WHAT ARE THE MOST COMMON ANIMAL MODELS IN DRUG DISCOVERY?

“Animal research and testing has played a part in almost every medical breakthrough of the last century. It has saved hundreds of millions of lives worldwide...” – Former UK Home Office minister Joan Ryan.

Albert Sabin, who developed the Polio vaccine said, "Without animal research, polio would still be claiming thousands of lives each year.”

Why animal models?

·         If you are going to study a human disease you can’t directly perform the initial work in humans; you have to develop a model. Some models may be in vitro – literally, in glass tubes – but as you learn more and more, you must eventually test ideas in vivo.  That means you have to have a way of producing the disease that allows you to study it.  As mentioned earlier, humans cannot be used for this purpose for ethical reasons, therefore – living animals are used as models.

·         New medicines require testing because researchers must measure both the beneficial and the harmful effects of a compound on a whole organism. A medicine is initially tested in vitro using tissues and isolated organs, but legally and ethically it must also be tested in a suitable animal model before clinical trials in humans can take place.  Therefore, a new drug or a surgical technique is tested in animals to make sure that it is safe and effective.

·         Chimpanzees share more that 99% of DNA with humans and mice share more than 98% DNA with humans.  There is a 67% similarity between the DNA of humans and earthworms.

[Please understand the difference between ‘Shared genes’ and ‘Working DNA’ – You end-up seeing different % similarity values, but do read it carefully if the discussion is done based on ‘Shared genes’ or ‘Working DNA’]

Are animals as model is proper in research? 

Ø  Although humans and animals may look different, at a physiological and anatomical level they are remarkably similar. Animals, from mice to monkeys, have the same organs (heart, lungs, brain etc.) and organ systems (respiratory, cardiovascular, nervous systems etc.) which perform the same functions in pretty much the same way.

Ø  Many basic cell processes are the same in all animals, and the bodies of animals are like humans in the way that they perform many vital functions such as breathing, digestion, movement, sight, hearing and reproduction.

Ø  In particular, mammals are essential to researchers because they are the closest to us in evolutionary terms. For example, many diseases that affect human beings also affect other mammals, but they do not occur in insects, plants, or bacteria.

Ø  Humans and animals share hundreds of illnesses, and consequently animals can act as models for the study of human illness.

o   For example, rabbits suffer from atherosclerosis (hardening of the arteries), as well as diseases such as emphysema, and birth defects such as spina bifida.

o   Dogs suffer from cancer, diabetes, cataracts, ulcers and bleeding disorders such as haemophilia, which make them natural candidates for research into these disorders.

o   Cats suffer from some of the same visual impairments as humans.

From such models we learn how disease affects the body, how the immune system responds, who will be affected, and more.

What are the advantages of animal models?

v  Animals also offer experimental models that would be impossible to replicate using human subjects.

v  Animals have a shorter life cycle than humans and as a result, they can be studied throughout their whole life span or across several generations. 

v  In addition, scientists can easily control the environment around animals (diet, temperature, lighting), which would be difficult to do with humans.

v  Animals can be fed identical and closely monitored diets. As with inbred mice, members of some animal species are genetically identical, enabling researchers to compare different procedures on identical animals.

v  Animals such as rats, mice, birds, rabbits, guinea pigs, sheep, fish, frogs, pigs, birds, dogs, cats, primates, among others are used in biomedical research.

v  Approximately 95% of these animals used in biomedical research are rats and mice specifically bred for research and 4.25% of these animals include rabbits, guinea pigs, sheep, fish, frogs, insects, and other species.  Most importantly, only 0.75% of the animals in research are cats, dogs, and primates.

