Summary
preclinical drug
research
2018
Semester 1 2020-2021
1st Master Biomedical sciences
University of Antwerp
1
,Table of contents
Inhoud
Table of contents .............................................................................................................................................................................................. 2
Exam information .............................................................................................................................................................................................. 7
1 Introduction to drug R&D and the pharmaceutical industry ................................................................................................................... 8
1.1 Introduction............................................................................................................................................................................... 8
1.2 Some facts and figures ............................................................................................................................................................. 8
1.3 Development costs and revenue cycle...................................................................................................................................... 9
1.4 Timelines of drug development ............................................................................................................................................... 11
1.5 Challenges for the pharmaceutical industry ............................................................................................................................ 12
1.6 Product positioning ................................................................................................................................................................. 13
1.7 Failure rates in drug development ........................................................................................................................................... 13
1.8 Drug targets............................................................................................................................................................................ 14
1.9 Biotech-derived medicines ...................................................................................................................................................... 15
1.10 Major pharmaceutical companies ........................................................................................................................................... 15
2 Drug discovery process ...................................................................................................................................................................... 19
2.1 Therapeutic modalities............................................................................................................................................................ 19
2.1.1 Types of therapeutics .................................................................................................................................................... 19
2.2 Current therapeutics ............................................................................................................................................................... 19
2.2.1 Conventional therapeutic drugs ..................................................................................................................................... 19
2.2.2 Biopharmaceuticals....................................................................................................................................................... 20
2.3 General principles of drug discovery process .......................................................................................................................... 22
2.3.1 Introduction ................................................................................................................................................................... 22
2.3.2 Current trends in drug discovery ................................................................................................................................... 24
2.4 Selection criteria for oral drug candidate (in early discovery phase) ........................................................................................ 24
2.4.1 Chemical characteristics ............................................................................................................................................... 24
2.4.2 Pharmacological ........................................................................................................................................................... 25
2.4.3 Pharmacokinetic ........................................................................................................................................................... 25
2.4.4 toxicological .................................................................................................................................................................. 25
2.5 Choosing the project ............................................................................................................................................................... 25
3 Target identification and validation ..................................................................................................................................................... 27
3.1 Drug target interaction ............................................................................................................................................................ 27
3.2 Strategies for finding new drug targets.................................................................................................................................... 28
3.2.1 Conventional strategies to find new drug targets ........................................................................................................... 28
3.2.2 New strategies for drug target identification .................................................................................................................. 29
3.2.3 New strategies for drug target validation ....................................................................................................................... 30
4 Lead finding and lead optimization (drug screening) ........................................................................................................................... 32
4.1 Introduction............................................................................................................................................................................. 32
4.2 Definitions............................................................................................................................................................................... 33
4.2.1 Hits ............................................................................................................................................................................... 33
4.2.2 Leads ............................................................................................................................................................................ 33
4.3 The drug candidate ................................................................................................................................................................. 34
4.4 Lipinski’s rule .......................................................................................................................................................................... 34
4.5 Hit and lead identification ........................................................................................................................................................ 35
2
, 4.5.1 Random (high throughput screening of compound libraries ........................................................................................... 35
4.5.2 Knowledge-based ......................................................................................................................................................... 35
4.6 Drug screening technologies .................................................................................................................................................. 35
4.6.1 Background .................................................................................................................................................................. 35
4.7 Practical: high throughput methods and set up ....................................................................................................................... 36
4.7.1 Compound logistics....................................................................................................................................................... 36
4.7.2 Assay development....................................................................................................................................................... 36
4.7.3 Automated screening .................................................................................................................................................... 36
4.7.4 Assay validation in high throughput .............................................................................................................................. 36
4.8 Screening assay types............................................................................................................................................................ 37
4.8.1 Cell-free in vitro system................................................................................................................................................. 37
4.8.2 Cell-based in vitro system ............................................................................................................................................. 39
4.9 Lead structure requirements ................................................................................................................................................... 40
4.9.1 Suitable molecular properties ........................................................................................................................................ 40
4.9.2 Favorable pharmacodynamics ...................................................................................................................................... 40
4.9.3 Acceptable pharmacokinetics........................................................................................................................................ 40
4.9.4 Chemical optimization potential..................................................................................................................................... 40
4.9.5 Patentability .................................................................................................................................................................. 40
5 Pharmacokinetics in drug discovery.................................................................................................................................................... 41
5.1 Introduction............................................................................................................................................................................. 41
5.2 Basics of pharmacokinetics .................................................................................................................................................... 42
5.2.1 Absorption .................................................................................................................................................................... 42
5.2.2 Distribution.................................................................................................................................................................... 43
5.2.3 Metabolisation............................................................................................................................................................... 44
5.2.4 Elimination .................................................................................................................................................................... 46
