BIOTECHNOLOGY DRUG PRODUCTS

byS. Adebayo, PhD

"The World is a book and those who do no travel read only a page"

Saint Augustine

INTRODUCTION Biotechnology encompasses any technique that

uses living organisms in the production or modification of products.

Thus, biotechnology products broadly refer to biopharmaceutical drugs generated through researches in cell biology, genetics and recombinant DNA technology.

Recombinant DNA (rDNA) and monoclonal antibody (MAb) are providing exciting opportunities for new pharmaceuticals development as well as new approaches to drug delivery.

INTRODUCTION…. During the last decade, over 359 biotechnology products

were developed by more than 40 pharmaceutical companies for indications ranging from hemophilia, sepsis, skin ulcers, and rheumatoid arthritis to cancer.

More than one-third of all biotechnology products are geared toward cancer therapy

More than 29 were under development for HIV, AIDS and AIDS-related diseases.

About 19 other products were being developed for autoimmune diseases.

Research and development network

Application Biotechnology derived drugs are being applied in

cancer therapy, HIV, AIDS and AIDS-related and autoimmune diseases

In diagnostic investigations

As blood substitutes, clotting factors, etc.

**Human insulin (approved in 1982) and human growth factor were the first set of biotechnology products to be applied for human therapy.

Biotechnology techniques and processes (Evans, P. R. Biotechnology and Biological Preparations in Encyclopaedia of PT vol. 1, 3rd edn.)

Recombinant DNA (rDNA) DNA constitutes the genes through which

cells reproduce and are able to maintain life.

DNA consists of two strands connected by bases adenine (A), guanine (G), cytosine (C) and thymine (T).

The order of base arrangement (in pairs) along the two strands of DNA constitutes a specific gene for a specific trait.

Recombinant DNA (rDNA) A typical gene consists of 100’s of bases arranged

in pairs according to the A-T and C-G rule. The pattern constitutes the DNA message for

maintaining cells and organisms and for building next generation.

Creation of new cell or organism involves unwinding and separation of the DNA strands and subsequent attachment to new bases within the cell according to the A-T/C-G rule.

Recombinant DNA (rDNA) DNA also codes for information required

for the production of the 23 amino acids.

The arrangement of these a.a. in a vast array of ways give rise to thousands of different proteins

When DNA molecules are selectively hydrolyzed using endonucleases, molecules of two different DNA’s can be joined (recombinant DNA).

Recombinant DNA (rDNA) Other techniques involved are

replication, separation, identification which permit the production of large quantities of purified DNA fragments.

Recombinant DNA technology allows

the removal of a specific piece of DNA out of a large complex molecule.

Recombinant DNA (rDNA) The ability to join two different pieces of

DNA together at specific sites within the molecule is achieved with two enzymes – a restriction endonuclease - DNA ligase.

This technology permits utilization of non-human cells (e.g. special strain of E. coli) to manufacture proteins identical to those produced in human cells.

Recombinant DNA (rDNA) DNA technology is been utilized to

diagnose diseases. This method utilizes small pieces of DNA

to search a cell for viral infection or genetic defect.

DNA probe has been applied in testing for infectious diseases, cancer, genetic defects and disease susceptibility.

Quality control tests in r-DNA technology

Evans, P. R. Biotechnology and Biological Preparations in Encyclopaedia of PT vol. 1 3rd edn

Approved rDNA biologicals (hormones)

Approved rDNA biologicals (growth factors)

Approved biologicals (blood factors and interleukins) (rDNA products)

Monoclonal Antibodies When an antigen molecule enters the body, an

immune response is initiated. An antigenic molecule may contain several

different antigenic determinants to which each line of β-lymphocytes will proliferate, secreting an immunoglobulin molecule that fits a

single antigenic determinant or part of it.

Monoclonal antibodies are produced by perpetuating the expression of a single β-lymphocyte

Monoclonal Antibodies (Cont.)Hybridoma technology permits

large quantity production of identical, monospecific antibodies.

Monospecific antibodies are produced by the fusion of β-lymphocytes, stimulated with a specific antigen, with immortal myeloma cells

Monoclonal Antibodies (Cont.) The resultant hybridomas are then

maintained in cultures and are capable of producing large amounts of antibodies.

From hybrid cells, a specific line or clone producing monospecific immunoglobulins can be selected.

Immunoglobulin F Large proportions of the currently

used antibodies belong to the immunoglobulin F (i.e. IgG) subclass.

The IgG molecule has a MW 150-180 kD and consists of two polypeptide chains connected by di-sulphide bonds.

Immunoglobulin F… The heavy and light chains consist of two zones:

the constant and variable domains. The constant domain amino acids are relatively

conserved among Ig’s of specific class (e.g. IgGi, IgM).

The variable domains of antibodies are highly heterogeneous and it is this domain that gives antibodies its binding specificity and affinity (Fig. 1)

Monoclonal antibodies

PSWC 2007 Post-Satellite EAPB and EUFEPS Workshop on Monoclonal AntiBodies

A schematic representation of the structural and functional domains of IgG.

