Lecture 3– 22 March 2016 - Jagiellonian Universitybiotka.mol.uj.edu.pl/zbm/handouts/2016/JD/03_Medical_biotechnology... · Mutiple cell types – both local and migrating are taking

Medical biotechnology:from chemical to biotechnological laboratory

Prof. Jozef Dulak

Email: [email protected]: www.biotka.mol.uj.edu.pl/zbm

Lecture 3– 22 March 2016

Aims of medical biotechnology

1.Prevention of diseases

2.Diagnostic of diseases

3.Treatment of diseases

Drug discovery

1. By chance (serendipity)

2. By rational design

Medical biotechnology in drug discovery

1. Classical drugs – small molecular compounds

1.1. Traditional way of discovery – eg. aspirin

- antibiotics

1.2. Screening of chemical libraries - current testing employs numerous biotechnological methods

2. Biotechnological drugs – generated using the biotechnological models

2.1. Monoclonal antibodies

2.2. Genetic engineering - recombinant proteins – in vitro production

- cloning- vaccine

- recombinant proteins – in vivo production

Drug developments – stages

It typically takes 10 to 15 years and an average of more than $800 million (includingthe cost of failures) to develop a new therapy. The process is rigorous and conductedin multiple stages:

* Generating and validating a new hypothesis; by modulating the function of achosen protein target, a related disease pathology could be symptomatically relieved,modified or prevented.

* Identifying chemical ‘lead’ compounds that modulate the function of the chosentarget and have the general physico-chemical and toxicological properties needed forthem to become drugs.

* Chemically optimising a chosen series of lead compounds to be selectively uniquefor the target and to have the appropriate pharmacokinetic, metabolic and safetyproperties.

At the completion of this stage a preferred compound is selected for pre-clinical safetytesting using animal models.

Stages of drug discovery

1. In silico analysis

2. Biochemical tests in vitro (on isolated enzymes)

3. Biochemical tests in vitro (in cell cultures)

4. Research on animals: pharmacokinetics and effectiveness

5. Clinical trials – volunteers and patients: several phases

7

The process of drug discovery

Dr Steve Carney, [email protected] Editor, Drug Discovery Today

Drug development and drug testing

1. Searching for the compound- screening – chemical libraries (often: HTS) - combinatorial design- chemical synthesis- testing on cell cultre- further modification to improve effectiveness

High throughput screening (HTS)

Source: Wikipedia

Medical biotechnology for drug discovery

Testing drugs

1. Cell cultures

2. Animal models

3. Human subjects – clinical trials

Stages of drug discovery

1. In silico analysis – bioinformatic design of chemical compounds- Testing their potential effectiveness and toxicity

2. Synthesis of hundreds or thousands of new compounds

2. Testing of compunds on eg. tumor cells – determination of toxicity, effect on proliferation etc.

Pancreatic cancer cells cultured in the presence of increasing concentration of tested compound

Control cells

The process of drug discovery and development

- Once a potential drug has been identified, animal testing is usually the first step,typically in two or more species, since drug effects vary across species. Many of thesestudies are ADME:

* absorption,

* distribution,

* metabolism (including toxicity/activity of metabolites),

* excretion.

- Additionally, animal models of particular diseases are used to test for efficacysignals that can guide further refinement of a drug or clinical testing.

- A drug that passes animal safety studies may move into human testing followingthe submission of an investigational new drug (IND) application to the FDA. Mosttrials of new products may begin 30 days after the agency receives the IND.

- Although animal efficacy results are important to drug development, they may beused to support FDA approval for human use only for biodefense products.Biodefense products can be tested for safety in humans, but not for efficacy.

Faculty of Biochemistry, Biophysics and BiotechnologyAnimal facility - SPF conditions

Micro-ultrasound in preclinical imaging

specifically developed for small animal research

can image tissue of around 3 cm below the skin

with frequencies ranging from 15 MHz to 80 MHz (clinical ultrasound systems range from 3-15 MHz)

high resolution - up to 30 µm (visualization of tiny vasculature in cancer angiogenesis); the higher the frequency the higher the imaging resolution; however, this is at the expense of lower penetration

this resolution can be further increased to 3-5 µm with the injection of microbubble contrast agents(visualization of capillaries)

Vevo 2100 high-frequencymicro-ultrasound system from VisualSonics

IVIS® Lumina from Caliper Life Sciences

In vivo luminescence measurements

Analysis of cells after transplantation (viability, proliferation,migration)

Analysis of gene expression (conditional, stable, tissue-specific)