5.2.5 Steady-state.................................................................................................................................................................. 47
5.2.6 One-compartment model .............................................................................................................................................. 47
5.2.7 Two-compartment model .............................................................................................................................................. 47
5.3 Drug transporters.................................................................................................................................................................... 48
5.4 Drug properties and PK characteristics ................................................................................................................................... 49
5.5 Determining the plasma concentration of your drug ................................................................................................................ 50
5.6 In silico prediction of DMPK properties ................................................................................................................................... 51
5.7 Physicochemical properties .................................................................................................................................................... 52
5.7.1 Charge state ................................................................................................................................................................. 52
5.7.2 Lipophilicity ................................................................................................................................................................... 52
5.7.3 Aqueous solubility ......................................................................................................................................................... 53
5.8 In vivo pharmacokinetics ........................................................................................................................................................ 53
5.8.1 Exploratory pharmacokinetics ....................................................................................................................................... 53
5.8.2 Whole body autoradiography ........................................................................................................................................ 53
6 Pharmacology .................................................................................................................................................................................... 54
6.1 Introduction............................................................................................................................................................................. 54
6.2 Screening for selectivity (in vitro) ............................................................................................................................................ 56
6.2.1 Saturation assay ........................................................................................................................................................... 56
6.2.2 Displacement assay ...................................................................................................................................................... 57
6.3 Pharmacological profiling ........................................................................................................................................................ 57
6.3.1 In vitro profiling on isolated tissues................................................................................................................................ 57
3
, 6.3.2 In vivo pharmacological profiling ................................................................................................................................... 58
7 Patenting in drug discovery ................................................................................................................................................................ 59
7.1 introduction ............................................................................................................................................................................. 59
8 Management of drug discovery........................................................................................................................................................... 59
8.1 Company strategies ................................................................................................................................................................ 59
9 Drug development .............................................................................................................................................................................. 61
9.1 Introduction............................................................................................................................................................................. 61
9.2 Operational plan ..................................................................................................................................................................... 61
10 Assessing drug safety ........................................................................................................................................................................ 64
10.1 Introduction............................................................................................................................................................................. 64
10.2 General overview of toxicity evaluation ................................................................................................................................... 65
10.3 Toxicity measures................................................................................................................................................................... 66
10.4 The ICHM3 guideline .............................................................................................................................................................. 67
10.5 General timing toxicity evaluation ........................................................................................................................................... 68
10.6 Types of adverse drug effects ................................................................................................................................................. 69
10.7 Animals used in tox studies .................................................................................................................................................... 70
10.7.1 Rodents ................................................................................................................................................................... 70
10.7.2 Non rodents ............................................................................................................................................................. 70
10.8 General toxicity studies........................................................................................................................................................... 71
10.8.1 Single-dose (acute toxicity) ...................................................................................................................................... 71
10.8.2 Repeated dose toxicity ............................................................................................................................................. 73
11 Safety pharmacology .......................................................................................................................................................................... 77
11.1 Current guidelines .................................................................................................................................................................. 77
11.2 Central nervous system .......................................................................................................................................................... 77
11.2.1 Irwin’s test ................................................................................................................................................................ 77
11.2.2 Accelerating rotarod test .......................................................................................................................................... 77
11.2.3 Tail flick latency test ................................................................................................................................................. 78
11.2.4 Morris swim maze .................................................................................................................................................... 78
11.3 Respiratory system ................................................................................................................................................................. 78
11.3.1 Restrained/whole body plethysmography ................................................................................................................. 78
11.4 Cardiovascular system ........................................................................................................................................................... 78
12 Genotoxicity ....................................................................................................................................................................................... 79
12.1 General outlining and rationale ............................................................................................................................................... 79
12.2 Classification of mutagens ...................................................................................................................................................... 80
12.3 Regulatory tests...................................................................................................................................................................... 81
12.3.1 Ames test ................................................................................................................................................................. 81
12.3.2 Mouse lymphoma test .............................................................................................................................................. 81
12.3.3 Chromosome aberration test .................................................................................................................................... 82
12.3.4 Micronucleus test ..................................................................................................................................................... 82
12.4 Additional tests for genotoxicity............................................................................................................................................... 83
12.4.1 Unscheduled DNA synthesis .................................................................................................................................... 83
12.4.2 Comet-test ............................................................................................................................................................... 83
12.5 Genotoxicity screening tests ................................................................................................................................................... 84
12.5.1 DEREK .................................................................................................................................................................... 84
12.5.2 Vitotox ...................................................................................................................................................................... 84
12.5.3 Ames-II .................................................................................................................................................................... 84
4
