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Acharya Nagarjuna University, Guntur 19

CHAPTER - I


INTRODUCTION

Chiral drugs are a subgroup of drug substances that contain one or more chiral

centers. More than one-half of marketed drugs are chiral (Millership, J., 1993). It is

well known that the opposite enantiomer of a chiral drug a lot differs considerably in

its pharmacological (Islam, M. M., 1997), toxicological (Wainer, I., 1993),

pharmacodynamic, and pharmacokinetic (Drayer, D., 1986; Midha, K. M., 1998)

properties. Therefore from the points of view of safety and efficacy, the pure

enantiomer is favored over the racemate in several marketed dosage forms. However,

the chiral drug is often synthesized in the racemic form, and it is often costly to

resolve the racemic mixture into the pure enantiomers. Currently, then, most chiral

drugs, including some ‘‘blockbuster’’ drugs, such as fluoxetine hydrochloride

(Prozac) and omeprazole (Losec), are still marketed as racemates. However, the

recent development is toward marketing more single-enantiomer drugs in addition,

many companies to increase its therapeutic efficacy and to broaden patent protection

(Di, Cicco. R., 1993) actively follow a racemic switch, which involves the

development of a pure enantiomer of a drug that is already marketed as a racemate.

The assertion whether to market the racemate or the enantiomer of a chiral drug is

mainly based on pharmacology, toxicology, and economics. From a pharmaceutical

perspective, the physical properties of both the racemate and the enantiomer should

be characterized in detail in order to develop a safe, efficacious, and unswerving

formulation, no matter whether the racemate or the enantiomer is chosen as the

marketed form. Furthermore, the chirality of a drug will also control the efficiency of

delivery, which has not been well predictable in the pharmaceutical field. Many

physical properties of a crystalline solid, such as density, solubility, dissolution

activities, stability, and mechanical properties, are governed by the crystal structure

(Leusen, F., 1999). Knowledge of the relationship between the crystal structure and

the physical properties, and their influence on drug release, may therefore provide a

basic understanding of the property-delivery correlation.


1. Enantiomers, racemic species, and diastereomers

Molecular chirality is a theory that was derived historically from the

distinction between the configurational isomers of asymmetric molecules, which was

discovered by Pasteur and reported in 1894. Configurational isomers are compounds

with the same molecular formula and the same substituent groups but with diverse

configurations. The asymmetric center in configurational isomers is called the chiral

center (Mislow, K., 1999). Configurational isomers, which are a separation of

stereoisomers and also termed optical or chiral isomers, may be classified as

enantiomers, racemic species, and diastereomers. Enantiomers are pairs of

configurational isomers that are mirror images of each other and yet are not

superimposable. Each enantiomer is homochiral, meaning that all the molecules have

accurately the same configuration. Diastereomers are pairs of compounds that contain

more than one chiral center, not all of which are superimposable. Enantiomers act

differently only in a chiral medium, such as when exposed to polarized light or when

participating in a chemical reaction catalyzed by a chiral catalyst, predominantly an

enzyme in the body. Diastereomers commonly show different physical properties,

even in an achiral environment. An equimolar mixture of opposite enantiomers is

termed a racemic species or racemate, and is heterochiral, meaning that the molecules

have special chiralities.

2. Molecular interactions of chiral molecules

The differences in properties between the enantiomer and the racemic

compound start from the differences in interactions between the same homochiral

molecules and those between the heterochiral molecules, and from the different

packing arrangements in the crystal structures. In the enantiomer, the molecular

interactions are homochiral interactions (R…R), which are defined as the

intermolecular nonbonded attractions or repulsions in assemblies of molecules of the

same chirality. However, the heterochiral interactions (R…S) in the racemate are

those between molecules of opposite chirality (Eliel, E.L., 1994; Li, Z., 1997). These

two interactions are implausible to be identical because they are diastereomerically

related. However, the difference is small enough to be ignored in the gaseous or

liquid state, or in an achiral solvent. In the solid state or in a chiral environment, the


difference is important to result in different physical properties between the racemate

and the corresponding pure enantiomer; this difference is termed enantiomeric

discrimination. Enantiomeric discrimination is observed when comparing the crystal

structure of the racemic compound with that of the corresponding enantiomer. The

difference between the homochiral and heterochiral interactions leads to different

structures, which in revolve lead to differences in physical properties and/or

biological actions. A natural extension of the idea of enantiomeric discrimination is

diastereomeric discrimination, which is a result of the difference between the

interactions of diastereomeric pairs, such as RI …RII and RI …SII, where I and II

represents diverse molecules (Stewart, M., 1982). Diastereomeric prejudice is the

basis of chromatographic separation of enantiomers by a chiral stationary phase.

3. Assertive considerations in drug development of stereoisomers

This chapter deals with several of the regulatory issues that need to be

considered at the time of developing a drug with chiral center(s). Drug Development

and the regulatory process the regulatory drug development practice for new drugs

has evolved over the years. Development of drug law dates back to 1906 when the

Federal Food and Drug act was enacted, but the FDA had no accountability in

premarketing evaluation of drugs. In 1962, for the first time, a drug had to be shown

to be effective. Subsequently, guidelines to format and content of the clinical and

statistical sections of the drug application were issued in 1988. Only then were there

attempts to distinguish dose–response relationships in adverse events, and to examine

the rates of adverse events in demographics (age, race, gender) and other subgroups

(metabolic status, renal function). Cumulative drug development eras could be

recognized as the era of safety, of efficacy, and now of the individualization of drug

therapy. In general, once a drug company (sponsor/applicant) has identified a

chemical for development as a drug, a considerable amount of research has to be

carried out before it can be approved for marketing as a drug. The areas of research

for a drug can generally be categorized into chemistry, pharmacology/ toxicology,

clinical (determining safety and efficacy), clinical pharmacology, and

biopharmaceutics, and for certain drugs also microbiology. Research data in all these

areas are summarized and submitted in a New Drug Application (NDA), which is


reviewed by the scientists in the regulatory agencies for correctness and adequacy. In

assessing suitability of the data, regulatory agencies spotlight on assuring that the

drug will be safe and effective, and adequate information will be available to

individualize the dose in particular populations (e.g., by gender, age, and race),

disease state (hepatic and renal insufficiency), and in the incidence of other drugs

(drug–drug interactions). These data are then summarized into a product label for a

drug approved by the regulatory agencies. Therefore the product label is luminous

foundation of information about the drug product. Readers are encouraged to refer to

the product labels and drug reviews available on the FDA Internet site

(http://www.fda.gov/cder/ drug/default.htm New Prescription Drug Approvals) for

newly approved racemates and enantiomers. This will give the reader with summary

of information that was considered in the approval of such drugs. For the

development of any drug, general scientific and regulatory principles remain the same

and are known to the drug development community. Presence of chiral center(s) adds

a unusual challenge to the known principles, especially for drugs that show

enantioselective pharmacokinetics (PK) and/or pharmacodynamics (PD).

4. Regulatory guidance

Traditional bases for regulatory considerations are the code of federal

regulations (CFR), the domestic guidances issued by drug regulatory agencies, the

International Conference on Harmonization of Technical Requirements for

Restoration of Pharmaceuticals for Human Use (ICH) guidance, the reviewer MAPP

(manual of policies and procedures), present scientific standards, and precedents. In

order to ease drug development and provide simplicity in the drug regulatory and

approval process, regulatory agencies have issued numerous topic-specific guidances

in the last decade. These guidance documents give current idea and universal

acceptable approaches. It should be noted that these documents are guidance and not

regulations. Therefore if the applicant wishes to choose a different path from that

outlined in guidance, it is acceptable as long as the data provided by the other

approach addresses the questions and concerns about the drug. Several guidances

issued by the regulatory agencies worldwide address many aspects of drug

development. Since technological advances allow the production of a single


enantiomer on a commercial level, the FDA and other regulatory agencies have

issued their policy with respect to new drugs that consist of more than one

stereoisomer. Technological advances give choices to the sponsors as to what path of

the development should be taken for a drug candidate with a chiral center. These

choices contain the development of a racemate versus an enantiomer or an enantiomer

of an already marketed racemate. General guidance for development of chiral drugs

has been issued by European, Canadian, and US regulatory agencies and will be

discussed in this chapter. The main guidance documents, which include information

on the development of chiral compounds, are as follows:

1. The FDA’s policy statement for the development of new stereoisomeric drugs,

issued by the FDA in 1992.