Evaluation of gene transfer efficiency (overexpression or quiescence)

In vivo investigation of protein interactions, their stability and function

Monitoring of the disease progression or effectiveness of therapy (for eg.tumor growth and metastasis, inflammation, infections)

Bioluminescence in preclinical imaging

Anti-science movement: against animal research

„Alternative” methods for animal research

1. „In silico” analysis

2. Cells culture in vitro

3. Incubation of fragments of human organs, – eg. skin

4. „organs on a chip”

http://alsn.mda.org/article/brain‐computer‐interface‐reads‐brainwaves

http://www.telegraph.co.uk/news/science/science-news/11364896/Brain-food-6-snacks-that-are-good-for-the-mind.html

Can huma brain be replaced by computermodels?

„it is sufficient to test the new compounds only in vitro…”

?....

Skin cells - keratinocytes

http://www.genoskin.com/en/ex-vivo-human-skin/nativeskin-model/

Culturing of keratinocytes and fragments of skin

Nature, 26th March 2015

Can we replace animal research ?

Organ on a chip

Źródło: Wikipedia i inne

Organs on the chips

Ist this lung. liver, heart?

Can we model myocardial infarction… ?

Experimental myocardial infarctionShortly before

ligation

Shortly afterligation

(pallor and regional wallmotion abnormality)

Day 7 post‐MI heart in longitudinal axis(Vevo2100 high frequency ultrasound)

Blood plasma

Swirski et al., Science Jul 31, 2009

Mutiple cell types – both local and migrating are taking part in healing of the heart after myocadial infarction

Wound 3 days after injury

Wound 5 days after the injury

Wound healing comprises not only theskin cells, but also the blood vesselformation, influx of cells from the bloodand mobilisation of cells from the bonemarrow

http://bme240.eng.uci.edu/students/07s/ngunn/wound_healing.html

Wound healing engaes the whole organism, not only the skin

Newcapillaryformationinresponsetowounding

„Human” on a chip

Wikipedia

Would we be ever able to reconstructthe complexity of animal/human organism?

No doubts there are differences between other animals and humans – but there arecountless similarities! The latter justfiy to test the unknown compounds firston experimental animals – no doubt, this is more ethical (is ethical) than testingdirectly on humans

Due to the existing differences, the compounds have to be tested not on one, but on a few animal species (particularly, when toxicity and potential teratogenecity isdetermined)

And after testing on animals, the drugs are first tested on the small number volunteersto determine the safe dose and detect any serious side effects, which might appear,even if animal research did not reveal them (NOTE: the differences in drug responseexist also between humans!)

No doubts animal research cannot reveal all potential dangers relatedto the new compound.

But it is better to eliminate the compounds, whis are toxic for experimentalanimals, as there is highly probable they will be also toxic for human.

Necessity of animal research

• In short the answer is yes• In fact there has been a significant reduction in animal

usage in the Pharma industry over the last few years as a result of the introduction of new technology and approaches

• More than 82% of the animals used for experimentation are rodents. Only 4% of experiments are performed in mammals other than rodents.

• There has been a fall of 16% in animal usage since 1987• The UK is probably the most regulated country in the

world with respect to animal experimentation• There are good reasons why companies want to

reduce animal usage, not least financial

Can we reduce the number of animals used in experiments?


The strongest evidence for therapeutic interventions is provided bysystematic review of randomized, double-blind, placebo-controlled trialsinvolving a homogeneous patient population and medical condition.

Clinical trials

Randomized: Each study subject is randomly assigned to receive eitherthe study treatment or a placebo.

Double-blind: The subjects involved in the study and the researchersdo not know which study treatment is being given.

Placebo-controlled: The use of a placebo (fake treatment) allowsthe researchers to isolate the effect of the study treatment.

Phases of clinical trials

disease

Some Phase II and most Phase III drug trials are randomized, double-blindand placebo-controlled

- In many trials (especially those for diseases lacking effective existingtreatment), one group of patients (or arm of the study) receives the drugbeing tested, while another group (the control group) receives a placebo thatlooks just like the drug and is administered the same way. Patients arerandomized (randomly assigned) to one or the other arm.

* A single-blind trial - A trial in which the healthcare provider knowswhether the patient is receiving the placebo or active drug, but the patientdoes not.

* A double-blind trial - A trial in which neither the patient nor thehealthcare provider knows whether the drug or placebo is beingadministered.

Especially for trials measuring efficacy, double-blinded, randomizedtrials are considered the gold standard.

