Monoclonal antibodies as drug targeting particulate carrier system

Drug Targeting

Monoclonal Antibodies Drug Targeting Particulate Carrier System PRESENTED BY GUIDED BY MS. KRUTIKA H. PARDESHI MR. S.B. KHATALE M.Pharm (Pharmaceutics) (Assistant professor) Sem -II, Roll no. 37

NDMVP SAMAJS COLLEGE OF PHARMACY, NASHIK

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Site specific drug delivery system - Introduction - Objectives - Drug targeting - Drug targeting strategies & Components - Carrier or markers

Monoclonal antibodies as drug targeting - Introduction - Role & Need of antibodies in immune system - Structure, types , Production - Drug conjugates - Advantages & limitations - Monoclonal antibodies Approved by FDA

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IntroductionTargeted drug delivery system is a special form of drug delivery system where the medicament is selectively targeted or delivered only to its site of action and not to the non-target organs or tissues or cells

It is a method of delivering medication to a patient in a manner that increases the concentration of the medication in some parts of the body relative to others. Targeted drug delivery seeks to concentrate the medication in the tissues of interest while reducing the relative concentration of the medication in the remaining tissues.This improves efficacy and reduce side effects.

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THE DRUG MAY BE DELIVERD:

To the capillary bed of the active sites. To the specific type of cell (or) even an intracellular region. Ex: Tumour cells but not to normal cells.

To a specific organ (or) tissues by complexion with the carrier that recognizes the target.

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ObjectiveTo achieve a desired pharmacological response at a selected sites without undesirable interaction at other sites, there by the drug have a specific action with minimum side effects & better therapeutic index. Ex- In cancer chemotherapy and enzyme replacement therapy.

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Reasons for Drug Targeting Drug instability

Low absorption

Short half-life

Large volume of distribution

Low specificity

Low therapeutic index6

Common Approaches of Targeted Drug DeliveryControlling the distribution of drug by incorporating it in a carrier systemAltering the structure of the drug at molecular levelControlling the input of the drug into bioenvironment to ensure a programmed and desirable biodistribution7

Important Properties Influencing Drug Targeting

DrugConcentration, Particulate location and Distribution,Molecular Weight, Physiochemical propertiesDrug Carrier InteractionCarrierType, Amount of Excipients, Surface Characteristics, size, DensityIn Vivo EnvironmentPH, Polarity, Ionic Strength, Surface Tension, Viscosity, Temperature, Enzyme, Electric Field

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Strategies for Drug Targeting10

Components for Drug Targeting

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Carriers or MarkersTargeted drug delivery can be achieved by using carrier system. Carrier is one of the special molecule or system essentially required for effective transportation of loaded drug up to the pre selected sites. They are engineered vectors, which retain drug inside or onto them either via encapsulation and/ or via spacer moiety and transport or deliver it into vicinity of target cell.12

Pharmaceutical carriers :

1. Polymers 2. Microcapsules 3. Microparticles 4. Lipoproteins 5. Liposomes 6. Micelles

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Monoclonal Antibodies Drug Targeting Particulate Carrier System

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INTRODUCTION

Antibodies are produced by a specialized group of cells called B-Lymphocytes.

When an foreign antigen enters the body due immune response B-Lymphocytes develops into plasma cells and liberates antibodies or immunoglobulin's of various types(Ig A, Ig D, Ig E, Ig G, Ig M).

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What are antibodies?An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target.

Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell.

Polyclonal antibodies are antibodies that are derived from different cell lines. They differ in amino acid sequence.

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Role of Antibody in Immune SystemEach Antigen has specific antigen determinants (epitopes) located on it. The antibodies have complementary determining regions (CDRs). These are mainly responsible for the antibody specificity.Each antigen has several different epitopes on it. They are recognised by many different antibodies. All these antibodies thus produced act on the same antigen. Hence these are designated as polyclonal antibodies.

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In general naturally produced antibodies are non-specific and heterogeneous in nature. Hence there are several limitations in the use of polyclonal antibodies for therapeutic and diagnostic purposes.Thus there is a need for producing monoclonal antibodies for different antigens.

Need to develop antibodies18

The structure of antibodies

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Types of monoclonal antibodies

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Monoclonal antibody production

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ImmunizationCell fusionSelection of hybridomaScreening ,Cloning and propagationCharacterization and storage

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1. ImmunizationImmunize an animal (mouse) by injecting with an appropriate antigen along with adjuvant.