2. Investigations of chiral active substances issued by a commission of the European

countries in 1994.

3. Stereochemical issues in chiral drug development, issued as the Therapeutic

Product Programme, Canada, in 2000.

For any drug development, one has to design the program and experiments to

answer the questions that need to be addressed ahead of a drug can be accepted for

marketing. While developing a drug with chiral center(s), the most important question

is the contribution of each isomer to the safety and efficacy when it is administered as

a racemate or as an enantiomer (if inter-conversion occurs). The decision to develop a

racemate or enantiomer is that of the sponsors. When developing a racemate, issues

associated to acceptable manufacturing controls of synthesis and impurities,

appropriate pharmacological and toxicological assessments, characterization of

ADME (absorption, distribution, metabolism, and elimination), and clinical

assessment should be addressed. The following paragraphs discuss each of these areas

in detail.

4.1 Chemistry

For every drug being developed, manufacturing and control procedures to

reassure the identity, quality, purity, and strength of the drug substance and the drug

product are vital. In addition, the following considerations for chiral drug substances

and drug products are obligatory.


4.2 Drug substance, drug product, and stability

For enantiomeric and racemic drug substances, stereochemically specific

identity tests and/or stereospecific assay methods should be developed and available.

In addition to measuring optical rotation, the Canadian guidance recommends to

incorporate melting point, chiral chromatography, optical rotary dispersion, circular

dichroism, and NMR using chiral shift reagents. The selection of the controls should

be based upon the product’s composition, the method of manufacture, and its stability

characteristics. The stability protocol for enantiomeric drug substances and drug

products should contain methods capable of assessing the stereochemical integrity of

the drug substance and drug product. If it is established that stereochemical

conversion does not occur, stereospecific assays might not be needed. Complete

description including the critical factors of the manufacturing process used to attain

the single enantiomer or racemate should be provided. In regulatory submissions to

the authorities in Europe and Canada, the identity and stereoisomeric purity of the

starting material, key intermediates, and chiral reagents should be established.

4.3 Labeling

The labeling should incorporate a unique established name and a chemical

name with the appropriate stereochemical descriptors.

4.4 Pharmacology / toxicology

4.4.1 Pharmacology

For racemates, the pharmacological activity of the individual enantiomers

should be characterized for the primary and any other significant effects, with respect

to potency, specificity, and most effect. To monitor in vivo interconversion and

disposition, the pharmacokinetic profile of each isomer should be characterized in

animals and later on compared to the pharmacokinetic profile obtained in Phase 1

clinical studies.


4.4.2 Toxicology

If toxicity other than that predicted from the pharmacological properties of the

drug occurs at comparatively low multiples of the exposure planned for clinical trials,

the toxicity study should be repeated with the individual isomers to discover whether

only one enantiomer was responsible for the toxicity. If toxicity of significant alarm

resides in a single isomer, the development of single isomer with the desired

pharmacological effect would be desirable. Where questions exist regarding the

definition of ‘‘significant toxicity,’’one should discuss the issue with the suitable

clinical division within the regulatory agency where the drug will be reviewed.

4.5 Impurity limits

The concentration of each isomer should be determined and limits clear for all

isomeric components, impurities, and contaminants for the compound tested

preclinically that are potential for use in clinical trials. The maximum allowable level

of impurity in a stereoisomeric product employed in clinical trials should not exceed

that present in the material evaluated in nonclinical toxicity studies.

4.6 Developing racemates or single enantiomers

The US, European, and Canadian guidance documents state that it is the

sponsor’s choice to develop a racemate or an enantiomer for a chiral compound.

However, regulatory agencies assess the justification provided by the sponsor in

reaching that choice. Therefore it is constructive to discuss the drug development plan

with the regulatory agency. It is essential to develop quantitative analytical assays for

quantitation of individual enantiomers in samples obtained from in vivo studies early

in drug development.

Let us discuss different scenarios that may arise from a racemate consisting of

two enantiomers. In this case, there could be four various situations with respect to

the PK and PD of the two enantiomers: same PK and PD (efficacy and toxicity)

profiles; different PK but same PD profile; same PK but different PD profile; or

different PK and PD profiles.


4.7 Clinical and biopharmaceutical

4.7.1 Concerns

It has been recommended that, if experiential differences in activity and

disposition of the enantiomers are minimal, racemates may be developed. However,

the development of a single enantiomer is particularly desirable when only one of the

enantiomers has a toxic or undesirable pharmacological effect. Further investigation

of the properties of the individual enantiomers and their active metabolites is

warranted if unexpected toxicity or pharmacological effect occurs with clinical doses

of the racemate. Signals of adverse events may be explored in animals, but human

testing may also be necessary. It should be documented that toxicity or unusual

pharmacological properties might reside not in the parent isomer, but in an

isomerspecific metabolite. In summary, both enantiomers should be evaluated

clinically, and the development of only one enantiomer should be considered when

both enantiomers are pharmacologically active but vary considerably in potency,

specificity, or maximum effect. However, the development of a single enantiomer

may not be warranted if one of the isomers is basically inert. When both enantiomers

are establish to carry desirable but different properties, the development of a mixture

of the two, not necessarily the racemate, as a fixed combination might be rational.

If a racemate is being studied, the pharmacokinetics of the two isomers and

their possible interconversion should be studied in Phase 1. Based on Phase 1 or 2

pharmacokinetic data in the target population, it should be potential to determine

whether an achiral assay or monitoring of just one enantiomer, where a fixed ratio is

confirmed, will be adequate for pharmacokinetic evaluation.

4.7.2 Developing an isomer

If a single isomer is being developed and no interconversion occurs,

enantiospecific assays need not be used. However, if the antipode is formed in vivo,

that should be considered as a metabolite and addressed accordingly. If a racemate

has been studied, and a single enantiomer is being developed, an abbreviated

pharmacology/toxicology assessment could be conducted, which would permit for the


use of the existing knowledge of the racemate. This would usually contain the longest

repeat-dose toxicity (up to 3 months), and the reproductive toxicity part II study in the

most sensitive species, using the single enantiomer being developed. These studies

should include a positive control group, which consists of treatment with the

racemate. If no differences in toxicity profiles were found between the enantiomer

and the racemate treatment groups, no further studies would be required. If single

enantiomer is more toxic, the explanation should be provided with supporting data,

and suggestions for human studies should be considered.

In humans, assessment should include the determination of interconversion

and the comparison of the PK profile of the individual isomer when administered

alone versus when administered as a racemate. Some examples of existing single

isomer approvals for previously marketed racemic drugs include escitalopram

(S-citalopram), esomeprazole (S-omeprazole), and focalin (d-isomer of

methylphenidate). Readers are encouraged to submit to their product labels and the

FDA reviews available on the FDA website

(http://www.fda.gov/cder/drug/default.htm New Prescription Drug Approvals) to gain

an understanding of what data were submitted for the approval of these drugs. In case

of rapid in vivo interconversion, regulatory guidance issued by the Therapeutic

Products Program point out that the antipode should be considered as a metabolite

during the drug development course.

4.7.3 Biopharmaceutics

There has been considerable discussion in the literature on the need to use

enantiospecific assays to evaluate bioequivalence. Guidance published by the FDA,

Bioavailability and Bioequivalence Studies for Orally Administered Drug Products

General considerations addresses this question and is summarized below (see also fig.

1.1 for a decision tree). For bioavailability studies, measurement of individual

enantiomers may be important. For bioequivalence studies, this guidance

recommends measuring of the racemate using an achiral assay. Measurement of the

individual enantiomers in bioequivalence studies is recommended only when all of

the following conditions are met: (1) the enantiomers exhibit different

pharmacodynamic characteristics; (2) the enantiomers show different


pharmacokinetic characteristics; (3) primary efficacy/safety activity resides with the

minor enantiomer; and (4) nonlinear availability is present for at least one of the

enantiomers. A guidance issued by the Therapeutic Products Programme states that in

common, when comparing solid dosage forms of similar type

Is PD (Either efficacy or toxicity) enantiospecific?

NoYes

Measure racemate Is PK Enantiospecific?

