Drug discovery and development

Drug discovery and development is and always has been the most exciting part of clinical pharmacology. It is my attempt to compile the basic concepts from various books, articles and online journals. Feel free to comment.

1. Drug Discovery & Development Dr. Prashant Shukla Junior Resident Dept of Pharmacology 1/27/2015 1

2. Definitions: Drug: A chemical substance of known structure, other than a nutrient or an essential dietary ingredient, which, when administered to a living organism, produces a biological effect. Discovery phase: Identification of a new chemical entity as a potential therapeutic agent. Development phase: Compound is tested for safety and efficacy for one or more clinical indications, and in suitable formulations and dosage form. 1/27/2015 2

3. An important change: The term “ drug discovery ” is classical and conventional. It should be replaced with the term “ drug invention ”. In the past, drugs were discovered as natural products and used as such. Now, drugs are sculpted and brought into being by pharmacologists in air conditioned labs. The term invention emphasizes this process. 1/27/2015 3

4. New Drug definition (CDSCO) According to Rule 122 E: 1/27/2015 4 (a)  A new substance of chemical, biological or biotechnological origin; in bulk or prepared dosage form; used for prevention, diagnosis, or treatment of disease in man or animal; which, except during local clinical trials, has not been used in the country to any significant extent; and which, except during local clinical trials, has not been recognized in the country as effective and safe for the proposed claim.

5. New Drug definition (CDSCO) 1/27/2015 5 (b) A drug already approved by the licensing authority, which is now proposed to be marketed with modified or new indications, dosage forms (including SR dosage form) and route of administration.

6. New Drug definition (CDSCO) 1/27/2015 6 (c) A fixed dose combination of two or more drugs, individually approved earlier, and now proposed to be combined for the first time in a fixed ratio, or if the ratio of ingredients in an already marketed combination is proposed to be changed, with certain claims, viz. indications, dosage form (including SR dosage form) and route of administration.

7. Stages of the New drug synthesis: Drug discovery : Candidate molecules are chosen on the basis of their pharmacological properties. Preclinical development : Non-human studies (e.g. toxicity testing, pharmacokinetic analysis and formulation) are performed. Clinical development : The selected compound is tested for efficacy, side effects and potential dangers in volunteers and patients. 1/27/2015 7

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10. Drug discovery Usual time duration: 2-5 years Usual no. of projects: 100 It consists of following components: Target selection and validation Lead-finding or Lead generation Lead optimisation Pharmacological profiling 1/27/2015 10

11. Reverse Pharmacology AKA target based drug discovery (TDD) A hypothesis is first made that modulation of the activity of a specific protein target will have beneficial therapeutic effects. The reverse pharmacology approach takes about 2 years to obtain a new drug candidate. It is much faster and more efficient than the classical approach, which takes about 5 years. 1/27/2015 11

12. Comparison: 1/27/2015 12

13. Properties of ideal drug target: 1/27/2015 13

14. Various targets of drug action 1/27/2015 14 The majority of drug targets are a) G-protein coupled receptors ( est total 5000) b) Nuclear receptors ( est total >150) c) Ion channels ( est total 1000) d) Enzymes ( est total uncertain) Currently we are exploiting only 120 distinct drug targets. The potential drug targets that remain to be exploited range from a few hundreds to several thousands.

15. Target identification strategies Gene Expression profiling: Genomics Focussed Proteomics Metabolic pathways analysis: Molecular Biology Phenotype analysis Genetic association 1/27/2015 15

16. Target identification strategies... Inverse Docking: It is a computational docking program in which a specific small molecule of interest is tested against a library of receptor structures. Bio informatics: It derives knowledge from computational analysis of biological data. It includes information stored in genetic code, patients statistics and scientific literature. 1/27/2015 16

17. In the earlier times, the complex biological responses of chemicals were first seen. And then the drug targets were explored for those chemicals. Now target identification has become the first step of drug discovery. Limitation: Drugs which do not act through receptors- Antacids, Osmotic diuretics, Alkylating agents, Psoralens and Activated charcoal can not be recognised. 1/27/2015 17