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RAT

Lifespan: Brown rat: 2 years, Black rat: 12 months

Gestation period: Brown rat: 21 – 24 days, Polynesian rat: 21 – 24 days

Mass: Brown rat: 230 g, Black rat: 110 – 340 g

MOUSE

Lifespan: African pygmy mouse: 2 years

Gestation period: House mouse: 20 days, African pygmy mouse: 20 days

Mass: House mouse: 19 g, African pygmy mouse: 3 – 12 g

                                                                                                   

GUINEA PIG

Lifespan: 4 – 8 years

Gestation period: 59 – 72 days (Adult)

Mass: 0.7 – 1.2 kg (Adult)

Length: 20 – 25 cm (Adult)

                                                               

FERRET

Lifespan: 6 – 10 years

Gestation period: 42 days; Mass: 0.7 – 2 kg; Length: 38 cm (Without Tail)

Gestation period: The period from fertilization of the egg (ovum) to the birth of the child.

ü  For example, 28 days study of a drug on an animal model has to be conducted – which animal model should we choose if we are given a choice between a mouse and a rat?           Mice / mouse (~20 g wt of each mouse) and (rat - ~120 g wt of each rat).                                   Each day blood sample from mouse might not be a good idea.  But each day blood sample from rat should not be a problem.  Therefore, choice would be rat.

ü  Though (for initial studies) mouse data is collected (as less amount of compound needed for this study) later rat data is needed

ü  Few studies cannot be done on rat – then they are switched to other animals according to disease of study – like dog, ferret, guinea pigs, rabbit.  

Compiled from several sources. 

WHAT DO THE LETTERS & NUMBERS AT THE END OF A PATENT PUBLICATION DENOTE?

Most often we see patent numbers ending with letters A B or C.  These letters carry a specific meaning.  These letter codes indicate the stage in the patent’s lifecycle.  The additional digit following the letter ‘A’, ‘B’ or ‘C’ carries extra information, but depends upon the exact examination and granting process which occurs in each country.

o   A – a published version of an application which has not yet passed examination;

Therefore if the document is an ‘A’ publication then it is not a patent at all, but an application that has yet to pass examination.

§  A1 document is the first publication of an application, which usually occurs at around 18 months from initial filing.

§  A2 document is second or subsequent publication of application, requested by applicant.

§  A9 document is the publication with correction of errors in either A1 or A2.

o   B – a published version of an application or patent which has passed examination, i.e. which is either a granted patent, or an accepted/allowed application; and

§  B1 document is a granted patent.  This document is not published as patent application earlier. That means after initial filing it was directly granted patent.

§  B2 document is a granted patent which was earlier published as patent application.

o   C – a published version of a patent which has been amended since it originally passed examination.

Therefore, for a pre-granted patent publication i.e. a patent application – publication number appears something in this form --- yyyy/nnnnnn-Ax

Whereas a corresponding granted patent will have a number of this form --- n,nnn,nnn-Bx

For more information, please see below links,

http://blog.patentology.com.au/2012/09/the-abcs-of-patent-publication.html; 

http://www.bios.net/daisy/patentlens/2354.html

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WHY DMSO IS SOLVENT OF CHOICE?

Ø  Dimethylsulfoxide (DMSO) is known for its

(i) capacity to dissolve compounds with a wide range of chemical properties

(ii) it has low volatility

(iii) relatively low toxicity to both tissue culture and technicians

(iv) it has good miscibility with water, and limited deleterious effects at low concentration upon bioassays.

Ø  Though DMSO has become the de facto solvent of choice for compound solubilization for HTS, DMSO has its shortcomings

(i) DMSO can act as oxidizing agent.  You can oxidize suitable compounds by leaving them in DMSO open to air    
                                                                                                            
(ii) DMSO is highly hygroscopic. 
                                                                                          
(iii) lipophilic compounds might not have appropriate solubility in DMSO (sometimes completely insoluble).

Ø  ADMET data is obtained for compounds as methanol stocks and enzyme assay most often are performed with DMSO stocks.  The reason for such solvent selection depends on what read-out you have.  

ADMET (fluorescence read out) – therefore, DMSO is not a compatible solvent.