No

Yes

In which enantiomer

does the major activity reside?Major

Is PK linear?Measure

enantiomer

Minor

Yes* No**

* Enantiomer ratios remain constant with change in input rate

** Enantiomer ratios remain change with change in input rate

Fig 1.1: When to use an enantiospecific assay for a racemate.

(e.g., two immediate release formulations), total drug concentrations can be

measured. When conducting a comparative bioavailability study of solid dosage

forms of different types, such as a modified release dosage form and an immediate

release dosage form enantioselective assays should be used. This guidance also calls

for meeting the bioequivalence criteria for each enantiomer for drugs whose rate of

release and/or absorption affect the in vivo enantiomeric ratio of the drug (e.g., the

nonlinear first-pass effect).

4.8 Clinical pharmacology

The role of clinical pharmacology during drug development is to optimize the

dose and dosing regime with the objective in mind of reducing the undesirable effects

and producing beneficial responses in a predictable method. To attain


individualization of dose, maximum knowledge of the factors contributing to the PK

and PD variability has to be acquired. As stated by Temple (Temple, R. J., 1996),

‘‘Clinical pharmacologists are trained to take a broad view of drugs, recognizing that

they not only have the pharmacologic property of foremost interest but regularly other

properties as well, that a ‘drug’ is really many drugs (isomers, active metabolites)

with diverse properties, and that the properties of drugs should influence how they are

dosed and used.’’ Planning the right type of studies to answer the questions that

prescribing physicians and patients reason will optimize the drug development

process.

Proper planning and asking the precise questions early in the drug

development development will save costs by minimizing the time and eliminating the

studies that do not contribute to the largely understanding of the drug being

investigated. The Office of Clinical harmacology and Biopharmaceutics at the FDA

has described its assignment as ‘‘to assure that an individual patient receives the right

drug, in the right dose at the right time and in the right dosage form.’’ To attain this

objective for every drug has its sole challenges, but one has to work toward achieving

this objective if patient and physician are to have maximum confidence in the drug

product. Recent significant advances in science related to pharmacogenomics, the

discovery of the function of transporters, and the importance by the regulatory

agencies on product quality issues present an important role for clinical

pharmacologist in the entire drug development process. It is now accepted that

enantiospecific ADME is more likely than not, and disease states could have

enantiospecific effects on the pharmacokinetics of drugs, which in revolve could have

significant crash on pharmacodynamic response. Therefore, when developing a chiral

compound, one should evaluate the need for enantiospecific determinations in

particular populations (e.g., age, gender, and race) and drug interactions. This choice

will be based on knowledge of enantiospecific assessments during early Phase 1

studies and the animal and in-vitro data available.


4.9 Regulatory conclusions

Regulatory principles for chiral drugs are the similar as for any other drug,

that is, to give safe and effective drugs based on sound scientific principles.

Appropriate information to regulate the dose/dosage regimen should be presented.

In general, for PK consideration of chiral compounds, the main difference

from other drugs is the decision whether to use an enantioselective or an achiral assay

to characterize the pharmacokinetics of each enantiomer or racemate, respectively. At

present, the marketing exclusivity period in the US, for developing a single isomer

from a earlier approved racemate, is 3 years. Various examples of chiral drugs in

recent times approved are citalopram, which is a racemate, and esomeprazole (S-

isomer of omeprazole), escitalopram (the S-isomer of citalopram), and focalin (the

dextrorotary isomer of methylphenidate), which are the single isomers of already

approved racemates. Product label and reviews on the FDA website for these drugs

provide excellent references and examples for readers interested in learning more

about the data required for development of chiral drugs.

5. Lenalidomide:

5.1 Chemical and physical properties

Lenalidomide (fig.1.2), a thalidomide analogue, is an immunomodulatory

agent with antiangiogenic and anti-neoplastic properties. The chemical name is 3-(4-

amino-1-oxo 1,3-dihydro-2H-isoindol- 2-yl) piperidine-2, 6-dione. The empirical

formula for lenalidomide is C13H13N3O3, and the gram molecular weight is 259.3.


Fig 1.2: 3-(4-amino-1-oxo 1,3-dihydro-2H-isoindol-2-yl) piperidine-2, 6-dione

5.2 Mechanism of action and clinical pharmacology

The mechanism of action of lenalidomide is not completely characterized.

Lenalidomide has anti-neoplastic, immunomodulatory and anti-angiogenic properties;

it inhibits the secretion of pro-inflammatory cytokines and increases the secretion of

anti-inflammatory cytokines from peripheral blood mononuclear cells. The drug

inhibits cell proliferation with varying effectiveness in some, but not all, cell lines. It

inhibits the expression of cyclooxygenase-2 (COX-2) but not COX- 1 in vitro.

Lenalidomide is successful in inhibiting the growth of MM cells obtained from

patients as well as Multiple myeloma (MM) cells. It also blocks the production of

cytokines, which are essential for cell growth in MM, including TNF-α �and IL-6.

5.3 Multiple myeloma

Multiple myeloma (MM) is a B cell malignancy characterised by excess

monotypic plasma cells in the bone marrow (BM) in connection with monoclonal

protein in serum and/or urine, decreased normal Ig levels and lytic bone disease. It is

the third most general blood cancer in the US affecting ~ 50,000 people. There are ~

15,000 new cases of MM diagnosed each year, and ~ 11,000 Americans (including


5500 men and 5300 women) were projected to die of MM in 2004. Cellular and

molecular substantiation suggests that MM cells are the transformed counterparts of

post germinal centre BM plasma blasts/plasma cells (Kuehl, W. M., 2002).

5.4 Role of the bone marrow milieu in multiple myeloma pathogenesis

The BM microenvironment plays a critical function in enhancing tumour cell

growth, survival, migration and drugresistance (Hideshima, T., 2003). Delineation of

the mechanisms mediating MM cell proliferation, survival and migration both

enhances the understanding of pathogenesis and provides the framework for the

identification and validation of novel molecular targets. MM cells home to the BM

and adheres to extracellular matrix proteins and BM stromal cells (BMSCs), which

not only localises tumour cells in the BM milieu but also has important functional

sequelae (Teoh, G., 1997). Specifically, binding of MM cells to the extracellular

matrix protein fibronectin triggers the upregulation of p27 Kip1 and cell adhesion-

mediated drug resistance (CAM-DR) (Hazlehurst, L.A., 2000), related directly to cell

contact and mediated independently of cytokines. In addition, the authors’ studies

have characterized MM cell binding to BMSCs as well as sequelae, including growth,

survival and drug resistance. Significantly, adhesion of MM cells to BMSCs not only

equally mediates resistance to drug-induced apoptosis but also triggers the paracrine

NF-k B-dependent IL-6, the major cytokine mediating MM cell growth and sur-

transcription and secretion of vival, in BMSCs (Hideshima, T., 2002). The authors

have also shown that MM cells that are localised in the BM milieu secrete cytokines,

such as TNF-α, TGF-β (Hayashi, T., 2004) and vascular endothelial growth reason

(VEGF) (Ankbar, B., 2000), which further upregulate IL-6 secretion in BMSCs.

Within the BM microenvironment, the authors and others have demonstrated that

cytokines mediate growth (IL-6, -6 and IGF-1) and migration (VEGF and stromal

cell-derived factor [SDF]-1α of MM cells and also activate Angiogenesis (Mitsiades,

C. S., 2004). Therefore, signalling cascades mediating these cytokine properties can

be targeted using novel therapeutic approaches.

5.5 Pharmacokinetics of lenalidomide

Even though thalidomide was exposed to be useful in different diseases, it is a

effective teratogen and causes a number of side effects including peripheral


neuropathy (Tsen, G. S., 1996), consequently, attempts were made to develop

thalidomide analogues that were more potent and had fewer adverse effects. This

drug discovery programme expands Lenalidomide.

Wu and Schreiber (Wu, A., 2004) have reported the pharmacokinetics of

lenalidomide in MM patients at the 2004 American Society of Clinical Oncology

conference. This study is a single centre, open-label, non-randomised, Phase I dose

escalation study in relapsed and refractory MM. The doses of lenalidomide used were

5, 10, 25 or 50mg/day p.o. for 28 days. Blood samples were collected before and at:

15, 30and 45 min; and 1, 1.5, 2, 2.5, 3, 4, 6, 8, 8, 10, 12, 18, 24, 48 and 72 h

following administration on days 1 and 28. No lenalidomide dose-limiting toxicity

(DLT) was observed at any of the dose levels within the first 28 days. Absorption of

lenalidomide was rapid on both days 1 and 28, with the time to reach maximum

concentration (tmax) ranging from 0.7 to 2h at all of the dose levels. Plasma

lenalidomide levels declined in a monophasic manner, with elimination half-life

ranging from 2.8 to 6.1h on days 1 and 28 at all four doses. No plasma accumulation

was observed on multiple dosing.