18. Target Validation To identify the most useful target among the various identified targets, Target Validation is done. Identified targets are analysed and compared for Ability to regulate biological and chemical process/ molecules in the body. Association with a specific disease. It is done using genetic knockout, knockdown and suitable disease models. 1/27/2015 18

19. Lead finding/ Lead generation Approaches to new drug molecule 1. NEWER TECHNIQUES Molecular modeling Combinatorial chemistry Biotechnology Genetic medicine Immunopharmacology 1/27/2015 19 BIOLOGICS or BIOLOGICAL COMPOUNDS

20. Molecular modelling AKA Rational drug designing . Aided by three dimensional computer graphics. Allows design of structure based on new & known molecules. Highly selective targeted compounds are created by enhancing desired properties of known molecules. 1/27/2015 20

21. Combinatorial chemistry It is systematic and repetitive covalent connection of set of different building blocks of varying structures to each other to yield a large array of diverse molecular entities. This process is Faster, more efficient and cheaper. Millions of compounds can be synthesized : Chemical Compound Library . 1/27/2015 21

22. Limitations of Combinatorial chemistry: Building and maintaining huge compound libraries is a costly business. Even the largest compound collection represents only a fraction of the number of “drug like” molecules that exists in theory- estimated to be about 10 60 . 1/27/2015 22

23. Biotechnology Therapeutic agents produced by biotechnology rather than conventional synthetic chemistry are called Biopharmaceuticals . Involve the use of recombinant DNA technology /genetic engineering to clone & express human genes. to produce large amount of hormones like insulin. 1/27/2015 23

24. Genetic medicine Transfer of Genetic material A single gene which is typical for gene therapy . Fragments of coding sequences (as in RNA modification therapy- MC being anti sense oligonucleotide strategy). Entire genome (as in the case of SSC and ESC therapy). Vectors are Viruses and Liposome-plasmid complex . Diseases addressed: Hereditary diseases like SCID, Haemophillia , etc. 1/27/2015 24

25. Immunopharmacology Deals with finding the Biological immune modifiers or Immuno -modulating agents that cause selective up-regulation or down-regulation of specific immune responses. Examples include: Rituximab - Anti CD 20 monoclonal antibody for RA. Adalimumab - Anti TNF- α inhibitor antibody for RA. 1/27/2015 25

26. Lead finding/ Lead generation Approaches to new drug molecule: 2. OLDER TECHNIQUES Animal models as human disease. Natural Products like plants , animals & micro- Organisms: Random screening approach Ethnobiological approach Traditional Medicines. Modification of structure of known drugs to develop “Me-too / derivative medications/ follow-up drugs ” drugs. 1/27/2015 26

27. Source of leads: Plants 1/27/2015 27 Papaver somniferum : Morphine Atropa belladona : Atropine Rauwolfia serpentina : Reserpine Digitalis lanata : Digoxin Strychnos toxifera : d- TC Pilocarpus microphyllus : Pilocarpine Salix alba (Willow bark): Aspirin Bark of Yew tree: Paclitaxel Cinchona tree: Quinine

28. Source of leads: Animals Kraits: α- Bungarotoxin Marine invertebrates: Arabinose nucleosides Cone snail toxin: Ziconotide ( Prialt ) Marine invertebrates: Bryostatin -like compounds Dinoflagellates : Saxitoxin 1/27/2015 28

29. Source of leads: Micro-organisms Streptomyces notatum : Penicillin Streptomyces venezuelae : Chloramphenicol Penicilium griseofulvum : Griseofulvin Streptomyces griseus : Streptomycin Streptomyces gradiae : Neomycin 1/27/2015 29

30. SCREENING The usual approach is to clone the target protein- the human form . This is because the sequence variation among species is associated with pharmacological differences and it is essential to optimise for activity in humans. An assay system is then developed to measure the functional activity of the target protein. 1/27/2015 30