,13 Reproduction toxicity .......................................................................................................................................................................... 85
13.1 Introduction............................................................................................................................................................................. 85
13.2 General outlining and rationale ............................................................................................................................................... 86
13.2.1 Embryofetal development toxicity study.................................................................................................................... 88
13.2.2 Fertility study ............................................................................................................................................................ 89
13.2.3 Pre-and postnatal development study ...................................................................................................................... 90
14 Carcinogenicity ................................................................................................................................................................................... 91
14.1 Introduction............................................................................................................................................................................. 91
14.2 Standard study protocol .......................................................................................................................................................... 91
14.3 Carcinogenesis ....................................................................................................................................................................... 91
15 Local tolerance ................................................................................................................................................................................... 92
15.1 Introduction............................................................................................................................................................................. 92
15.2 Irritation and corrosion testing ................................................................................................................................................. 92
15.2.1 Draize rabbit skin test ............................................................................................................................................... 92
15.2.2 Alternative models for skin irritation and corrosion.................................................................................................... 93
15.2.3 Eye irritation ............................................................................................................................................................. 93
15.3 Skin sensitization testing ........................................................................................................................................................ 94
15.3.1 In vivo magnussen and kligman (GMT) test.............................................................................................................. 94
15.3.2 In vivo local lymph node assay (LLNA) ..................................................................................................................... 94
15.4 Phototoxicity testing ................................................................................................................................................................ 94
16 Immunotoxicity ................................................................................................................................................................................... 95
17 Paediatric drug development .............................................................................................................................................................. 96
17.1 Introduction............................................................................................................................................................................. 96
17.2 Pharmacokinetic differences in children .................................................................................................................................. 97
17.2.1 Absorption................................................................................................................................................................ 97
17.2.2 Distribution ............................................................................................................................................................... 98
17.2.3 Metabolism .............................................................................................................................................................. 98
17.2.4 Elimination ............................................................................................................................................................... 98
17.3 Importance of knowledge of ontogeny .................................................................................................................................... 98
17.4 Juvenile animal studies ........................................................................................................................................................... 99
18 Pharmaceutical development ........................................................................................................................................................... 101
18.1 Introduction........................................................................................................................................................................... 101
18.2 Dosage forms ....................................................................................................................................................................... 101
18.2.1 Solutions ................................................................................................................................................................ 101
18.2.2 Suspensions .......................................................................................................................................................... 102
18.2.3 Emulsions .............................................................................................................................................................. 102
18.2.4 External preparations ............................................................................................................................................. 103
18.3 Preformulation studies .......................................................................................................................................................... 103
18.4 Solubility ............................................................................................................................................................................... 103
18.5 Stability & particle size/morphology ...................................................................................................................................... 104
18.6 Micelles ................................................................................................................................................................................ 105
18.7 Liposomes ............................................................................................................................................................................ 105
18.8 Cyclodextrins ........................................................................................................................................................................ 105
18.9 OROS technology (osmotic controlled release and oral delivery system) .............................................................................. 106
18.10 Nasal delivery .................................................................................................................................................................. 106
19 Biopharmaceuticals .......................................................................................................................................................................... 107
5
, 19.1 Introduction........................................................................................................................................................................... 107
19.2 Earlier biopharmaceuticals.................................................................................................................................................... 109
19.3 Differences between pharmaceuticals and biopharmaceuticals ............................................................................................ 109
19.3.1 Manufacturing ........................................................................................................................................................ 110
19.3.2 . Pharmacokinetics ................................................................................................................................................. 113
19.3.3 Regulatory ............................................................................................................................................................. 114
19.3.4 . Immunogenicity .................................................................................................................................................... 115
19.3.5 . Delivery ................................................................................................................................................................ 115
19.4 Vaccines............................................................................................................................................................................... 116
19.4.1 Vaccine production................................................................................................................................................. 116
19.5 Antibodies............................................................................................................................................................................. 119
19.5.1 . Hybridoma technology.......................................................................................................................................... 119
19.5.2 .Antibodies structure .............................................................................................................................................. 119
19.5.3 .Antibody classes ................................................................................................................................................... 120
19.5.4 Mechanism of action .............................................................................................................................................. 120
19.5.5 IgG subclasses ...................................................................................................................................................... 121
19.5.6 Nomenclature......................................................................................................................................................... 123
19.5.7 . Engineering and design........................................................................................................................................ 123
19.5.8 .Alternative formats ................................................................................................................................................ 126
20 Examenvragen ................................................................................................................................................................................. 128
6
,Exam information
Only the slides are the course material and must be learned for the exam. This
summary is based on these slides. There is a book meant as background
information, but it’s not expected that you have to learn this book (Drug Discovery
& Development by Raymead G. Hill). The course is taught by professor Maes,
professor Delputte and Professor Van Cruchten.