MOST GENERAL TYPES OF TOXICITIES WE ENCOUNTER

MOST GENERAL TYPES OF TOXICITIES WE ENCOUNTER

Mechanism based pharmacology:                                                                                        

ü  A molecule provides desired therapeutic effect by activation of a biological target; however the activation of the same biological target causes unwanted effect also.

ü  Here, there should be a balance between good effects vs. bad effect.  

Formation of reactive metabolites:

ü  We should avoid chemically reactive functional groups in the molecules.  We should also bear in mind the kind of metabolites a drug molecule might end-up with depending on the functional groups on the molecule.  With experience one can make an educational guess of the fate of certain molecules bearing a particular functional group. 

ü  The best way of avoiding reactive metabolites is avoid functional groups which have been shown to cause a problem in the past. This is why you will see very few drugs containing, for example, a nitro group. However, some functional groups are worse than others.

ü  Such reactive metabolites can lead to hepatotoxicity and genotoxicity.

Activation of other receptors, including hERG:

ü  Along with biological target of interest, sometimes the drug molecules can activate other receptors/enzymes – we call this as ‘’off-target toxicity’.

ü  Therefore, before moving onto preclinical studies – these molecules are tested for biological activity against similar biological targets.   For example, our biological target of interest is ‘KINASE A’, then we screen the molecule against several other KINASES which fall under the family of ‘KINASE A’ family.

ü  Potency (and therefore dose) is important as we are looking for a safety margin, i.e. the absolute potency at another receptor is less important than how much less than the potency at the primary receptor it is.

ü  hERG:  The receptor is a potassium ion channel located in cell membranes in the heart, which opens and closes to allow potassium ions to flow out of the cells.  Arrhythmia is a lack of rhythm in the heart beat. A delay of the T wave by 5-10 milliseconds can cause lack of control of the heartbeat, which may lead to a fatal arrhythmia.  This is obviously something to be avoided.

Interactions with other substances:

ü  Many patients take poly-medication.  One substance can affect the metabolism of another.  It’s not just drugs interacting with each other that can cause a problem – they may also interact with foodstuffs, alcohol or herbal medicines.

ü  Drugs are mainly cleared by a group of hepatic enzymes called cytochrome P₄₅₀s or CYPs. CYPs can be further sub-divided into smaller classes of enzymes, all of which have their own structure-activity relationships. The most common CYPs to cause problems are known as 3A4, 2C9, 1A2, 2D6 and 2C19. Compounds with low oral bioavailability and high first pass metabolism are most susceptible to interaction with other medicines which affect CYPs.   The effect of inhibiting these enzymes is that they are then unavailable to metabolize other compound.

Idiosyncratic toxicity:

ü  Idiosyncratic toxicity’ is term to include other toxic effects that we don’t currently understand.

ü  Note that increased potency reduces the possibility of this.

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Cytotoxicity / Hepatotoxicity test & CYP450 Inhibition Profiling

Cytotoxicity / Hepatotoxicity test 

What about the toxicity of the test compound? Is the compound too toxic to be therapeutically useful?

The in vitro cytotoxicity test with primary hepatocytes is used to identify the cytotoxic potential of a test substance. The relative cell viability upon incubation with test article compared to the solvent control is determined (single point).

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Hepatocyte cells are incubated for 24 hours with known toxic and non-toxic compounds at a range of different concentrations. At the end of the incubation period the cells are loaded with the ATP-liteTM 1step ATP monitoring reagent and scanned using an automated plate reader with luminescence detection to determine the number of active/live cells.

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CYP450 Inhibition Profiling

Will the compound of interest lead to drug-drug interactions?  Does the compound of interest inhibit a key enzyme responsible for oxidative metabolism?

·         Cytochrome P450s (CYPs) are a superfamily of heme-containing enzymes that mediate the inactivation and metabolism of many drugs as well as endogenous substances.

·         Compounds that inhibit P450s may cause the toxic accumulation of other substrates.

