Drug Development Process

• Basic concepts in toxicology and metabolism are studied in Science of Medicines 2.
• Isolda Romero-Canelon is a doctor at the University of Birmingham

Drug Development Process

• The five drug development phases are discovery and development, preclinical research, clinical research, FDA review, and FDA post-market safety monitoring.
• Discovery and development involves about 10,000 compounds, preclinical research involves 250 compounds, clinical research involves 5 compounds, FDA review involves 1 compound.
• Target validation in drug development requires ~1.5 years of cycle time with ~3% cost per new molecular entity (NME).
• Compound screening requires ~1.5 years, consuming ~6% cost per NME.
• Lead optimization requires ~1.5 years, with ~17% cost per NME.
• Pre-clinical tests take ~1 year and ~7% cost per NME.
• Phase I requires ~1.5 years, incurring ~15% cost per NME with a 66.4% probability of success.
• Phase II requires ~2.5 years, costing ~21% of NME with a 48.6% probability of success.
• Phase III requires ~2.5 years, taking up ~26% NME costs and achieving a 59% probability of success.
• Approval to launch requires ~1.5 years at ~5% cost per NME.
• Drug failures can stem from toxicity, PK/bioavailability issues, clinical efficacy, or clinical safety concerns.

Profile of Drug Toxicity

• Drug toxicity can manifest in various organ systems, e.g., the heart, brain, lungs, stomach, kidneys, liver, etc
• In the heart drug toxicity can cause blood pressure changes, effects on cardiac rhythm, and thrombosis.
• In the brain seizure and suicide can be caused by drug toxicity
• In the lungs drug toxicity can cause respiratory suppression, constriction, and inflammation
• In the stomach drug toxicity can cause bleeding, diarrhoea, and motility effects
• In the kidneys drug toxicity can cause renal injury
• In the liver drug toxicity can cause liver injury

Causes of Drug Toxicity

• Drug toxicity can be target-driven, arising from the drug's intended mechanism.
• Off-target effects, secondary pharmacology, can cause drug toxicity.
• Generation of reactive metabolites, especially in the liver can cause drug toxicity
• Drug interactions can cause drug toxicity
• Target-driven toxicity arises when modulating the intended biological target causes unavoidable side effects.
• Even a 'clean' agent can show target driven toxicity when some targets are difficult to modulate without causing side effects
• Anti-cancer drugs must kill cells to be effective and cancer cells are not that different to the host cells
• The binding characteristics & PK of a drug can help to distinguish efficacy & toxicity which is tough to predict
• There is no drug which is 100% targeted
• Off-target toxicity arises when a drug interacts with unintended targets due to the presence of over 20,000 proteins and many other macromolecules in the human body.
• Hydrophilic compounds bind predominantly through bonding interactions, meaning there is a high demand for ideal target interactions.
• Lipophilic compounds bind predominantly through entropic effects and pushed out by water into an environment that is less unfavoured.
• Interaction with protein may be much less specific, though the compound still needs to fit in the binding site.

Off Target Toxicity Example

• Ibuprofen, an NSAID discovered in 1961, carries an increased risk of gastric bleeding/ulceration.
• 2nd generation NSAIDs, including diclofenac and naproxen, were discovered/developed in the 1970s.
• COX enzymes discovered in 1988 led to the discovery of the biological target of NSAIDs, – inhibilitons leading to reduction in synthesis of prostaglandins
• COX-2 inhibition is linked to anti-inflammatory effects while COX-1 inhibition is believed to effect gastric lining, resulting in side effects
• Merck patented rofecoxib, a selective COX2 inhibitor in 1993 with potential for improved pain relief and side effect profile
• Clinical studies in 1999 showed the COX2 selective inhibitors offer superior risk/benefit profile to NSAIDs
• Rofecoxib was launched and marketed as Vioxx by Merck
• Rofecoxib was withdrawn in 2004 due to monitoring showing an increased risk of cardiovascular side effects including heart attacks.
• Heart attacks are now believed to be linked to COX inhibition
• In November 2007, Merck agreed to settle Vioxx suits for $4.85 Billion to settle thousands of lawsuits
• In the case of NSAIDs inhibition of COX1 can be considered secondary pharmacology – a biological target/effect of the drugs which is not linked to its efficacy
• A more selective drug that has less off target effects will have a safer overall profile
• Can we predict/understand or even test selectivity against over 20,000 proteins?

Metabolism

• Metabolism is a key factor in understanding off-target toxicity
• Phase 1 metabolism include Oxidation, Reduction, and Hydrolysis
• Phase I metabolism involves oxidation, reduction, and hydrolysis.
• Aliphatic or aromatic hydroxylation, N or S-oxidation, and N-, O-, S-dealkylation happen in Oxidation
• Nitro reduction and Carbonyl reduction can happen in reduction
• Esters, amides, or phosphates become acids/alcohols/amines or substituted hydrazines in hydrolysis
• Metabolites may still have activity at the target (or at others)
• Metabolites like this are called "active metabolites"
• Cytochrome P450s are the main class of proteins involved in Phase 1 metabolism and account for approximately 60% of commonly prescribed drugs.
• ~1000 isoforms are known, with over 100 found in humans.
• Cytochrome P450s carry out Phase I oxidations in liver cells, they are also present in the intestine
• These are membrane-bound Haeme-containing proteins coordinating FeII/III at the active site
• Lipophilic molecules will bind more quickly into the CYP450
• More lipophilic drugs are more rapidly cleared
• The rate of oxidation is determined by the stereo-electronics of the oxidation and the concentration of the drug-CYP complex.
• There are many isoforms (protein with different shaped active sites); the only consistent effects are solvent-based/entropic.
• The binding is a combination of enthalpic and entropic effects
• Certain compounds can act as inhibitors of CYP enzymes, such as unhindered aromatic nitrogens.
• CYP inhibitors typically bind to the metal centre of the heme. Inhibition of cytochrome P450s can cause changes in exposure which leads to drug interactions
• If a patient is taking drugs metabolised by CYP, the exposure to these drugs will increase
• This is the most common cause of drug-drug interactions and is a common issue for drugs with low TR
• Two types of CYP inhibition are inhibition of cytochrome P450s leading to changes in exposure and ion channel inhibition related to the non-metabolised form

