state

Piroxicam

Levemopamil HCl

βCD

HPβCD

Effective complexation at low pH

Enhancement of solubility was 3 fold with charged drug and 525 fold with the neutral form

97

98

Temperature Sulindac βCD Increase in temperature, decrease in

stability constant

99

43

Table 1.6: Summary of literature on cyclodextrins based on effect of preparation methods

Drug CD Effect Reference

Ibuproxam E, β, γCDs With β and γCDs, spray drying and sealed heating resulted in true complexation and

kneading was ineffective.

100

Nimesulide βCD Drug dissolution was higher with kneading than with co-evaporation.

101

Sulfamethoxazole βCD and

HPβCD

Increased dissolution rate with solid complexes

prepared by freeze drying

102

Glibenclamide βCD Superior dissolution with ground mixture, physical mixture and kneaded product

103

44

Table 1.7: Review on applications of CDs in oral delivery

Effect CD Drug Reference

↑ Bioavailability by ↑ in

solubility and dissolution rate

βCD

HPβCD

SBE7βCD

MβCD

Ketoprofen, Griseofulvin, Terfenadine

Albendazole, Ketoprofen, Phenytoin

Spiranolactone

Albendazole

104-106

71,106,107

108

71

↑ Permeability HPβCD Flutamide 109

↑ Intensity or duration of therapeutic activity

βCD

HPβCD

Terfenadine, Tolbutamide

Tolbutamide, Amylobarbitone

106,110

110,111.

↑Gastro intestinal stability ZCD

HPβCD

Digoxin

Rutin

112

113

↑ Buccal bioavailbility SBE7βCD

HPβCD

Danazol 114,115

45

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