Clinical trials

Drug approval process- If a therapy succeeds in clinical trials, the next step is applying for approval withthe FDA by filing a new drug application (NDA). This application can run hundredsor thousands of pages and includes details on the product’s structure,manufacturing, lab testing and clinical trials.

- FDA has a goal of acting on a priority review products (those addressing unmetmedical needs) by six months after the application receipt. For a standard reviewproduct, the agency’s goal is a 10-month review.

- In weighing an NDA, particularly for a novel product, the FDA may seek theguidance of one of its independent advisory committees. Each committee has 10-15members and includes experts and representatives of the public. The committeeshost public meetings, often attracting media coverage, at which the pros and cons ofthe products in question are presented and debated, culminating with arecommendation either for or against approval. Advisory committeerecommendations are non-binding, however. The final regulatory decision restswith the agency.

The drug development pyramid

www.iconplc.com

www.sciencedirect.com

70%o.k.

33%o.k.

25-30%o.k.

(in total: ~6%)

The process of drug discovery and development (1)

• Although these figures are a little out of date* (they are probably worse now with the introduction of HTS (high throughput screening), it gives an idea of how wasteful the process is.

• For every 30,000 compounds synthesized– 2000 (6.7%) enter preclinical development– 200 (0.67%) enter phase 1 trials– 40 (0.13%) enter phase 2 clinical trials– 12 (0.04%) enter phase 3 clinical trials– 8 (0.027%) are approved– 1 (0.003%) makes a satisfactory ROI (simply: income…)

*Christine A. Shillingford and Colin W. Vose Effective decision-making: progressing compounds through clinical development DDT Vol. 6, No. 18 September 2001


The process of drug discovery and development (2)

- The design, or protocol, of clinical trials varies tremendously, depending on thenature of the product, the patient population, and efficacy of existing treatments.

- Some drugs, for very rare and devastating diseases, have been approved afterstudies in only a handful of patients; others, often products for milderconditions and/or for which therapies are already available, must be tested inthousands of patients to win approval.

time:1.7-fold costs:

19.6-fold

The process of drug discovery and development

Costs of drugs development (1)

• This is quite a hard question to answer, but it is estimated that it cost on average $800,000,000 to develop a new drug

• Although not confirmed, estimates for development of a new drug are now in the order of $0.5 - 2 billion**

*DiMasi JA, Hansen RW, Grabowski HG. J Health Econ. 2003 22(2):151-85

**Adams C, Brantner V (2006). Health Aff (Millwood) 25 (2): 420–8

Dr Steve Carney, [email protected] Managing Editor, Drug Discovery Today

• Broken down, the cost broadly works out at – Around $335 million in the preclinical phases

– Around $467 million in the clinical trial phases

– Around $100 million in Post approval costs


Costs of drugs development (2)

• Attrition at late clinical trial phase is very expensive and can be disastrous for smaller companies

• It is important to point out that in the last few years, some compounds have been pulled out late because it was thought that they would not make a return on investment (ROI)….

• Clearly just getting a drug on the market is not a case of the goose laying the golden egg


Further costs of drugs development (3)

Modified from Human Molecular Genetics 2 2nd ed. New York and London: Garland Science; c1999.

History of drug discovery

examples

-When the lack of correct clinical trials resulted in tragic side effects

Homework: read the story of thalidomide.

- at the exam there will be questions concerning the thalidomide!


Aspirin

Acetylsalicylic acid

-A derivative of salicylic acid, originally isolated from willow bark (Lat. Salix)- 1828 – named salicin

-Felix Hoffmann – obtained acetyl salicylic acid on 10 August 1897 and offered the concept to Bayer company – which marketed Aspirinin 1899


Aspirin discovery – another version of history

W. Sneader, British Medical Journal 2000: 321: 1591-4

Modified from Human Molecular Genetics 2 2nd ed. New York and London: Garland Science; c1999. J.Balasubramaniam

www.uninet.edu

Mechanisms of action of anti-inflammatory drugs


Mechanisms of aspirin action – inhibition of cyclooxygenase-1 & -2

N. Chiang & CN Serhan, Discovery Medicine, 2009


Mechanisms of aspirin action – inhibition of cyclooxygenase- 1 & 2

N. Chiang & CN Serhan, Discovery Medicine, 2009


Aspirin

1. Analgesic to relieve minor aches and pains2. Antipyretic to reduce fever 3. Anti –inflammatory

Other activities/indications

1. anti-platelet – inhibits the production of thromboxane 2. May be effective at preventing certain types of cancer (lung, colon & breast)3. some claims on prevention against Alzheimer disease


Painkillers – non steroidal antiinflammatory drugs

Paracetamol – acetaminophenN-acetyl-para-aminophenol-APAP

Paracetamol – is not classified as NSAID – has little anti-inflammatory activity

- blocks COX-2 mostly in the central nervous system

Released to the market in 1956

Known in USA as Tylenol

In 2001, NSAIDs accounted for 70,000,000 prescriptions and 30 billion over-the-counter doses sold annually in the United States

Do not give your get paracetamol!