Injection of antigens at multiple sites are repeated several times for increased stimulation of antibodies.

3 days prior to killing of animal a final dose is given intravenously.

Spleen is aseptically removed and disrupted by to release the cells.

By density gradient centrifugation lymphocytes are separated from rest of the cells.

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2. Cell fusionLymphocytes are mixed with myeloma cells and is exposed to PEG for a short period.

The mixture is then washed and kept in a fresh medium.

The mixture contains hybridomas, free myeloma cells, and free lymphocytes.

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3. Selection of hybridomaThe above mixture is cultured in HAT medium for 7-10 days. Due to lack of HGPRT enzyme in myeloma cells, salvage pathway is not operative. Hence free myeloma cells are dead.As the lymphocytes are short lived they also slowly dissappear.Only the hybridomas that receives HGPRT from lymphocytes are survived.Thus hybridomas are selected by using HAT mediumSuspension is diluted so that each aliquot contains one cell each. These are cultured in regular culture medium, produced desired antibody.

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4. Screening, Cloning and PropagationScreening is done for antibody specificity.Common tests like ELISA and RIA are used for this.The single hybrid cell producing the desired antibody are isolated and cloned.Usually two techniques are commonly employed for this

a) Limiting dilution method: Suspension of hybridoma cells is serially diluted so the aliquot of each dilution is having one hybrid cell. This ensures that the antibody produced is monoclonal.

b) Soft agar method: In this method the hybridoma cells are grown in soft agar. These form colonies and the colonies are monoclonal in nature.

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5.Characterization & storageBiochemical and biophysical characterization are made for desired specificity.It is important to note the monoclonal antibody is specific for which antigenMAbs must be characterized for their ability to withstand freezing and thawing.

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Drug conjugatesToxin conjugates (immunotoxins) EX: diphtheria toxin, Ricin have been conjugated to the tumor specific antibodies A-chain is cytotoxic and B-chain is non-specific. Hence B-chain is removed and the toxin is conjugated to tumor specific antibody. Thus we increase the specificity of the toxins by using MAbs as active drug targeting systems.

Drug immunoconjugates: Agents like chlorambucil, methotrexate and doxorubicin are conjugated with tumor specific antibodies. Ex: doxorubicin-BR96 immunoconjugate for Lewis antigen found on the surface of tumor cells.

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Advantages of monoclonal antibodiesThey are homogenous in nature.They are specific to a particular antigen with a particular epitope. Ex:Rituximab (Rituxan, anti-CD20) is a good example this antibody is used for the treatment of lymphoma.

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LimitationsAs they are specific to a particular antigen, they cannot distinguish molecule as a whole.Some times they cannot distinguish groups of different molecules. Ex:- presence of retro viruses as a part of mammalian chromosomes is not distinguished.

The presence of some of these viruses is detected in hybridomas. This poses a great danger since there is no guarantee for MAb produced is totally virus free.For this reason US food and drug administration insists that MAb for human use should be totally free from all pathogenic organisms including viruses.

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Monoclonal Antibodies Approved By FDA

AntibodyTargetIndicationTrastuzumabHER2Breast CancerBevacizumabVEGFLung CancerCetuximabEGFRColorectal carcinomaPanitumumabEGFRColorectal carcinoma

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Reference:

Lachman/Liebermans The Theory and Practice of Industrial Pharmacy, Editors: Roop Khar, SP Vyas, Farhan Ahmad, Gaurav Jain, Chapter 25 Targeted Drug Delivery Systems, Pg. No. 907-943, 2014.

R. Panchagnula and C. S. Dey, Monoclonal Antibodies in Drug Targeting, Journal of Clinical Pharmacy and Therapeutic, 22, Pg. No. 7 19, 1997.

Gupta Manish and Sharma Vimukta, Targeted drug delivery system: A Review, Research Journal of Chemical Sciences, Vol. 1 (2), Pg. No. 135 138, May 2011.

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R. Panchagnula and C. S. Dey, Monoclonal Antibodies in Drug Targeting, Journal of Clinical Pharmacy and Therapeutic, 22, Pg. No. 7 19, 1997.

Gupta Manish and Sharma Vimukta, Targeted drug delivery system: A Review, Research Journal of Chemical Sciences, Vol. 1 (2), Pg. No. 135 138, May 2011.

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