5.6 Preclinical studies of lenalidomide

5.6.1 Overview of anti-MM activity of lenalidomide

Lenalidomide could inhibit MM cell growth by several various mechanisms.

First, lenalidomide has a direct effect on MM cells to induce G1 growth arrest or

apoptosis of even drug-resistant cells (Mitsiades, N., 2002). Second, lenalidomide

inhibits the adhesion of MM cells to BMSCs, and in that way can overcome CAM-

DR. Third, lenalidomide inhibits bioactivity and / or secretion in MM cells and / or

BMSCs of cytokines (e.g., IL-6, -1� and -10, and TNF-), which increase MM cell

growth, survival, drug resistance, migration and expression of adhesion molecules.

Importantly, lenalidomide is ~ 50,000-fold more potent than thalidomide at inhibiting

TNF-α -/IL-1� -triggered cytokine secretion from mononuclear cells stimulated with

lipopolysaccharide (LPS) invitro (Muller, G. W., 1999). Fourth, VEGF and bFGF are

secreted by MM cells and/or BMSCs, and lenalidomide may inhibit the activity of

VEGF, bFGF, and angiogenesis in MM. Finally, lenalidomide acts against MM in the


course of immunomodulatory effects, such as the augmentation of action of cytotoxic

Tcells and natural killer (NK) cell-associated secretion of IL-2 and IFN-γ

5.6.2 Direct antitumour activities

Although the direct antitumour activity of lenalidomide has not been fully

delineated, several studies have examined the molecular mechanisms mediating

sequelae of lenalidomide. The authors’ preceding studies established that

lenalidomide induces G0/G1 growth arrest associated with pCip upregulation and/or

apoptosis that are mediated via caspase-8 activation. Lenalidomide inhibits LPS-

mediated induction of COX-2 and prostaglandin (PG)-E production by a post-

transcriptional mechanism in RAW 364.7 cells (Fujita, J., 2001), suggesting that the

antitumour activity induced by lenalidomide may also be due to an inhibition of

COX-2 and PGE 2. Lenalidomide inhibits NF-K B activity in MM cell lines, which is

consistent with reports that thalidomide inhibits DNA binding activity of the p50–p65

NF-KB heterodimer triggered by TNF-α and IL-1β in the Jurkat cell line (Keifer, J. A.,

2001) and peripheral mononuclear blood cells (PBMCs) (Rowland, T. L., 2001). As

NF-KB plays an important role in cell-cycle regulation, cell survival, anti-apoptosis

and cytokine production in MM (Hideshima, T., 2001), the inhibition of NF-KB

activation by lenalidomide may also enhance or restore sensitivity to other

chemotherapeutic agents. Specifically, it was demonstrated that MM cell adhesion-

mediated upregulation of IL-6 is mediated via NF-KB activation. Recently, Stewart

et.al in 2004 reported pharmacogenomic studies suggesting that hyper activation of

the Wnt signaling antagonist DKK-1 is related with response to the

immunomodulators thalidomide and lenalidomide. Furthermore, β-catenin expression

is downregulated by lenalidomide in MM cell lines.

Dexamethasone combination treatment with lenalidomide induces at least

additive cytotoxicity in MM cells 22, this effect is associated with the activation of

dual apoptotic signalling cascades as dexamethasone induces caspase-9 (Chauhan, D.,

2001), and lenalidomide triggers caspase-8 activation. Recently, enhanced anti-MM

activity of rapamycin, a specific mammalian target of rapamycin (mTOR) inhibitor,

in combination with lenalidomide has been reported (Raje, N., 2004). In this study,

the combination of rapamycin plus lenalidomide overcomes drug resistance in MM


cell lines that are resistant to conventional chemotherapy. Interestingly, these drugs

target differential signalling cascades, including the ERK and PI3K/Akt pathways,

individually and in combination, suggesting the molecular mechanism by which they

interfere with MM growth and survival.

5.6.3 Immunomodulatory activities

A unique feature of the antitumour effect of thalidomide and lenalidomide is

their ability to modulate and potentiate host immune responses against MM. Several

studies have demonstrated the effects of lenalidomide on peripheral blood

lymphocytes. Co-culture of naive splenocytes with anti-CD3 monoclonal antibody

and IMiD1 (Actimid directly costimulates T cells and increases Thelpertype 1(TH1)-

type cytokines. Most excitingly, IMiDs augment cytocell lines and autologous MM

cells, which is associated with increased serum levels of IL-2. Although thalidomide

/IMiDs induce IL-2 secretion from T cells, the way in which these compounds induce

IL-2 production from T cells has not been fully defined. Importantly, the authors’

recent studies demonstrated that lenalidomide significantly costimulates a

proliferation of CD3+ T cells induced by CD3 ligation, immature dendritic cells or

mature DCs (SI: 2.6). T-cell proliferation triggered by DCs is abrogated by CTL

antigen 4-immunoglobulin (CTLA-4-Ig). Lenalidomide also overcomes the inhibitory

effects of CTLA-4-Ig on Epstein-Barr virus and influenza-spe-cific CD4 and CD8 T-

cell responses that are measured by cytokine capture and enzyme-linked

immunosorbent spot (ELISPOT) assays. Importantly, lenalidomide triggers tyrosine

phosphorylation of CD28 on T cells followed by activation of NF-KB. Furthermore,

the authors demonstrated that IMiDs facilitate the nuclear translocation of nuclear

factor of activated Tcells (NF-AT)-2 and activator protein-1 via an activation of

PI3K/Akt signalling, with resultant IL-2 secretion. IMiDs enhance both NK cell

cytotoxicity and antibody-dependent cell-mediated cytotoxicity induced by

triggering IL-2 production from T cells (Hayashi, T., 2005). Therefore, these studies

define the molecular mechanisms by which lenalidomide triggers NK cell-mediated

cytotoxicity against MM cells, further supporting their therapeutic use in MM.


5.6.4 Adverse effects

The general side effects of lenalidomide treatment found in Phase II clinical

trials of relapsed refractory MM are summarised in, the most common toxicities

related with thalidomide (e.g., constipation, neuropathy and tremors) were not

observed. Toxicities associated with lenalidomide were largely haematological and

reversible. The most common grade 3 or higher adverse actions during lenalidomide

therapy were neutropoenia and thrombocytopoenia. Grade 4 neutropoenia occurred in

2 of 34 (5.8%) patients treated with lenalidomide 15mg b.i.d. versus 4 of 68 (5.9%)

patients treated with lenalidomide 30mg/day. Grade 4 thrombocytopoenia occurred in

2 (5.8%) of 34 patients with lenalidomide 15mg b.i.d. versus 2 of 68 patients (2.9%)

treated with lenalidomide 30 mg/day. DVT was reported in 1 patient on lenalidomide

30mg/day and 2 patients (5.8%) treated with lenalidomide 15mg b.i.d. Sedative or

neuro- logical toxicities were not observed in most of these studies (Richardson, P.

G., 2003). The differences in the side-effect profile between thalidomide and

lenalidomide may reflect distinct patterns of antiangiogenic, cytokine-related,

microenvironmental and immunomodulatory activity, relatively than distinct separate

mechanisms of action. Further study may finally lead to the development of safer and

additional effective analogues for utilisation in MM therapy.


Name Thalidomide Lenalidomide

Empirical

Formula C13H10N2O4 C13H13N3O3

Molecular

weight 258.2 259.3

Chemical

structural

Thalidomide has 2

oxo Groups in

phthaloyl ring

Lenalidomide has amino

group at 4 th location and

single oxo group in

phthaloyl ring

Effects on T-

cell propagation

Thalidomide

reproduce T cell

propagation and

increases IFN-γ and

IL-2 production

Lenalidomide is 100–1000

times more potent in

stimulating T cell

proliferation and IFN-γ and

IL-2 production than

thalidomide

Adverse

effects

Thalidomide has

higher incidence of

side effects like

sedation, neuropathy

and constipation.