STATINS – inhibitors of HMG-CoA reductase –3-hydroxy-3-methyloglytarylocoenzyme A reductase

• converts HMG-CoA to mevalonate• one of the enzyme in cholesterol synthesis pathway

Statins


Inhibition of HMG-CoA reductase by statins

STATINS


Akira Endo and Masao Kuroda (Japan) started research on inhibitorsof HMG-CoA reductase in 1971 - certain microorganisms may produceinhibitors of this enzyme to defend themselves against other organisms, as mevalonate is a precursor of many substances required by organisms for the maintenance of their cell wall (ergosterol) or cytoskeleton (isoprenoids)

• mevastatin - first agent isolated from Penicillium citrinum• next lovastatin, the first commercially marketed statin, from the Aspergillus terreus have been obtained (approved by the FDA - August 1987). • mevastatin is the source for production of pravastatin

Statins history


Angiogenesis Anti-inflammatory

Immunomodulatory

Anti-oxidant

Vascular cytoprotection

Anti-thrombotic

Endothelial function

Plaque stability

STATINS

Pleiotropic effects of statins

Side effects of statins actionRhabdomyolysis

◦Muscle pain, especially in the shoulders, thighs or lower back.◦Muscle weakness or trouble moving arms or legs.◦Abdominal pain.◦Nausea or vomiting.◦Fever, rapid heart rate.◦Confusion, dehydration, fever, or lack of consciousness.

the risk of developing rhabdomyolysis from statin therapy is very low, around 1.5 for each 100,000 people taking statins. Rhabdomyolysis ormilder forms of muscle inflammation from statins can be diagnosedwith a blood test measuring levels of the enzyme creatinine kinase.

Cerivastatin was voluntarily withdrawn from the market worldwide in 2001, due to reports of fatal rhabdomyolysis.

During postmarketing surveillance, 52 deaths were reported in patients using cerivastatin, mainly from rhabdomyolysis and its resultant renal failure

Risks were higher in patients using fibrates, mainly gemfibrozil (Lopid), and in patients using the highest (0.8 mg/day) dose of cerivastatin.

The frequency of deadly cases of rhabdomyolysis with cerivastatin was 16 to 80 times higher than with other statinsAnother 385 nonfatal cases of rhabdomyolysis were reported.

A case of cerivastatin


Drug discovery and pharmaceutical industry

Blockbusters

- The blockbuster drug is defined as one with peak annual global sales exceeding $1 billion (market definition):


Blockbuster business model-Pharmaceutical industry is dominated by the blockbuster model, in whichcompanies built their operations around a few products.

Top 10 blockbuster drugs (of ~120 on the market) - $64 bln in 2007.

* Lipidor (Atorvastatin, Pfizer) - $13.7 (2007) - mega blockbuster (more than$10 billion)

* Plavix (Clopidogrel, Sanofi-Aventis) - $8.1 (2007)* Enbrel (Etanercept, Amgen) - $5.5 (2007)

-From medical point of view blockbuster drug is:

* single compound effective in most or all patients, who have particularcondition

* labeled for use by the general population* prescribed for a chronic condition (thus, providing long-term sale)


-In fact the blockbuster drug can be effective e.g. in 40-60% of the generalpopulation. As long as there is no adverse effect in the remainingpatients, physicians are able to prescribe them on the trial-and-errorbasis.

- Clinical trials must be large enough (that means very expensive), toshow clear efficacy, even if large proportion of a general population doesnot respond to the drug.

Blockbuster business model


Blockbusters drugs in 2011

2. Plavix (clopidogrel) - $ 6.8 billion 3. Nexium (esomeprazole) -$ 6.2 billion4. Abilify (aripiprazole) - $ 5.2 billion 5. Advair (fluticazol/salmeterol) –

$ 4.6 billion 6. Seroquel (quetiapine) - $4.6 billion7. Singulair (montelukast) - $ 4.6 billion 8. Crestor (rosuvastatin) - $ 4.4. billion 9 . Cymbalta (duloxetine) - $ 3.7 billion 10. Humira (adalimumab) - $3.5 billion

Vioxx (Rofecoxib, Merck) - blockbuster drug prescribed for artretic pain,withdrown from the market because of increased risk of stroke andcardiovascular events in some patients.