Human anti-mouse antibodies Most of the monoclonal antibodies in clinical trials

have been derived from mice A patient exposed to the antibodies typically

develops detectable levels of antibody responses to the foreign monoclonal antibody within two to four weeks.

On giving additional doses, a typical allergic reaction is elicited – i.e. chills, urticaria, wheezing – and the antibody is rapidly cleared from the serum.

Man-made antibodies Man-made antibodies overcome the lack

of intrinsic antitumour activity and immunogenicity of many murine monoclonal antibodies.

They retain the binding specificity of the original rodent antibody determined by the variable regions but can potentially activate human immune system through their human constant regions.

Man-made antibodies… Smaller fragments that contain

immunoglobulin binding sites e.g. F(ab)2 and Fab′ do not contain the lower binding domain of the molecule.

A smaller sized molecule tends to be less immunogenic but may have greater tumour penetration ability than the larger structure (Fig 2).

Approved biologicals (Mabs)

A historical timeline of therapeutic monoclonal antibody approvals in the United States relative to the development of antibody generation technologies.

Polymerase Chain Reaction Biotechnology process involving substantial amplification

(> 100,000 fold) of a target nucleic acid sequence (a gene). It is an enzymatic reaction which occurs in 3 steps:

DNA is denatured to separate the 2 strands

A nucleic acid primer is hybridized to each DNA strand at a specific location within the nucleic acid sequence

DNA polymerase enzyme is added for extension of the primer along the DNA strand to copy the target nucleic acid sequence.

Gene Therapy Exogenous genetic material is transferred

into somatic cells to correct an inherited or acquired defect.

It may also be used to introduce a new function or property into cells.

Gene therapy has been applied to diseases such as cystic fibrosis, hemophilia, sickle cell anemia and diabetes.

Gene Therapy… Ethical consideration does not permit intervention

with germ cells. Self renewing stem cells are used for therapeutic

transfer of genetic materials into end-state differentiated somatic cells

Since stem cells can renew themselves, the inserted gene will remain in place throughout subsequent generation of differentiated cells or tissue population.

Gene Therapy For example, a patient’s cells (T-lymphocytes) are

harvested and grown in the laboratory.

A gene from a viral carrier (e.g. moloney murine leukemia virus) is supplied which starts to produce the missing protein necessary to correct the deficiency.

The genes with extra functional cells are then returned to the patient and the normal protein is produced and released thereby alleviating the disease.

Gene Therapy… Gene therapy has been used to treat

adenosine deaminase deficiency (ADA). The gene coding for ADA is found on chromosome 20.

Gene deletion and point mutation lead to loss of or severe reduction in ADA enzymatic activity resulting in clinical presentation of severe combined immunodeficiency syndrome (SCID) causing death in childhood or adolescence

Nucleotide Blockade/Antisense technology This technology focuses on the study of

function of specific proteins and intra cellular expression.

The sequence of a nucleotide chain that contains information for protein synthesis is called the sense sequence; nucleotide chain complementary to the

sense sequence is called the antisense sequence.

Nucleotide Blockade/Antisense technology… Antisense drugs recognize and bind to the

nucleotide sense sequence of specific messenger RNA (mRNA) molecule and prevent the synthesis of unwanted proteins and actually destroying the sense molecules in the process.

When antisense RNA is introduced into a

cell by cloning technique, the specific gene of interest is cloned in an expression vector in the wrong orientation.

Nucleotide Blockade/Antisense technology…A complementary mRNA is thus

created to match an abnormal mRNA.

When the 2 mRNA strands complex together, translation to form the disease-producing protein is prevented.

Products of Biotechnology Anticoagulant drug –Lepirudin

(Refludan®)

Antisense drugs – Fomivirsen sodium injection is approved for local treatment of cytomegalovirus (CVM) retinitis in patients with AIDS

Efavirenz (Sustiva®) – A non-nucleoside reverse transcriptase inhibitor and the first anti-HIV drug to be approved by FDA for once daily dosing in combination with other anti-HIV drugs.

Products of Biotechnology (Cont.) Clotting factors - Kogenate®,

Recombinate®) – Recombinant anti-hemophiliac factor indicated for the treatment of classical hemophilia A in which there is a demonstrated deficiency of clotting factor (Factor VIII )

Growth factor – Becaplemin (regranex®)

Systemic growth hormone - Somatropin recombinant (Humatrope®)

Products of Biotechnology (Cont.)… Colony stimulating factors -

Erythropoietin Interferon

Vaccines – Genetically engineered vaccines use a synthetic copy of the protein coat of a virus to “fool” the body immune system into mounting a protective response.

Approved biologicals (interferons)

Approved biologicals (enzymes)

Approved biologicals (vaccines and liposomes)

Approved biologicals (tissue and cell engineering)

Approved biologicals (blood derivatives and natural extracts)

Exercise (5 % of CW) Identify ANY 2 products on T&T Drug market, Hospital or

clinics

Specify the Biotech method of making the product

Specify the dosage form and dosage amount

Specify the route of administration

Specify the storage recommendation

State any special warning, recommendation & advice to patient

THANK YOU FOR YOUR ATTENTION

BIOTECHNOLOGY DRUG PRODUCTS