Lenalidomide has lower

occurrence of adverse

effects namely sedation,

constipation and

neuropathy than

thalidomide.

Teratogenecity Thalidomide is a

known teratogen.

Lenalidomide is not

teratogenic in rabbit

models

Table 1.1: Differences between thalidomide and lenalidomide


5. Enantioseparaion techniques:

Fig 1.3: Techniques used for the separation of enantiomers

The most important techniques for the separation of enantiomers are shown in

fig 1.3. Enantiomer separation methods, with an importance on separation by chiral

inclusion complexes and crystallization, biological methods, preparative liquid and

gas chromatographic methods have been reported. It is not surprising that rigorous

efforts have production of enantiomerically pure drugs. Though, the usual method of

separating the optical isomers of racemic compounds has always been complicated

and costly. Chiral separation can be used to simultaneously produce in the same way

enantiomers or it can be used in ways that generate only one enantiomer. In the mode

of single-isomer recovery, the chiral technology also selects one of the isomers, but in

Sensors

Liquid-liquid Extraction

Capillary Electrophoresis

Chromatography

Crystallization

Membranes

Asymmetric

Biotransformation

Enantioseparation

Technique


addition it deliberately racemizes the other isomer, and recycles it into the selection

process, thus ultimately producing the one wanted isomer. Alternatively, the

conventional method of separating the optical isomers of racemic compounds

involves the preparation of diastereomeric intermediates, which can then be separated

from each other by differential crystallization (Lorenz, H., 2007), hydrolysis and

purification. The resolution of N-methylamphetamine was achieved with the

resolution agents O, O′-dibenzoyl- (2R, 3R)-tartaric acid monohydrate (DBTA) and

O, O′-di-p-toluoyl- (2R, 3R)-tartaric acid (DPTTA). After incomplete diastereomeric

salt formation, the unreacted enantiomers were extracted by supercritical fluid

extraction (Kmecz, I., 2004). Inclusion complexation of a racemic compound with a

chiral congregation compounds which gives chiral host-chiral guest inclusion

compounds, from which the chiral guest can be obtained. When this separation is

combined with distillation technique, for example, enantiomer separation can be

accomplished by partial distillation in the presence of a chiral host compound. This

represents a current and green practice of enantiomer separation (Fumio, T., 2004).

Successful techniques such as chiral diastereomeric, enzyme, and chromatography

resolution are now accompanimented by a new technique, which meets the

challenging criteria of being easy to screen, rapid, and with little capital cost.

Membranes extraction technology for enantiomer separation utilizes enantioselective

inclusion complexation via a chirally selective host molecule coupled to organic

solvent nanofiltration for host separation. Then it is recycled, to afford competent

practical way to resolve diastereomeric mixtures resulting in separation, and

resolution, of greater than 95 % of racemic mixtures. So, it is a fast and capable

method for enantiomer separation. Chromatography is the most renowned way of

enantiomers separation. A potential preparative scale separation technique, which has

fascinated awareness, recently is to resolve such substances by chromatographic

techniques, using chiral stationary phases, which work basically by a lock-and-key

mechanism. Industrial scale separations have been successfully accomplished using

simulated moving bed (SMB) procedure. If cross-linked, immobilized enzymes are

useful organic reagents, then so are enzymes ligated to a solid support and packed

into chromatographic columns. Not only proteins, but also all biologically derived


materials, because of their production from enantiomerically pure building blocks

such as amino acids, and sugars, are appropriate as stationary phases in chiral

chromatography. The stationary phases discover applications in gas chromatography

(GC), high performance liquid chromatography (HPLC), and supercritical fluid

chromatography (SFC). A number of chiral stationary phases for the separation of

enantiomers in drugs and biological compounds have previously been developed, and

their employ is presently widespread (Inagaki, S., 2008). Recent trends in

enantioseparation of chiral drugs have been reported (Chankvetadze, B., 2004).

Separations of molecules with multiple chiral centers are more difficult since chiral

stationary phases are normally good at separating enantiomers but not at separations

of diastereoisomers. Separations of molecules with multiple centers can, however, are

achieved by careful choice of operating conditions.

6.1 Enantioselective HPLC analysis

There are mainly two options for chiral HPLC analysis to be exact direct and

indirect approach (Write, D. T., 1992). The direct chiral high performance liquid

chromatographic technique, with reference to application in enantiospecific drug

analysis was reported (Valliappan, K., 2005). In the indirect approach, drug

enantiomers are derivatized with an enantiopure chiral reagent to form a pair of

diastereomers, which may be then separated on a conventional chromatographic

column, since diastereomers exhibit different physicochemical properties. In the

direct method, transient relatively than covalent diastereomeric complexes are formed

between the drug enantiomers and a chiral selector present either added to the mobile

phase or coated/bonded to the surface of a silica support. The technique relying on

chiral stationary phases are preferred as they present specific advantages over indirect

methods. There is no need to chemically manipulate the analytes, interference with

sample matrix, chiral purity of the chiral stationary phase does not require be known,

rapid analysis, method can be readily scaled to commercial production, online

coupling with MS or NMR permits structure discovery.

High-performance liquid chromatography is a powerful apparatus for the

enantioselective separation of chiral drugs (Mehta, A. C., 2008). However, the

selection of an appropriate chiral stationary phase and suitable operating conditions is


a bottleneck in method development and a time- and resource-consuming task.

Multimodal screening of a small number of CSPs with extensive enantiorecognition

abilities has been recognized as the most excellent approach to achieve rapid and

reliable separations of chiral compounds (Huybrechts, T., 2007). Supercritical fluid

chromatography coupled to a hybrid mass spectrometer (Q-Tof) equipped with

electrospray ion source has been used to separate and characterise a wide range of

pharmaceutical racemates. Supercritical fluid chromatography is best known for

chiral separation and, in general, is a greater chromatographic technique for chiral

separation than normal phase liqiuid chromatography. SFC-MS does not have the

troubles one would encounter in normal phase liquid chromatography (Garzotti, M.,

2002).

6.2 Capillary electrophoresis and enantiomer separation

Capillary Electrophoresis has become major analytical tool for separation of

charged analytes due to the enhanced separation efficiency. In addition, the CE

method when used in combination with a micellar pseudostationary phase offered a

number of advantages for separation of neutral analytes, called micellar electrokinetic

chromatography (MEKC). Scientists have recently employed chiral polymeric

surfactants for superior enantiomeric separations of racemic mixtures using MEKC-

Fluorescence anisotropy (McCarroll, M. E., 2001). The use of Fluorescence

anisotropy to probe chiral recognition has been reported (Warner, I. M., 2003).

Enantiomer separation of chiral pharmaceuticals by CEC has been reported with

open-tubular capillaries (o-CEC) [coated with a thin film containing cyclodextrin

derivatives, cellulose, proteins, poly-terguride or molecularly imprinted polymers as

chiral selectors], with packed capillaries (p-CEC) [typical chiral HPLC stationary

phases such as silica-bonded cyclodextrin or cellulose derivatives, proteins,

glycoproteins, macrocyclic polyacrylamides and molecularly imprinted polymers are

used as chiral selectors] or with monolithic capillaries prepared by in situ

polymerization into the capillary (Wistuba, D., 2000). Enantioresolution of basic

compounds with human serum albumin by means of affinity EKC (AEKC)-partial

filling technique depends on the hydrophobicity, polarity, and molar volume of

compounds (Martínez-Gómez, M. A., 2004). The application of CE for


enantioseparation of enantiomers of chiral drugs has paying concentration amplified

in the last decade (Nele, M., 2004). Capillary electrophoretic method has been

developed for the enantioselective analysis of amisulpride in pharmaceutical

formulations, using beta-cyclodextrin sulfate as the chiral selector (Landers, J. P.,

2007). Modified microemulsion as a CE technique has been applied to the chiral

separation of atropine, scopolamine, ipratropium and homatropine (Musenga, A.,

2008). A comparison between chiral cyclodextrin-modified microemulsion

electrokinetic chromatography (CDMEEKC) and cyclodextrin-modified micellar

electrokinetic chromatography (CD-MEKC) for the enantiomeric separation of

esbiothrin was reported (Bitar.Y., 2007). Excellent enantioseparation of profens has

been achieved using macrocyclic glycopeptide antibiotic, eremomycin, as a chiral

selector in CE (Chu, B. L., 2008). Enantiomeric separation of primaquine, an

antimalarial drug, was achieved by cyclodextrin-modified micellar electrokinetic

capillary chromatography (Prokhorova, A. F., 2009). A chiral microemulsion

electrokinetic chromatography method has been developed for the enantiomeric

separation of 3,4-dihydroxyphenylalanine, its precursors phenylalanine and tyrosine,

and the structurally allied substance methyldopa (Zhang, C., 2002). The achievements

of enantioseparation of adrenergic drugs and application of liquid chromatography

and capillary electrophoresis methods in clinical and pharmaceutical analysis were

reported (Zheng, J., 206). The feasibility of using a new and more versatile polymeric

chiral surfactant, i.e., poly (sodium N-undecenoxy carbonyl-L-leucinate was

investigated for simultaneous enantioseparation and detection of eight structurally

similar β-blockers with tandem UV and MS detection and the CMEKC−ESI-MS

method was more suitable as a routine procedure for highthroughput separation of β-

blockers with high sensitivity (Dung, P. T., 2008).

