Pharmacokinetics and Pharmacodynamics of biotechnological products

Pk and Pd of biotechnological products

1. PHARMACOKINETICS AND PHARMACODYNAMICS OF BIOTECHNOLOGICAL PRODUCTS PRESENTED BY : ARIF NADAF PRESENTED TO DR. SANJULA BABOOTA DEPARTMENT OF PHARMACEUTICS SPER, JAMIA HAMDARD, NEW DELHI.

2. TABLE OF CONTENT INTRODUCTION PK AND PD OF PROTEINS AND PEPTIDES PK AND PD OF MONOCLONAL ANTIBODIES PK AND PD OF OLIGONUCLEOTIDES GENE THERAPY VACCINES

3. INTRODUCTION Biotech Drug: Biotechnologically derived drug products including proteins, peptides, antibodies, and antibody fragments, as well as antisense oligonucleotides and DNA preparations for gene therapy. Peptide and protein drugs, as well as oligonucleotides and DNA, now constitute a substantial portion of the compounds under preclinical and clinical development in the global pharmaceutical industry. The pharmacokinetic and pharmacodynamic properties of a biotech drug determine the relationship between administered dose, resulting systemic exposure, and subsequent pharmacologic response. In the last two decades, an increasing fraction of pharmaceutical R&D has been devoted to biotechnology-derived drugs (biotech drugs) – large molecules such as soluble proteins, monoclonal antibodies, and antibody fragments, as well as smaller peptides, antisense oligonucleotides, and DNA preparations for gene therapy.

4. PROTEINS AND PEPTIDES Proteins are the large organic compounds made up of amino acids arranged in linear chain attached by a peptide chain. Proteins are made up of 50 or more amino acids Peptides are the short polymers formed from the linking in defined order of amino acids. Peptides consists of 2-50 amino acids Proteins and Peptides also have various roles in pathological conditions such as cancer and diabetes. Therefore, the use of proteins and Peptides as therapeutic agents is considered as an attractive approach to combating various diseases. small numbers of peptide- and protein-based therapeutics have long been used in medical practice (e.g., calcitonin or glucagon),

5. Cont.. Advances in biotechnology and their application in drug development have propelled peptides and proteins based therapeutics Recombinant human insulin, which was approved in 1982, was the first of these biotechnologically derived drug products The lack of systemic bioavailability is mainly caused by two factors: high gastrointestinal enzyme activity, (peptidase and protease) low permeability through the gastrointestinal mucosa. Due to the lack of activity after oral administration, administration by injection or infusion is frequently the preferred route of delivery. (intravenous (IV), subcutaneous (SC), or intramuscular (IM) administration) other non-oral administration pathways have also been utilized, including nasal, buccal, rectal, vaginal, transdermal, ocular, or pulmonary drug delivery

6. DISTRIBUTION The problem of tissue accumulation of potentially toxic metabolites does not exist for protein drugs because the catabolic degradation products (amino acids) are recycled in the endogenous amino acid pool. Biodistribution studies for peptides and proteins are performed primarily to assess targeting to specific tissues to identify the major elimination organs. The volume of distribution of a peptide or protein drug is determined largely by its physicochemical properties (e. g., charge, lipophilicity), protein binding, dependency on active transport processes.

7. Cont.. Receptor-mediated specific uptake into the target organ can result in therapeutically effective tissue concentrations despite a relatively small volume of distribution. Example: Nartograstim , (recombinant derivative of the Granulocyte-Colony Stimulating Factor (G-CSF)), is characterized by a specific, dose-dependent and saturable tissue uptake into the target organ bone marrow, presumably via receptor-mediated endocytosis

8. FACTORS AFFECTING DISTRIBUTION Molecular size Distribution rate is inversely correlated with molecular size. Due to their large size – limited mobility through biomembranes . site-specific and target-oriented receptor-mediated uptake Eg. recombinant human vascular endothelial growth factor ( rhVEGF ) Proteins binding Active tissue uptake and binding to intra- and extravascular proteins, however, increase the volume of distribution, ( eg. atrial natriuretic peptide (ANP).