The exam will only be in written format and you will get a scientific insert of a
marketed drug. The subjects on this insert have to be discussed and explained on
the exam, this is the MAIN focus.
7
,1 Introduction to drug R&D and the
pharmaceutical industry
1.1 Introduction
Creation of drugs is based on the simple idea that one starts from a molecule
(simple or complex), which gets formulated into a drug in a certain form (e.g. tablet,
injection, suspension). The formulation of your drug will be important because the
formulation will drive the absorption and distribution. So, the drug must be in
the right form for max efficacy.
Chemicals = chemicals are small molecules with a certain molecular structure
(e.g. paracetamol, aspirin, fluoxetine, omeprazole). These small molecule drugs
were the ones that were the most popular.
Natural products = Complex molecules (vinblastine, paclitaxel, artemether) that
originate from for example plants.
Biologicals = Biologicals are large (sometimes complex) molecules like insulin,
interferon, or EPO. (see chapter X)
1.2 Some facts and figures
The biopharma industry is a costly industry. The price per employee tops that of
other industries (eg. semiconductors, computers & electronics).
On baby Pia and gene therapy
• Baby Pia was born with SMA disease and needed gene therapy (single
treatment) to survive.
• Novartis had a drug Zolgensma, but it cost 2 million dollars for a single
treatment.
• Of course, the parents couldn’t afford this, so a crowdfunding was started.
• The minister of health would not reimburse this drug and thus her and
Novartis were in the fireline of the public.
• Gene therapy is very expensive and directed to a small population.
• Novartis explained why this was so expensive: the drug takes into account
all the money they spent on the creation.
8
, The left figure explains that there is
a change over time in what pharma
companies gain. If a company had
a blockbuster years ago, they
would profit off of it for years. This
is not the case anymore.
While the sales have gone up, the
R&D costs have also raised as
well, all while the number of the
approved drugs by the FDA goes
down.
The number of approved drugs has gone down because of a higher attrition
rate in pharma companies. E.g. oncology or the central nervous system (CNS)
are tricky disciplines to find a drug that targets one specific receptor or a cell
population, and there will be a lot of side effects. So there is more failure of
compounds.
Blockbuster drugs = Popular drugs that have sales of 1 billion dollars of
revenue per year. These are drugs that generate sufficient sales to drive new
research for the companies.
An example of a blockbuster drug is humira, a mAb for juvenile arthritis. Such
biologicals gain a high revenue. This is why large pharmaceutical companies
are buying up smaller companies that focus on biologicals because they expect
it to give the most revenue later on. In this way, they can also extend their drug
portfolio.
1.3 Development costs and revenue cycle
Depending on the therapeutic area, a drug will cost 1-2 billion dollars to bring to
the market. The cost to bring a new drug to the market shows a continuous
increase over the years.
People are getting older in the 21st century, so the older group forms a big target
to sell drugs to. Diseases linked with age (Alzheimer, Parkinson, osteoporosis,
prostate cancer, arthritis) are popular research domains to discover drugs in. The
sales of drugs against these age-related diseases has also continuously
grown.
9
, Because a pharmaceutical company is still a business, it will invest in drugs that
are profitable. Some indications are just not profitable, like neglected tropical
diseases, orphan diseases and pediatric diseases. (exam question)
Neglected tropical diseases like leishmaniasis are not profitable. A company will
not invest, unless it is for an incentive (subsidies or a good public image). Orphan
diseases are diseases that only have a small population of patients, and this is
also not something a company will invest in. Pediatric diseases are a small
population and in clinical trials it is very dangerous if something goes wrong. So
regulators have actually enforce the companies to think about pediatric
diseases; include a preclinical plan in drug discovery. If you don’t have an
approved preclinical plan, then they don’t get approval for the drug!
In this graph you see that the costs for the preclinical development are okay in
comparison to clinical development. The aim at the end is that you can
compensate the cost by the leftover years that you bring the drug to the market
while you have patent protection. The steep decrease at the end means that the
patent is expired. A patent expires after 20 years, unless you get an extension
(max 5 years). When your patent expires, your competitors will make use of this
and create generics. (Exam question)
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