6.3 Simulated moving bed (SMB) for chiral separation of drugs

In chromatography, the simulated moving bed technique is a alternative of

high performance liquid chromatography. Although chiral HPLC is being a standard

technique, but it is a non-continuous process and has high solvent consumption and

small productivity. To overcome this problem, SMB technology has been developd to

pharmaceuticals chemicals, particularly to enantiomer separation. S-bupivacanine


with the pharmacological activity of epidural anaesthesia was isolated and separated

from R-bupivacanine by SMB chromatography (Miriam, Z., 2009). The new CSP

was found to be efficient for the enantioseparation of chiral drugs (Han, S. K.,).

Simulated counter-current moving bed systems have been applied to

enantioresolution trials on a series of racemic nonsteroidal antiflammatory drugs

(NSADs), anti asthmatics and β-blocker drugs (Yu, H. W., 2003). Concurrently,

study on integrated processes involving both adsorption and crystallisation is in

progress to resolve more difficult drugs. The enantiomeric resolution of the racemic

mixture of α-ethyl-2-oxo-1-pyrrolidineacetamide was carried out by simulated SMB,

using at least three columns filled with chiral stationary phase (Emile, C., 2000).

6.4 Nano-chiral technology and enantioseparation

Chiral, nanoscale science and technology was reviewed relating to

nanotechnology in the service of asymmetric synthesis, chiral separations, and

analysis (Zhang, J., 2005). Nano-chiral technology describes the nanoscale

approaches to chiral technology such as chiral separation and detection and

enantiomeric analysis (Bag, D. S., 2008; Sancho, R., 2009). Scientists have carried

out chiral separations using an antibodybased nanotube membrane. These membranes

are based on alumina films that have cylindrical pores with monodisperse nanoscopic

diameters of size 20 nanometers. Further, Silica nanotubes were chemically

synthesized within the pores of these films, and an antibody that selectively binds one

of the enantiomers of the drug was attached to the inner walls of the silica nanotubes.

These membranes selectively transported the enantiomer that specifically binds to the

antibody, relative to the enantiomer that has lower affinity for the antibody (Lee, S.

B., 2002). New LC separation techniques and applications contain a chiral separation

based on single walled carbon nanotubes conjugated with bovine serum albumin

(Ward, T. J., 2008). Scientists studied the potentiality of nano-liquid chromatography

(nano-LC) for the enantiomeric resolution of both basic and acidic compounds of

pharmaceutical interest using a vancomycin-modified silica stationary phase

(D’Orazio, G., 2005).


6.5 Kinetic resolution-catalyzed by enzymes

Enzymes have competed well with chemical methods for resolution. In kinetic

resolution-catalyzed by lipases, only one enantiomer of a chiral reactant fit to the

active site properly and is able to undergo the reaction while the second enantiomer is

left and in enantiomerically pure form (Ghanem, A., 2008). Dynamic kinetic

resolution is a powerful tool to transform a racemic mixture into one enantiomer. This

approach overcomes the restriction of the maximum 50% yield in a kinetic resolution

by combining it with an in situ racemization of the substrate. Recently, the coupling

of enzymes and transition metals for dynamic kinetic resolution of a variety of

molecules has attracted considerable attention and a deeper understanding of the

compatibility of these two catalysts has been achieved (Martín-Matute, B., 2007). An

enzymic membrane reactor consisted of a lipase immobilized polymeric membrane an

organic phase dissolving ester and an aqueous phase was reported for the optical

resolution of racemic ibuprofen ester (Long, W. S., 2005).

6.6 Gas chromatographic–mass spectrometric method and enantioseparation

Separation of the enantiomers of ibuprofen was achived by a gas

chromatographic–mass spectrometric method using selected ion ionization and

tandem mass spectrometry on a chiral capillary column.

7. Classification of chiral Stationary phases

Chiral stationary phases can be classified according to their interaction

mechanism with a solute in a classification system first proposed by Wainer (Wainer,

I. W., 1987) in 1987, Type I CSPs differentiate enantiomers by formation of

complexes based on attractive interactions, e.g. hydrogen bonds, π-π interactions or

dipole stacking. Type II CSPs use a combination of attractive interactions and

inclusion complexes to produce a separation. Most type II CSPs is based on cellulose

derivatives. Type III CSPs rely on solute entering chiral cavities to form inclusion

complexes. The most common is the CD-type of column. Type IV CSPs separate by

means of diastereomeric metal complexes. This is also called chiral ligand exchange

chromatography. Type V CSPs are proteins, and separation depends on a mixture of

hydrophobic and polar interactions


7.1 Dinitrobenzoyl phases (Pirkle CSP)

Known also as donor-acceptor or brush columns, Pirkle columns (Mehta, A.

C., 1998) are able to resolve enantiomers based on enhanced binding of one

enantiomer to the CSP. This forms a diastereomeric complex through a combination

of π-π bonding, hydrogen bonding, steric interactions and / or dipole stacking. For

enantiomers to be separated on brush-type CSPs, the analytes must exhibit the

necessary three interaction sites. Major interaction sites are classified as π-π

interaction, supply hydrogen for hydrogen bonding, such as π-electrons, hydroxyl or

ether oxygens or amino groups, steric interaction sites, or sites for electrostatic

interaction. Usually, brush-type CSPs utilizes π-π interaction in the recognition

development.

One conscientious Pirkle CSP (fig.1.4) is the CSP based upon 3,5-

dinitrobenzoylphenylglycine. This type of CSP, termed a π-electron acceptor,

commonly is effective for resolving aromatic enantiomers that are considered good π-

electron donors participating in π-π interactions. The DNBPG phase also contains two

acidic hydrogen and two basic carbonyl groups that can hydrogen bond with materials

such as amines, amides, or hydroxyls a second type of Pirkle column contains π-

electron donating species in the

Fig 1.4: Pirkle-type 3,5-dinitrobenzoylphenylglycine (DNBPG) CSP

CSP. The π-electron donor CSPs were rationally designed to separate amines,

amino acids, amino alcohols, carboxylic thiols and acids, especially 3,5-dinitrophenyl

urea and carbamate derivatives of amines and alcohols.


A third type of Pirkle column is the hybrid π-electron acceptor-donor CSP. A

important example is the Whelk-O 1 columns. The Whelk-O 1 column incorporates a

π- acid and π-base, and is competent of resolving enantiomers that contain either π-

acid or π-base groups. The Whelk-O 1 column can be used in both reverse and

normal-phase modes. However, enantio stability and selectivity of this phase are

generally much superior under normal conditions and not under reversed-phase

conditions where HPLC drug analysis is usually performed. And even with the

present selection of brush-type CSPs, it is not viable to separate all enantiomers. The

performance of the Whelk-O 1 CSP may even be lower as compared to that of other

polysaccharide-based columns (Magora, A., 2000).

7.2 Protein bonded phases

Composed of L-amino acids as chiral subunits, proteins are chiral polymers

that can undergo stereoselective interactions with a huge number of

pharmacologically vigorous compounds. Hence, protein-based CSPs show to have a

broad application in the meadow of drug bioanalysis. This property is especially

pronounced for the serum proteins α1-acid glycoprotein (AGP), human serum

albumin (HSA) and ovomucoid (OV) important to the commercial development of

three chiral stationary phases for HPLC (Guebitz, G., 1990). Using a HSA stationary

phase, the preparative separation of benzodiazepinones has been achieved (Felix, G.,

1995). The direct separation of some neutral, base and acidic compounds was

possible on an OV CSP (Iredale, J., 1991). Even though protein bonded phases have a

high selectivity, they have a low capacity, as the load of protein on the column is low.