9. METABOLISM AND ELIMINATION peptides and protein drugs are eliminated by metabolism via the same catabolic pathways as endogenous or dietary proteins, resulting in amino acids that are reutilized in the endogenous amino acid pool for de-novo biosynthesis of structural or functional body proteins. Non-metabolic elimination pathways such as renal or biliary excretion are generally negligible for most peptides and proteins

10. Cont.. A. Proteolysis Proteolytic enzymes such as proteases and peptidases are ubiquitous throughout the body. metabolism is not only limited to the liver, kidneys, and GIT, but also include the blood and other organs and tissues. proteases and peptidases are also located within cells, intracellular uptake is more an elimination rather than a distribution process B. Gastrointestinal Elimination GIT is the major site of metabolism for orally administered peptides and proteins. Pre-systemic metabolism is the primary reason for their lack of oral bioavailability.

11. Cont.. C. Renal Elimination For parenterally administered and endogenous peptides and proteins, the kidneys are the major elimination organ if the peptide/protein size is less than the glomerular filtration limit of ~60 kDa , In addition to size–selectivity, glomerular charge–selectivity has also been observed anionic polymers < neutral polymers < cationic polymers. D. Hepatic Elimination An important first step in the hepatic metabolism of proteins and peptides is uptake into the hepatocytes. Small peptides may cross the hepatocyte membrane via passive diffusion if they have sufficient hydrophobicity. Uptake of larger proteins, such as t-PA (65 kDa ), is facilitated via receptor-mediated transport processes. Substrates for hepatic metabolism include insulin, glucagon, and t-PAs

12. Cont.. E. Receptor-Mediated Endocytosis A substantial fraction of a peptide and protein dose can be bound to receptors. This binding can lead to elimination through receptor- mediated uptake and subsequent intracellular metabolism. The endocytosis process is not limited to hepatocytes, but can also occur in other cells, including the therapeutic target cells.

13. Monoclonal Antibodies

14. INTRODUCTION 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. Therapeutic antibodies distinguished as mAbs (and derived products) and polyclonal antibodies mAbs are more important compared to polyclonal antibodies in drug development and applied pharmacotherapy due to their favorable properties and higher clinical success rates

15. Cont.. Therapeutic mAbs: A monoclonal antibody (mAb) is an antibody (immunoglobulin molecule) that is produced by recombinant DNA technology with the result that all molecules produced are identical in structure. These mAbs are of high purity and are produced in sufficiently large quantities for use as therapeutic agents. The first report of the treatment of a tumor patient with a mAb was published in 1980 The first extracted mAbs were produced using a method called the hybridoma technique, and emanated from murine B cells.

16. TYPES OF MONOCLONAL ANTIBODIES Class Example Year of Approval Murine Muromonab CD3 1886 Chimeric Rituximab 1997 Humanized Alemtuzumab 2001 Human Adalimumab 2002

17. ABSORPTION OF mAbs Due to their high molecular mass (and other reasons), the vast majority of mAbs that have been approved or are currently in clinical development are administered by intravenous (IV) infusion . Consequently, extravascular routes have been chosen as alternatives, including subcutaneous administration (SC; e.g., adalimumab, efalizumab) intramuscular administration (IM; e.g., palivizumab) Oral administration has been investigated and applied to a much smaller extent because of the chemical structure of classical mAbs: High molecular mass prevents permeation in GIT Acidic pH of stomach denatures glycoprotein mAbs

18. Cont.. The mechanism of absorption after SC or IM administration is thought to occur via the lymphatic system. The mAbs enter the lymphatic system by convective flow of interstitial fluid into the porous lymphatic vessels. The molecular mass cut-off of these pores is >100-fold the molecular mass of mAbs. From the lymphatic vessels, the mAbs are transported unidirectionally into the venous system. As the flow rate of the lymphatic system is relatively low, mAbs are absorbed over a long time period after administration. The resulting tmax is much later.

19. DISTRIBUTION OF mAbs Distribution of mAbs in the body is poor. Rate Limiting Factors are the high molecular mass and the hydrophilicity/polarity of the molecules. Distribution throughout the body has been visualized for conjugated mAbs using imaging techniques such as single photon emission computed tomography (SPECT).