31. Desired characteristics of the assay: Should run automatically (if possible, with an optical read out e.g. Fluorescence or optical absorbance). Should be in a miniaturised multiwell plate format- for reasons of speed and economy. Robotically controlled assay has become the standard starting point for most drug discovery projects. e.g. High through put screening 1/27/2015 31

32. Virtual screening (VS) It is based on the computationally inferred or simulated real screening. Advantages compared to laboratory experiments are: 1.low costs. 2.Investigate compounds that have not been synthesized yet. 1/27/2015 32

33. Virtual screening (VS)... 3. VS can be used to reduce the initial number of compounds before using expensive HTS methods. 4.The number of possible virtual molecules available for VS is much higher than those available for HTS. Disadvantage is that it can not substitute the real screening. 1/27/2015 33

34. Two types of approaches used in virtual screening Target based virtual screening (TBVS), or Receptor based virtual screening Ligand based virtual screening (LBVS), or Similarity based virtual screening. 1/27/2015 34

35. Target based virtual screening (TBVS) Exploits the molecular recognition between the ligand and a target protein information about the target. Selection of chemical that has high affinity for the target’s active site. Structural information can be determined by Nuclear Magnetic Resonance(NMR) or X-ray diffraction. 1/27/2015 35

36. Target based virtual screening (TBVS) TBVS relies on 3D structures of protein targets and on 3D databases of chemicals. TBVS allows the identification of structurally novel ligands that may present interaction modes similar to the already known ligands . Even new interaction pattern identification with different parts of the target’s active sites. This methodology uses virtual filtering of all available ligands in a suitable database. 1/27/2015 36

37. Ligand based virtual screening (LBVS) There is no structural information about the target. The screening focuses on physical and chemical based searches among the ligands . Through pharmacophore pattern matching. On similarity searching using descriptors that may be 1D, 2D or 3D. 1/27/2015 37

38. Chris Lipinski’s rule of five Linear descriptors (1D) are used to identify chemicals which do not violate any rules for solubility and permeability: H-bond donors <5. Molecular weight <500. Partitioning coefficient (Log P) <5. H-bond acceptors <10 (=5×2). The “ rule of five ” name came from the cutoffs all being multiples of five. But there are only four rules. 1/27/2015 38

39. Ligand based virtual screening (LBVS) The complete structure of the ligands can be considered in the quantitative structure-activity relationships (QSAR) methods. QSAR methods can make accurate prediction of the relative conformation and alignment of the ligands . LBVS are more limited than TBVS since it is biased by the properties of the already known ligands for a given target. 1/27/2015 39

40. High through put screening (HTS) The “ Real Screening ”. It is the process of testing a large number of diverse chemical structures against disease targets to identify “hits”. Compared to traditional screening methods, HTS is characterised by: Simplicity Rapidness High information harvest Based on ligand -target interaction principle 1/27/2015 40

41. High through put screening... Various technologies used for HTS are: Fluorescence Nuclear Magnetic Resonance (NMR) Affinity chromatography Surface plasmon resonance DNA microarray HTS can analyse around 10,000-100,000 samples/day. 1/27/2015 41

42. End results of screens: Hit : A molecule with confirmed concentration-dependent activity in a screen, and known chemical structure. Progressible hit : A representative of a compound series with activity via acceptable mechanism of action and some limited structure-activity relationship information. 1/27/2015 42

43. Lead Optimisation The aim of this stage is: Increase the potency of the compound on its target. Increase its selectivity. Increase its metabolic stability. Usually one project out of five passes this stage. 1/27/2015 43

44. Lead Optimisation... Various steps: Identification of the Pharmacophore (relevant groups on a molecule that interact with a receptor and are responsible for the biological activity). Functional group modification : Modification of the group may enable or disable certain biological effects. 1/27/2015 44

45. 1/27/2015 45 3. Structure-Activity relationship : Some of these features are important for the activity and the others are not. (1) NH 2 and sulfonyl (R) should be para . (2) NH 2 should be unsubstituted . (3) Benzene ring should not be replaced by other ring systems.