Cerivastatin (Baycol, Lipobay, Bayer) - blockbuster drug prescribed forlowering cholesterol, withdrawn from the marked because of increased riskof muscle breakdown and kidney failure.

Rezulin (Troglitazone, Warner-Lambert) - blockbuster drug prescribed forinsulin resistance, withdrawn from the market because of increased risk ofhepatotoxicity.

Blockbusters withdrawn from the marketWithdrawning caused by side-effects:

On November 23, 2000, the results of the Vioxx Gastrointestinal OutcomesResearch study, known as VIGOR, were published in the NEJM.

This randomized, controlled trial showed that VIOXX, an inhibitor ofcyclooxygenase-2 that had been marketed since May 1999, was associatedwith fewer gastrointestinal complications than naproxen, a standardnonsteroidal antiinflammatory drug.

Unexpectedly, the VIGOR study also showed that the patients who weregiven rofecoxib had four times as many myocardial infarctions as those whowere given naproxen.

This finding of a significant increase in the risk of myocardial infarction wasan early signal of a potentially serious safety problem with rofecoxib.Nonetheless, sales remained robust. By the time of rofecoxib’s withdrawalfrom the market in September 2004, after a placebo-controlled studyconfirmed its cardiovascular risk, more than 100 million prescriptions hadbeen filled in the United States.

Vioxx

VIGOR trial

Cumulative incidence of the primary end point of a confirmed uppergastrointestinal event among all randomized patients

Risk of myocardial infarction(MI) or stroke associated with Rofecoxib use

Vioxx

Merck, the manufacturer of Vioxx, has an excellent reputation within thedrug industry and supports many products, such as vaccines, that aremedically essential but not very profitable.

Vioxx

The company funded VIGOR and appropriatelysought to publish its results in a prestigiousmedical journal. In advance of the committee’shearing, Merck cooperated voluntarily withrequest for information, providing more than20,000 pages of internal company documents.

Merck also voluntarily sent a senior executive totestify at the hearing and answer the committee’squestions.

On February 7, 2001, the Arthritis Drugs Advisory Committee of the FDA met todiscuss the VIGOR study. At this meeting, Merck argued that the significant increasein the rate of myocardial infarction was explained by a protective effect of naproxen,not by any inherent risk posed by its drug. However, the advisory committee votedunanimously that physicians should be made aware of VIGOR’s cardiovascularresults.

The next day, Merck sent a bulletin to its representatives: "DO NOT INITIATEDISCUSSIONS ON THE FDA ARTHRITIS ADVISORY COMMITTEE...OR THERESULTS OF THE ...VIGOR STUDY." It advised that:

- if a physician inquired about VIGOR, the sales representative should indicate thatthe study showed a gastrointestinal benefit and then say, “I cannot discuss the studywith you.”

- representatives should show those doctors who asked whether Vioxx causedmyocardial infarction a “The Cardiovascular Card” prepared by Merck’s marketingdepartment, which provided a misleading picture and did not include any data fromthe VIGOR study. Instead, it presented a pooled analysis of preapproval studies, inmost of which low doses of Vioxx were used for a short time.

Vioxx

Persistent physicians who sought additional information about the cardiovasculareffects of Vioxx were directed to send inquiries to the company’s headquarters.Merck’s response to these physicians highlighted the information from theCardiovascular Card.

There was a disparity between the evidence-based perspective provided byscientific journals and expert committees, on the one hand, and the sales pitchused by the company’s field staff, on the other.

The company instructed its sales representatives, for example, to provide only certainapproved study results to doctors. Approved scientific studies were defined as thosethat provide “solid evidence as to why [doctors] should prescribe the products fortheir appropriate patients.” By contrast, those studies that raised safety questionsabout drugs were considered background studies. Distributing the results of abackground study was “a clear violation of Company Policy.”

The company also trained its representatives to identify speakers for educationalevents who were “opinion leaders” who could provide “favorable” views of thecompany’s products to other doctors.

Vioxx

More on drug discovery and personalized medicine during the next lectures

Worth to read…


Next lecture – 5th April

Documents

Lecture 3– 22 March 2016 - Jagiellonian Universitybiotka.mol.uj.edu.pl/zbm/handouts/2016/JD/03_Medical_biotechnology... · Mutiple cell types – both local and migrating are taking