20. METABOLISM AND ELIMINATION OF mAbs Therapeutic mAbs show different elimination pathways, including catabolic proteolysis , irreversible binding to antigen , and irreversible binding by anti-idiotype antibodies . A. Proteolysis MAbs undergo proteolytic degradation similar to that of physiological immunoglobulins. Degradation will be more rapid for more nonhuman antibodies. There is a clear relationship between the human fraction of the mAb and the half-life. The lower the nonhuman fraction, the longer the half-life murine (few days) < chimeric < humanized < human (few weeks) B. Binding to Antigen Binding of mAbs not only affects distribution (similar to the situation for small molecule drugs) but also reflects another means of elimination. Binding of the Fab region to the antigen with high affinity must be regarded as almost irreversible. The antigen–antibody complex, if located on the surface of a cell, will be internalized and subsequently degraded .

21. Cont.. C. Binding to Anti-Idiotype Antibodies A third elimination pathway occurs if anti-idiotype antibodies are formed as an immune response of the human body to the administration of mAbs. Following repeated administration, anti-idiotype antibodies are usually observed after one to two weeks, The formed anti-idiotype antibodies almost irreversibly bind the therapeutic anti- bodies and therefore, by neutralization, eliminate them from the body. D. Clearance As glomerular filtration has an approximate molecular size limit of 20–30 kDa , mAbs do not undergo filtration in the kidneys due to their relatively large size. renal elimination in total is uncommon or low for mAbs. Biliary excretion of mAbs has been reported only for IgA molecules, and only to a very small extent. Therefore, total clearance (CL) usually does not comprise renal or biliary clearance.

22. Cont.. The extent of the adverse reaction strongly depending on several factors: Type of mAb: the lower the extent of “humanization”, the more anti-idiotype antibodies are likely to be formed. Dosage regimen: single doses rarely evoke a strong immunological response, whereas multiple dosing often results in anti-idiotype antibody formation. Route of administration: SC injection has a higher incidence of forming anti- idiotype antibodies than IM or IV administration. Patient’s genetics: patients with autoimmune diseases or prior anti-antibody production are more likely to produce an immune response.

23. PHARMACODYNAMICS OF MABS Three different pharmaco-dynamic principles of action can be distinguished for mAbs depending on the type of antigen and the antigen–antibody interaction. lysis or apoptotic activity, coating activity, inactivating activity. The majority of mAbs exert their pharmacodynamic activity by Fc-mediated ADCC/CDC activation or delivery of toxic substances to the targeted cell. In some cases “only” a coating of the antigen-binding site by the antibody takes place, which will lead to a “down-regulation” of the antigen. The pharmacodynamic principle is to block the antigen-binding activity to its receptor and thereby inactivate the target antigen function. The binding of the therapeutic antibody to the target antigen finally leads to agglutination and neutralization of the antigen.

24. THERAPEUTIC AREAS OF mAbs Efficacious mAbs are used in a variety of therapeutic indications such as Cancer, Rheumatoid Arthritis, Crohn’s Disease, Psoriasis, Organ Transplantation, Asthma, Infectious Diseases, Cardiovascular Diseases,

25. Antisense Oligonucleotides

26. INTRODUCTION Antisense compounds are short synthetic oligonucleotides, usually between 15 and 25 nucleotides in length, designed to hybridize to RNA. Upon binding the target RNA, the oligonucleotide prevents translation of the encoded protein product in a sequence-specific manner. Decreasing gene expression by selectively blocking mRNA translation can be accomplished using single-strand antisense molecules.

27. ABSORPTION Antisense oligonucleotides are administered parenterally in the majority of reported in-vivo studies: intravenous (IV), intraperitoneal (IP), or subcutaneous (SC) Parenteral administration allows complete systemic availability. Non-parenteral administration of antisense oligonucleotides is only made possible with the aid of novel formulations intended to overcome barriers to absorption

28. DISTRIBUTION The highest concentrations of oligonucleotides in all species studied were found in kidney , liver , spleen , and lymph nodes , but oligonucleotides can be measured in almost every tissue, except the brain , at 24 h after IV administration. A plasma concentration–time profile that is poly-phasic with rapid distribution half-life (^1 h) and long elimination half-life reflecting slow elimination from the tissues. High binding to plasma proteins (>90 % across species). Plasma clearance is dominated by distribution into the tissues. Long tissue elimination half-life cleared by nuclease-mediated metabolism.