46. 4. Structure modification to increase potency and therapeutic index: Homologation: a homologous series is a group of compounds that differ by a constant unit, usually CH 2 . B. Chain branching C. Ring-chain transformation Affects (1) lipophilicity , (2) interaction with the enzyme or receptor. It could increase or decrease drug potency and therapeutic index. D. Bioisosterism . 1/27/2015 46

47. 5. Quantitative structure-activity relationships (QSAR-rational drug design) Based on the fact “the biological properties of compounds are a function of its physico -chemical parameters”. Fundamental physicochemical parameters Electronic effects: Hammett equation Lipophilicity effects: Hansch equation Steric effects: Taft equation 1/27/2015 47

48. 6. Molecular graphics-based drug design : To find a structure match, a computer technology called DOCKING is used. It is the computer-assisted movement of a terminal-displayed molecule into its receptor. Docking algorithms deal with ligand conformation prediction and orientation within the target active site. It predicts the various forces acting between target and ligand . Scoring function is a mathematical function to rank protein- ligand complexes according to their predicted binding affinity. 1/27/2015 48

49. The main problem is that lead optimisation often seems impossible despite much ingenious and back- breaking chemistry. It is because lead compounds, like anti social teenagers, refuse to give up their bad habits. In other cases, the compounds although they produce the desired effects on the target molecule and have no other obvious defects, fail to produce the desired effects in animal models of disease. This implies that the target is not a good one. 1/27/2015 49

50. Out of the above steps, Target identification and Lead Finding is often carried out in academic research laboratories. Screening for biologic activity and chemical modification of a known active molecule are usually carried out in industries due to their high costs. Translational research/ medicine or Experimental medicine : The process of moving from the basic science laboratory to the clinic. It involves the pre clinical and clinical steps of drug discovery. 1/27/2015 50

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52. Pre clinical development Usual time duration: 1.5 years Usual no. of Compounds: 20 The aims of pre clinical testing are: Pharmacokinetics Short term toxicology Formulation Synthesis scale up 1/27/2015 52

53. Work falls in four categories: 1. Safety Pharmacology : Pharmacological testing to check that the drug does not produce any hazardous side effects.* 2. Preliminary toxicological testing to eliminate genotoxicity and to determine the maximum non-toxic dose of the drug (usually when given daily for 28 days, and tested in two species). 1/27/2015 53

54. Work falls in four categories: 3. Animal studies: Pharmacokinetic testing i.e. studies on absorption, metabolism, distribution and elimination in laboratory animals like Mice, chicken, monkeys, and guinea pigs. 4. Chemical and pharmaceutical development : Feasibility of large- scale synthesis and purification Stability of the compound under various conditions To develop a formulation suitable for clinical studies. 1/27/2015 54

55. Animal studies: components Toxicology studies : Extended programme in animals studies. These could be acute, sub acute or chronic toxicity studies. Acute: 24-48 hours Sub Acute: Few weeks Chronic: For months 1/27/2015 55

56. Animal studies: components... Mutagenicity studies : comprises of in vitro tests where either unicellular organisms or tissue cultures are exposed to the drug. Following battery of tests is used: Ames test in S. Typhimurium . Cytogenetic assay in mammalian cells. Micronucleus assay in rodent hematopoietic cells. First two tests should be completed before Phase I study commences. But the battery should be complete in all respects before Phase II studies. 1/27/2015 56

57. Animal studies: components... Carcinogenicity studies : Two animal species with low incidence of spontaneous tumours are used. 3 doses- high, low and intermediate are employed. The study usually lasts for most of the animal’s life. Detailed autopsy and histological examination are performed at the end of the study. 1/27/2015 57

58. Animal studies: components... Reproductive studies : Extensive studies of a potential drug in the pregnant animals is mandatory. PK variations and plasma conc. of the drug in mother and foetus are recorded. Aims: Teratogenic potential Effect on gametes, uterine growth, parturition, post natal development and lactation. 1/27/2015 58

59. Animal studies: End parameters 1. Therapeutic dose = Lethal dose(LD 50)/ Effective dose (ED 50) 2.Maximum Tolerated dose (MTD) 3.Minimal lethal dose ( LD 10 ) 4. No adverse effect level (NOAEL) dose : It is the largest amount of drug which causes no detectable adverse effect with regards to morphology, physiology, growth, reproduction and life span of the organism exposed to drug. 1/27/2015 59

