Monoclonal antibodies as drug targeting particulate carrier system

Monoclonal antibodies as drug targeting particulate carrier system

1. 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 SAMAJ’S COLLEGE OF PHARMACY, NASHIK 1

2. 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, type’s , Production - D rug conjugates - Advantages & limitations - Monoclonal antibodies Approved by FDA CONTENT 2

3. Introduction “Targeted 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. 3

4. 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. 4

5. Objective To 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. 5

6. Reasons for Drug Targeting Drug instability Low absorption Short half-life Large volume of distribution Low specificity Low therapeutic index 6

7. Common Approaches of Targeted Drug Delivery Controlling the distribution of drug by incorporating it in a carrier system Altering the structure of the drug at molecular level Controlling the input of the drug into bioenvironment to ensure a programmed and desirable biodistribution 7

8. Important Properties Influencing Drug Targeting Drug Concentration, Particulate location and Distribution, Molecular Weight, Physiochemical properties Drug Carrier Interaction Carrier Type, Amount of Excipients, Surface Characteristics, size, Density In Vivo Environment PH, Polarity, Ionic Strength, Surface Tension, Viscosity, Temperature, Enzyme, Electric Field 8

9. 9

10. Strategies for Drug Targeting 10

11. Components for Drug Targeting 11

12. Carriers or Markers Targeted 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

13. Pharmaceutical carriers : 1. Polymers 2. Microcapsules 3. Microparticles 4. Lipoproteins 5. Liposomes 6. Micelles 13

14. Monoclonal Antibodies Drug Targeting Particulate Carrier System 14

15. 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). 15

16. 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. 16

17. Role of Antibody in Immune System Each 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. 17

18. 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 antibodies 18

19. The structure of antibodies 19

20. Types of monoclonal antibodies 20

21. Monoclonal antibody production 21

22. Immunization Cell fusion Selection of hybridoma Screening , Cloning and propagation Characterization and storage 22

23. 1. Immunization Immunize 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 . 23

24. 2. Cell fusion Lymphocytes 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. 24

25. 3. Selection of hybridoma The 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 medium Suspension is diluted so that each aliquot contains one cell each. These are cultured in regular culture medium, produced desired antibody. 25

26. 4. Screening, Cloning and Propagation Screening 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. 26

27. 5.Characterization & storage Biochemical and biophysical characterization are made for desired specificity. It is important to note the monoclonal antibody is specific for which antigen MAbs must be characterized for their ability to withstand freezing and thawing. 27

28. Drug conjugates Toxin 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. 28

29. Advantages of monoclonal antibodies They 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. 29

30. Limitations As 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. 30

31. Monoclonal Antibodies Approved By FDA Antibody Target Indication Trastuzumab HER2 Breast Cancer Bevacizumab VEGF Lung Cancer Cetuximab EGFR Colorectal carcinoma Panitumumab EGFR Colorectal carcinoma 31

32. Reference: Lachman /Lieberman’s 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. 32

33. 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. 33

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