Therefore, care should be taken not to overload the column especially when the

optimal solution is required.

7.3 Chiral phases with inclusion effects

7.3.1 Cyclodextrin bonded phases

Cyclodextrin (Lee, C. K., 1999) is a natural macrocyclic polymer of glucose

that include from six to twelve D- (+)-glucopyranose units, which are bound via α-

(1,4)-linkages. They are chiral, toroidal-shaped molecules with all the glucose units in

a C-1 (D) chair conformation. The structure of β-cyclodextrin (β-CD, with seven

glucose units) is shown in fig.1.5.


Fig 1.5: Structure of β-CD

It contains the secondary hydroxyl groups on the C-2 and C-3 of the glucose

units. The primary hydroxyl groups, attached to C-6 of the glucose unit, are on the

opposite end of the CD, forming a smaller opening. Thus, the CD molecule is shaped

like a truncated cone with the secondary hydroxyl side more open than the primary

hydroxyl side. While the primary hydroxyl groups on the truncated end can rotate to

partially block the cavity, the secondary hydroxyl groups are held relatively rigid. The

interior of the cavity consists of two rings of C-H groups with a ring of glucosidic

oxygens between. Therefore, the interior is relatively hydrophobic in comparison with

polar solvents such as water, while the mouth of the CD cavity is hydrophilic (Harata,

K., 1998; Connors, K. A., 1997).

A greek letter is used to indicate the number of glucose units per CD. For

example, α is for cyclohexaamylose, β for seven cycloheptaamylose, γ for eight

cyclooctaamylose and so on. During reversed-phase condition, inclusion complex

formation, in which the hydrophobic part of the analyte enters the CD cavity, is


largely responsible for selectivity and retention. Under normal phase condition

(Armstrong, D. W., 1990), the hydrophobic mobile phase occupies the CD cavity and

analyte interactions are primarily with the hydroxyl groups at the mouth and base of

the CD.

Of the three commercially available native CD bonded stationary phases, β-

CD has been the largely flourishing in chiral separations. β-CD bonded stationary

phases usually need a structure with at least two aromatic rings. Structures with

naphthyl or biphenyl moieties generally afford the tight fit to β-CD cavity. The large

γ-CD bonded stationary phase has been shown to separate successfully, in reversed-

phase mode, enantiomers containing fused rings such as pyrenes or multiple fused

ring moieties.

7.3.2 Crown-ether phases

Macrocyclic polyethers are known as crown ethers. They have the capacity to

form strong complexes with metal cations and substituted ammonium ions. Such a

chiral host is able to discriminate between enantiomeric ammonium compounds, such

as D, L-amino acids, and many compounds bearing a primary amino group near the

chiral center. For instance, the enantiomeric resolution of a number of

phenylalkylamines, namely racemic cathinone, amphetamine, norephedrine, and

norphenylephrine were achieved on an S-18-crown-6-ether chiral stationary phase

(Aboul-Enein, H. Y., 1997). Due to multiple hydrogen bonds shaped between the

ammonium group and the ether oxygen, for steric reasons a less stable complex with

one of the enantiomers will be formed. This phase should thus be operated with an

aqueous acidic mobile phase to form the ammonium ion.

7.3.3 Chiral ligand exchange phases (CLEC)

First reported by Davankov and Rogozhin (Davankov, V.A., 1971) in 1971,

using resin packings in which amino acid residues were bonded, CLEC involves the

reversible arrangement of metal complexes by coordination of substrates that can

work as ligands to the metal ion. In ligand exchange phases, an amino acid such as L-

proline is bonded to silica gel and the resulting phase is treated with copper sulphate

solution. The separation is based on the formation of an enantioselective ternary

complex between amino acid, copper ion and the analyte on the stationary phase. The


differences in stabilities between complexes with D- and L- forms of the analyte

conduct to the separation of the enantiomers. The stability of such complexes is

extremely reliant on the transition metals used. pH, ionic strength of the mobile phase

and temperature are factors that influence the selectivity of the separations. The

mixed ternary complexes result from numerous equilibria existing in the

chromatographic columns, and these equilibria depend on the conditions of the

separations such as pH and temperature (Kuganov, A., 2001; Aboul-Enein, H. Y.,

2003). For the separation to be successful the example molecule must be a bidentate

ligand for Cu2+ or other metal ions. The drawback of this technique is the complexity

of the aqueous mobile phase, which contains buffers and essential copper ions.

Advantages include good stability and high selectivity values.

7.4 Polysaccharides as the successful and competent chiral selectors:

Polysaccharide based chiral selectors are the paramount in the chiral

separation science due to their extraordinary recognition capabilities (Zhang, T.,

2005), which resulted in the chiral separations of many compounds (Okamoto, Y.,

1989). First of all, in 1951, Kotake et al. used cellulose paper for enantiomeric

resolution of amino acids and since then polysaccharides have been recognized as

potential chiral selectors. The main polysaccharides are cellulose, amylose, chitosan,

xylan, curdlan, dextran and inulin (Yashima, E., 1997) but these could not be used as

commercial CSPs because of their lowresolution capacities and handling problems

(Okamoto, Y., 1997). That is why the derivatives of these polymers were synthesized.

Among these, cellulose and amylose approved to be the best polymers because of

their good abundance and good capabilities for chiral resolution. Both

polysaccharides contain glucose units and polymeric chains of D- (+) glucose units

are joined through b-1, 4 and a-1, 4 linkages in cellulose and amylose, respectively.

The degree of polymerization of cellulose is in the ranges from 200-14,000 units of

glucose. Similarly, more than 1000 glucose units are found in amylose. Each glucose

unit has chair conformation with 2- OH, 3-OH and 5-CH2OH groups all in equatorial

position as shown in fig. 1.6a. The chains of these units lie side-by-side in a linear

fashion in case of cellulose and helical in amylose and, hence, amylose provides more

chiral grooves for enantiomeric resolution. Therefore, amylose is improved chiral


selector in comparison to cellulose (Ronden, N. G., 1993). The three dimensional

structures of amylose and cellulose are shown in fig. 1.6b indicating the helical and

more distinct grooves in amylose than cellulose. As discussed above native amylose

and cellulose are not good chiral selectors, and, hence different workers synthesized

their derivatives. Most successful and applicable derivatives of cellulose and amylose

are tri-esters and tri-carbamate (Shibata, T., 1989). Okamoto et al. in 1984 prepared

tri-esters and tri-carbamates of cellulose and amylose and tested them for chiral

resolution. Later on, other derivatives of cellulose and amylose were also synthesized

and used for the enantiomeric resolution (Kubota, T., 2003).

Fig 1.6: Chemical structures of (a): cellulose and (b): amylose

polymers


7.4.1 Status of commercial polysaccharide CSPs in the market

Presently, amylose and cellulose derivatives in use are tris-benzoate, tris- (4-

methyl benzoate, trisphenylcarbamate, tris- (3,5-dimethylphenylcarbamate, tris-(R)-1-

phenylethylcarbamate, tris- (S)-1-methylphenylcarbamate etc. They have been

commercialized by special names such as Chiralcel and Chiralpak for cellulose and

amylose, respectively, by Daicel Chemical Industries, Tokyo, Japan. Recently, some

other industries such as Kromasil, Macherey Nagel, Knauer and Sepaserve have also

introduced some chiral columns. The chiral columns commercialized by these

companies are in normal and reversed phases separately and respectively. The trade

names of chiral columns of Kromasil are Amy Coat and Cellu Coat in normal phase

and Amy Coat RP and Cellu Coat RP in reversed phase respectively. The commercial

CSPs supplied by Macherey Nagel are Nucleocel-Alpha, Nucleocel-Alpha-S,

Nucleocel-Alpha-RP-S, Nucleocel-Delta, Nucleocel- Delta-S, Nucleocel-Delta-RP

and Nucleocel-Delta-RP-S having tris-3, 5- (dimethylphenyl)-carbamate derivatives

of amylose in alpha and cellulose in delta configurations respectively. The RP

represents reversed phase while S denotes small particle size (5 mm). On the other

hand Knauer introduced Europak 01 amylose-tris- (3,5-dimethylphenylcarbamate)

and Eurocel 01 cellulose-tris- (3,5-dimethylphenylcarbamate chiral pak columns

respectively, which can be used in both normal and reversed phases respectively.