29. METABOLISM Nucleases are the enzymes which metabolize oligonucleotides. While distribution to tissues relies on the mechanism of plasma clearance, whole body clearance is the result of metabolism and the excretion of low mol. Weight oligonucleotides.

30. ELIMINATION AND EXCRETION ASOs are highly bound to plasma proteins, which prevents their glomerular filtration and limits urinary excretion The elimination of 2'-MOE partially modified ASOs is attributed to slow (but continuous) nuclease-mediated metabolism in tissue, followed by ultimate excretion of these shortened metabolites in the urine. Minor urinary or fecal excretion of the intact drug.

31. PHARMACODYNAMICS

32. Gene Therapies

33. INTRODUCTION Gene therapy refers to the introduction of new genetic material of therapeutic value into somatic cells. It is a powerful method for the treatment of diseases for which classical pharmacotherapy is unavailable or not easily applicable. Example: Patients who suffer from rare genetic disorder lipoprotein lipase (LPL) deficiency have abnormally high levels of triglycerides and very low-density lipoproteins (VLDL) causing pancreatitis and cardiovascular disease. The LPL gene has been incorporated into a recombinant adeno-associated virus. Gene therapy face several challenges. gene delivery, sufficient extent and duration of stable gene expression, and safety.

34. APPROACHES TO GENE THERAPY A variety of delivery systems (also known as “ vectors ”) have been evaluated. The function of the vector is to transverse the biological barriers for reaching its attended target, usually the nucleus. Two main approaches have been used for in vivo delivery of rDNA. Virus based approach non viral approach

35. A. VIRUS BASED APPROACH It involves the replacement of viral replicating gene with transgene and then packaging the rDNA into viral particle. The recombinant virus can then infect target cell and transgene is expressed; though the virus is not capable of replicating. Both retrovirus has been used successfully in gene delivery RNA virus that have ability to permanently insert their genes into chromosomes of the host cells and DNA viruses (which remain outside the host chromosome)

36. B. NON VIRAL APPROACH The transgene is engineered into plasmid vector, which contains gene expression control region. Chemical vectors include Polycationic carriers such as liposomes (lipoplexes) Polymers (polyplexes) These carriers avoid the DNA size limitations and immunogenicity associated with viral vectors.

37. CHARACTERISTICS OF IDEAL GENE DELIVERY An ideal gene delivery system should fulfill the following criterias Packaging of DNA Migrating through blood capillary vessels Targeting Cellular uptake Tracking to the nucleus Effective gene therapy depends on several conditions. The vector must be able to enter the target cells efficiently and deliver the corrective gene to the nucleus without damaging the target cell. The corrective gene should be stably expressed in the cells, to allow continuous production of the functional protein. Neither the vector nor the functional protein produced from it should cause an immune reaction in the patient.

38. PROBLEMS IN GENE THERAPY It is also difficult to control the amount of functional protein produced after gene therapy, and excess production of the protein could cause side effects, although insufficient production is more typically observed. the physical and chemical properties of DNA and RNA molecules, such as size, shape, charge, surface characteristics, and the chemical stability of these molecules and delivery systems. In vivo problems may include bioavailability, distribution, and cellular and nuclear uptake of these macromolecules into cells. Moreover, naked DNA and RNA molecules are rapidly degraded in the body.

39. Vaccines

40. INTRODUCTION A vaccine is a biological preparation that provides active acquired immunity to a particular disease. Vaccines are like a training course for the immune system. They prepare the body to fight disease without exposing it to disease symptoms. The terms vaccine and vaccination are derived from  Variolae vaccinae  (smallpox of the cow), the term devised by Edward Jenner to denote cowpox. In 1798, he described the protective effect of cowpox against smallpox.