60. Animal studies: End parameters... NOAEL is calculated in at least three species with one being a rodent. NOAEL is extrapolated to humans. 5. Human equivalent dose (HED) = Animal NOAEL x ( W animal / W human ) 1-b where W is the weight in Kg and b is a correction factor (equal to 0.67) used to convert mg/kg to mg/m 2 1/27/2015 60

61. Animal studies: End parameters... HED (mg/kg) = Animal Dose (mg/kg) x [Animal K m  / Human K m (37)]* 6. The dose to be used for initial human studies is called “ Maximum recommended starting dose (MSRD) ” calculated as MRSD = HED/ Safety factor ( S f ) S f allows the interspecies variability in drug disposition. Default S f is 10. 1/27/2015 61

62. Good laboratory practices (GLP) It is defined by the OECD (Organization for Economic Co-operation and Development) principles as “a quality system concerned with the organizational process and the conditions under which non-clinical health and environment safety studies are planned, performed, monitored, recorded, archived and reported.” 1/27/2015 62

63. Good laboratory practices (GLP)... 1/27/2015 63

64. Limitations of pre clinical testing 1.Toxicity testing is time consuming and expensive. 2-6 years may be required to collect and analyse data on toxicity for testing in humans. 2. Large no. of animals may be needed to obtain valid pre clinical data. It raises ethical issues. 1/27/2015 64

65. Limitations of pre clinical testing... 3.Extrapolations of therapeutic index and toxicity data from humans are reasonably predictive for many but not for all toxicities. 4. For statistical reasons, rare adverse effects are unlikely to be detected in preclinical testing. 1/27/2015 65

66. Alternatives to animals: 1/27/2015 66 Micro-organisms Cell components Cell cultures Tissue cultures Tissues slices Isolated & perfused organs Computer modelling In silico ADMET Physiologically Based PK simulations

67. 5 R’s Reduce the no. of animals used to a minimum. Refine the way that experiments are carried out so that the effect on the animal is minimized and animal welfare is improved. Replace animal experiments with alternative (non-animal) techniques wherever possible. Rehabilitation When death is not the end point. Reuse Whenever and wherever possible. 1/27/2015 67

68. What after preclinical phase?? Once the preclinical trials are over, sponsors are required to submit the “ Investigational New Drug ” application. It contains information regarding: Preclinical data: PK, PD & Toxicological Manufacturing data: Composition, Manufacturing process, Stability & Shelf life. Protocol of clinical trials 1/27/2015 68

69. In Silico drug design or CADD 1/27/2015 69 Target Ident . Target Validation Lead Ident . Lead Opt Preclinical Tox Clinical trials Bioinformatics Inverse docking Protein pred. Target druggabilty Tool compound design Lib. Design Docking scoring De novo design QSAR 3 D- QSAR Struc . based opt. In silico ADMET PBPK simulations No options

70. Drug Discovery: Past & Present Criterion Last century Present era Drug development Predominantly compound centred Predominantly target centred Source of Leads Natural Compounds Natural + Synthetic compounds Time taken Many years Few years First step Finding appropriate biological response Finding appropriate drug target Safety Lesser safe More safe Screening Animal disease models Virtual Screening/ HTS 1/27/2015 70

71. Criterion Last century Present era Animal use Frequent Relatively less Ethical Considerations Less stringent More Stringent No. of Compounds explored Less More No. of Targets explored Less More Change in term Drug discovery Drug Invention 1/27/2015 71 Drug Discovery: Past & Present

72. Future Prospects 1/27/2015 72

73. Summary: 1/27/2015 73

74. 1/27/2015 74 Target identification Target validation Lead finding OLDER TECHNIQUES NEWER TECHNIQUES Lead Optimisation Preclinical Development Clinical Development VS HTS HITS LEAD COMPOUND

75. Thank you for your patience! 1/27/2015 75