7.4.2 Application

As discussed above polysaccharide CSPs have gained a excellent reputation in

the chiral world. They are very efficient and competent to work under normal,

reversed and prohibited organic mobile phase modes, and that is why they have

extensive range of applications. These CSPs are offered as coated which can be used

in normal and reversed phase modes but not on the same column. But, recently,

immobilized CSPs have been developing, which are capable to work under banned

organic mobile phase modes enhancing the range of enantiomeric recognition

capabilities. Polysaccharides have been used very often in HPLC as CSPs along with

few reports as mobile phase additives. However, the research papers are accessible on

these chiral selectors in capillary-electrochromatography, sub- and supercritical fluid

chromatography and thin layer chromatography. Briefly, the applications of these


phases for enantiomeric resolution of pharmaceuticals and drugs are discussed in the

following sections.

8. Preparative separations:

Jansen et al. (Jansen, J. M., 1994) reported semi-preparative enantiomeric

separation of a series of reputed melatonin receptor agents using triacetylcellulose as

chiral stationary phase and observed that first eluting enantiomer was around 99%

pure. Magri et al. in 2005 reported semipreparative chiral separations of some novel

atropisomeric quaternary and ternary 1,2-disubstituted 1,4,5,6-

tetrahydropyrimidinium salts. The authors compared the experimental data in order to

establish the factors influencing the extent of the barriers and with those

corresponding to the parent amidines. The chiral columns used OD-R, OJ-R, and AD-

RH with mobile phases of acetonitrile and water in different combinations. De

Veredas et al. in 2006 reported a simulated moving bed chromatographic chiral

separation of a baclofen precursor by using polysaccharide carbamate as chiral

stationary phase (cellulose tris- (3,5-dimethylphenylcarbamate) at semi-preparative

scale. The method was competent of providing high purity enantiomers. (Collina, S.,

2006) was reported semipreparative scale resolution of N, N-dimethyl-3- (naphthalen-

2-yl)- butan-1-amines on Chiralcel OD column.

In addition to the coated CSPs, immobilized chiral phases have been used for

semi-preparative separations in liquid chromatography (Cirilli, R., 2006) described

semi-preparative chiral separations of mianserin and a series of aptazepine derivatives

on immobilized polysaccharide based chiral stationary phases (Chiralpak IA). The

non-conventional dichloromethane based eluents have expanded the chiral resolving

capability of the immobilized Chiralpak IA to perform mg-scale enantioseparations

with an analytical size column. The authors assigned complete configuration of the

separated enantiomers by comparing their chiroptical data with those of structurally

related mian serin. Furthermore, the same (Cirilli, R., 2006) reported semi-preparative

chiral resolution of 3,4- dihydropyrimidin- 4(3H)-one derivatives on Chiralpak IA

column. The non-standard solvents such as ethyl acetate, methyl tertbutyl ether, or

dichloromethane were used. The authors reported mg-scale separations and used

further for chiroptical properties.


9. Plasma protein binding

In drug discovery, drug-protein binding data (fraction unbound drug, fu) can

be used (Di, L., 2003) to enhanced understand in vivo pharmacokinetic (PK) profile

such as volume of distribution, half-life and clearance (Sebille, B. J., 1990), to design

best possible dose regimes and estimate safety margins (Lindup, W. E., 1987), to

interpret pharmacodynamic (PD) data, as it is generally accepted that only unbound

drug (free concentration) is pharmacologically active or accountable for a preferred in

vivo efficacy. It was estimated that about 40% development compounds fail to reach

market due to poor pharmaceutical properties. This drives pharmaceutical companies

to profile drug-like properties as early as possible in order to increase the success rate

of compounds to the market. Therefore, the capability to screening of drug-protein

binding becomes an important concern in drug discovery, even in early ADME in

modern drug design. So far, equilibrium dialysis is the preferred and most widely

used technique for protein binding measurement in most of pharmaceutical

communities, because it offers accurate binding data due to the fact that the drug

binding to plasma proteins is analyzed at equilibrium. However, such a traditional

equilibrium dialysis method, as depicted in fig. 1.7, is labor-intensive and time-

consuming with limited sample throughput capacity, as well as relatively large

plasma sample spending, which considerably limits screening a large number of

compounds. Thus, development of different new methods and technologies is highly

advantageous to increase throughput and reduce costs. Development of high

throughput assays or novel pharmaceutical approaches while limiting the increasing

costs enables the advance of improved in vitro models to accelerate drug designs. In

this review, we will summarize recent developments with emphasis on high

throughput technologies and new approaches applicable for drug-protein binding

screening as outlined in fig. 1.7.


Fig 1.7: Traditional low throughput equilibrium dialysis versus high

throughput screening technologies.

• ED: equilibrium dialysis;

• LCMS: liquid chromatography mass spectrometry, immobilized

• HSA column: immobilized human serum albumin;

• ACE: affinity capillary electrophoresis;

• FA: frontal analysis. Comparative Analysis of HSA and AGP Binding

HSA and AGP are abundant proteins in plasma, which primarily governs the

whole plasma protein binding. It has been generally supposed and confirmed that

acidic drugs bind strongly to HSA, while basic and neutral drugs bind more to AGP

(Jia, Z. J., 2002; Israili, Z. H., 2001) as shown in Table 1.2, the binding affinity

constants of basic compounds to HSA are greater than AGP.


Compound Lidocaine Imipramine Propranolol Chlorpromazine

PKa 7.79 9.52 9.55 9.12

LogKaHSA (M-1) 2.85 3.26 3.34 4.05

fu(HSA)% 71.0 49.0 44.3 13.5

LogKaAGP (M-1) 4.50 4.74 5.67 6.70

fu(AGP)% 65.4 54.1 15.6 1.9

fu(HSA�AGP)

% 51.6 34.6 13.0 1.7

fu(plasma)% 50.0 18.0 21.0 3.0

Table 1.2: Comparative binding affinity and fraction unbound of HSA and AGP

Data from pharmacokinetic database: Goodman & Gilman 1996.

9.1 High throughput assays for drug-protein binding screening

Equilibrium dialysis with standard 96-well plate and LC-MS/MS in recent

times, throughput of the traditional equilibrium dialysis method has been improved by

implementation of a 96-well format. Kariv et al. presented a 96-well equilibrium

dialysis device for plasma protein binding measurement and validated three drugs,

propranolol, paroxetine, and losartan, with low, intermediate, and high binding

properties, respectively (Kariv, I., 2001; Kariv, I., 2002)). They concluded that high-

throughput 96-well equilibrium dialysis device showed good correlation to the

traditional method and it is compatible with the HTS format for automated liquid

handling and bioanalytical mass spectrometry. Banker et al. also presented a novel

96-well format dialysis apparatus for measuring plasma protein binding (Banker, M.

J., 2003) which is a vertical design having advantages over current 96-well dialysis

on the market in terms of surface-to-volume ratio and reduction of non-specific


binding (NSB). The device, made of Teflon material, is not only easy to get to by a

robotic system for easy automation, but is also reusable. In addition, a semi-

automatic, high-throughput, 96-well plate ultrafiltration has also been employed to

rapidly evaluate plasma protein binding of new chemical entities (Fung, E. N., 2003).

It is well known that ultrafiltration is not suitable for measuring highly bound

compounds due to the NSB effect. However, this drawback seems to be overcome by

a newly modified ultrafiltration methodology, i.e., mixing of control plasma retentate

with the filtrate, thus eradicating the NSB effect (Taylor, S., 2006). Currently, several

96-well equilibrium dialyzers are also commercially available from Linden

Bioscience (rapid equilibrium dialysis device, Ricerca). Obviously, the application of

96-well format dialyzer get better throughput as compared to the conventional single

chamber based devices. However, the assay is usually based on single compound

measurements, hence they are not amenable for screening large sets of compounds. It

should be addressed that, for each compound, a number of samples from both buffer

and plasma sides has to be analyzed by LCMS, which essentially limits throughput of

the whole assay.


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