41. TYPES OF VACCINES

42. PHARMACODYNAMICS 1. DNA VACCINES DNA vaccines are made up of small strands of DNA, a gene, encoding the antigen of interest. The Gene is attached to a plasmid for delivery into the body. The Plasmid is used so that the body does not degrade the foreign gene before it can provoke an immune response. Once administered the DNA are taken up by host cells which produce the S-Protein, and show the antigen on its cell surface, thus stimulating an antibody and T cell response. 2. m RNA VACCINES RNA vaccines consist of an mRNA encoding the antigen of interest. This is placed in a Lipid Nanoparticle (LNP) vehicle. The LNP prevents the mRNA degradation by the host until it is taken up by the cell. Once administered, the RNA are taken up by host cells. The intra-cellular lipases degrade the LNP exposing the mRNA. The mRNA is then translated into the S-protein, and is on its cell surface, stimulating an antibody and T cell response.

43. 3. VIRAL VECTOR VACCINES Viral vector vaccines are similar to live-attenuated vaccines in that they use a harmless virus or an attenuated virus known as a vector. However, the attenuated virus carries a foreign gene in their genome representing the antigen of interest. When the virus infects a cell, they administer this foreign gene into the cell. The cell then transcribes and translates the gene to produces the antigen, and display the antigen on the cell surface to stimulate an immune response. 4. LIVE ATTENUATED VACCINES: Live attenuated vaccines contain a live but less infective form of the pathogen. These vaccines have all the components of the original pathogen, but they possess mutations that reduce their ability to replicate inside the body, so they will not reproduce natural infection. It is a proven vaccine technology used to vaccinate people against many infections such as polio, tuberculosis and chicken pox. 5. INACTIVATED VACCINES: Evolving from live-attenuated vaccines that are able to (slowly) replicate in the body, inactivated vaccines contain a whole pathogen that is killed or inactivated by chemical, heat or radiation. This eliminates the possibility of the pathogen replicating and possibly causing infection, yet the vaccine still has all the components of the original pathogen to induce a memory response. Various inactivated vaccines are available to vaccinate people against infections such as cholera and hepatitis A. PHARMACODYNAMICS

44. 6.VIRAL SUBUNIT VACCINES: Subunit vaccines take parts of the pathogen (antigens) that simulate an immune response and inject them into the body. Most subunit vaccines consist of proteins from the pathogen, but they can also be fragments of bacterial toxins (toxoids) or pathogenic components such as the cell wall. Subunit vaccines produce strong antibody responses as the antigens are collected, processed and presented to B cells to stimulate antibody production. 7. VLP VACCINES (VIRUS LIKE PARTICLE VACCINES) This type of vaccine contains molecules that mimic the virus but are not infectious and, therefore, not a danger. VLP has been an effective way of creating vaccines against diseases such as human papillomavirus (HPV), hepatitis and malaria. Virus-like particles (VLPs) are nanostructures (lipids NPs, dendrimers and fullerenes) that resemble the structures of viruses. They are composed of one or more structural proteins that can be arranged in several layers and can also contain a lipid outer envelope. VLPs trigger a high humoral and cellular immune response due to their repetitive structures. A key factor regarding VLP safety is the lack of viral genomic material, which enhances safety during both manufacture and administration. PHARMACODYNAMICS

45. REGULATORY ISSUES AND CHALLENGES The major issues and challenges that the biotech industry faces are as follows: – High Level of Risk Many Biotech companies are given patents for their products that are produced by them so that they can recover all the expenses that they have invested in the process of Research & Development. Once their patent expires, the products have a wider popularity in the market as compared to their competitors. The main problem which arises over here is that the investors have to weigh a factor that this period leads to a huge risk due to delayed results through Research & Development.  Level of Affordability The second big challenge faced by biotech industries is the level of affordability. Many drugs require a huge scale of amount and also many firms do not trust the R&D methods used by these industries in order to make a proper product for the use of the public at large.  Problem of Privacy Another main issue which we face in this industry is the problem of privacy. Many data are stolen by the rival industry and hence they use it in their product in order to gain huge profits in the markets. This is also a huge challenge for the industries and thus they need to protect it really very strictly.

46. REFERENCES Pharmacokinetics and Pharmacodynamics of Biotech Drugs Principles and Case Studies in Drug Development Edited by Bernd Meibohm . Applied Biopharmaceutics & Pharmacokinetics (Seventh Edition) Leon Shargel, Andrew B.C. Yu