·         CYP inhibition profiling examines the effects of a test compound on the metabolism of other known enzyme substrates of the five primary drug human metabolizing CYP: 1A2, 2B6, 2C9, 2D6, 3A4.

·         The levels of the CYP isoform marker substrate and metabolites are measured in the presence and absence of a test compound by LC/MS/MS.

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In an assay similar to the metabolic stability assay, liver microsomes are used to determine the CYP450 inhibition profile of test compounds by measuring the % metabolism of a known substrate. Microsomes (and NADPH regenerating system) are dispensed into a 96well plate containing a substrate and test compound (10 µM), and the reaction is allowed to proceed for 0.5 hours at 37°C with shaking. The reaction is quenched by the addition of MeOH, centrifuged and the amount of product is measured by LC/MS/MS.

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ION CHANNELS (hERG)

Ion channels open and close to allow Na+, K+, and Ca+2 ions that are electrically charged to flow in and out of the cell.

Each of this channel has right shape and size with the corresponding ions to fit through.  For example, a K+ ion can pass perfectly through a potassium channel and Na+ ion through sodium channel.  

However, a K+ ion is incapable of passing through a sodium channel, similarly a Na+ ion is incapable of passing through a potassium channel.

Movement of these ions into and outside of the cell is what causes the electrical activity in the heart.  This then spreads across the cells in the heart from top to bottom making them to contract and pump blood.

hERG K+ channel is responsible for length of QT interval.  When the channel is hERG blocked then K+ ions are delayed in leaving the heart muscle.

What is hERG?

·         hERG (the human Ether-à-go-go-Related Gene) is a gene (KCNH2) that codes for a protein known as K_v11.1, the alpha subunit of a potassium ion channel.

·         The hERG channel is a voltage-gated potassium channel and, like other voltage-gated potassium channels, it is highly selective for potassium and is a tetramer formed of four subunits, each containing six transmembrane domains.

·         hERG is expressed in multiple tissues and cell types, including neural, smooth muscle and tumour cells.  However, hERG is most highly expressed in the heart, and this is where its function-and dysfunction-is best understood.

What is its function?

·         This potassium ion channel (sometimes simply denoted as 'hERG') is best known for its contribution to the electrical activity of the heart that coordinates the heart's beating.

·         The human ether-a-go-go related gene (hERG) encodes the inward rectifying voltage gated potassium channel in the heart (IKr) which is involved in cardiac repolarisation.

Why should we be interested in hERG?

·         When this channel's ability to conduct electrical current across the cell membrane is inhibited or compromised, by rare mutations in some families, it can result in a potentially fatal disorder called long QT syndrome.  Inhibition of the hERG current causes QT interval prolongation resulting in potentially fatal ventricular tachyarrhythmia called Torsade de Pointes (TdP).

·         hERG K⁺ channels are also remarkably susceptible to block by a wide range of drugs, which in turn can cause drug-induced long-QT syndrome (LQTS or TdP) and an increased risk of sudden death. This has made hERG inhibition an important antitarget that must be avoided during drug development.

What can be done?

·         To reduce the risk of a drug candidate failing in preclinical safety studies because of blockade of hERG channels and associated QT prolongation, it is important to screen compounds for activity on hERG channels early in the lead optimization process

·         Although binding assays do not measure hERG function, they offer a rapid, robust and inexpensive measure of the ability of compounds to interact with the hERG channel.  Combined with electrophysiology on a few benchmark compounds to characterize the nature of the compound-channel interaction, binding assays can be used as an efficient means to develop the structure-function relationship of hERG interactions within a structural class of test compounds. This strategy should facilitate the development of new small molecule drugs with improved cardiovascular safety profiles.

·         The hERG channel inhibition assay is a highly sensitive measurement which will identify compounds exhibiting cardiotoxicity related to hERG inhibition in vivo. It is important to note, however that not all compounds which inhibit hERG activity in vitro will proceed to cause cardiotoxicity in vivo. The relevance of the in vitro data will be dependent on other factors such as the plasma concentrations reached in vivo.