Terfenadine

• Terfenadine was launched in 1985 as the first non-sedating antihistamine to treat allergic rhinitis without side effects of earlier antihistamines
• It was discontinued in the mid 1990s
• The reduction in drowsiness is predominantly due to the fact that the compound does not enter the CNS to the same extent as the earlier drugs, a good example of how pharmacokinetics can allow separation of efficacy and side effects
• First pass metabolism of terfenadine is high, there is limited systemic exposure to terfenadine itself, and the main circulating species will be the carboxylic acid metabolite (an active metabolite) after oral dosing is done
• Terfenadine is safe when taken alone, it can lead to serious cardiac side effects when taken in combination with drugs that inhibit cytochrome P450 enzymes, such as ketoconazole.
• Terfenadine has ~1000x higher affinity than the metabolite for the hERG channel, a potassium channel with a key role in cardiac signalling
• Terfenadine interacts with human ether-a-go-go related gene
• hERG= 'human ether-a-go-go related gene'
• The first is that the inhibition of cytochrome P450s leads to changes in exposure and the second is the ion channel inhibition related to the non metabolised form
• Since terfenadine has been discontinued, the metabolite fexofenadine is now used directly

Reactive Metabolites

• Drugs can become reactive metabolites, and these are typically removed by our bodies
• The liver is the most common target for small molecule toxicity, these cases can be linked to generation of reactive metabolites
• The reactivity of a metabolite correlates strongly with radical stabilisation.
• There are groups within metabolites which are commonly metabolised (structural alerts, or 'known toxicophores')
• There are two common approaches taken to avoid toxic reactive metabolites in drug production: exclude chemical functionalities undergoing metabolic activation and screen for reactive metabolite formation
• Aromatic rings may have direct oxygenation without H abstraction by epoxidation
• Quinones are usually removed by conjugation to glutathione (GSH), though they can also react with proteins
• Paracetamol can become a reactive metabolite that is usually removed from the system by conjugation to glutathione (GSH).
• GSH acts as a general antioxidant in the body.
• GSH conjugation is used to remove reactive oxygen species, thus is depleted by paracetamol
• Once GSH levels are depleted, NAPQI levels can accumulate leading to non-specific alkylation of proteins on the liver.
• Proteins may now be seen as exogenous by the immune system, leading to an inflammatory response which directly attacks the liver itself.
• Depletion of GSH can result in generation of reactive oxygen species (ROS) leading to direct cellular toxicity, possibly following mitochondrial toxicity.
• Although Atorvastatin can become a toxic metabolite, it is very effective and administered at very low doses, meaning these toxic metabolites are at too low levels

Metabolism - Phase II

• Phase II metabolism includes glucuronidation, sulfation, and glutathione conjugation.
• Alcohol, phenol, amine becomes Sulfation
• Halo-cpds, epoxides, arene oxides, quinone-imine becomes Glutathione conjugation
• Carboxylic acid, alcohol, phenol, amine becomes Glucurondation
• In some cases glucuronides can undergo 'migration' to form a stable gucuronide that reacts with proteins, either direct phase 2 or phase 2 after direct phase 1 activation
• In some patients the levels of glucuronide may be high and lead to extreme immune response to alkylated protein
• This can lead to lower liver injury but only in a fraction of patients; termed idiosyncratic (type-B) toxicity.

Mutagenicity

• Compounds can potentially induce genetic mutations, and present a risk of causing cancer by binding to/reacting with DNA
• Animal carcinogenicity studies tend to come later in development.
• AMES/MLA/Micronucleus are models of in vitro mutagenicity
• These are sensitive: predict ~80% of compounds that show in vivo mutagenic effects.
• Threshold / margin arguments do not exist for DNA-binding chemicals.
• Often run in the presence of microsomes (as metabolic activation may be needed)
• Although some actives are predicted, a significant portion do not show mutagenic effects in vivo
• Many aromatic amines are positive in the Ames assay
• Anilines can be positive in the Ames Assay with ~40% active
• Heteroaromatic amines can be positive in the Ames Assay with ~20% active
• Release of masked anilines can also occur via in vivo metabolism
• Mutagenicity can often be attributed to the presence of a masked aromatic amine.

Learning Outcomes

• Basic mechanisms of drug-mediated toxicities
• Relationship between ligand binding, selectivity and lipophilicity
• Pharamcokinetics/metabolism explain CYP inhibition in DDIs
• Common 'toxophores' in compounds and link to toxicity mechanisms
• Toxaphores are functional groups on molecules that are responsible for it's toxicity.

Table of common Toxophores

• Electron rich aromatic rings/phenols lead to reactive metabolites/liver damage
• Aromatic amines (anilines) lead to genotoxity
• Carboxylic acids lead to idiosyncratic liver toxicity
• Unhindered aromatic Nitrogens lead to CYP inhibition leading to DDIs
• Highly lipophilic compounds lead to Increased risk of promiscuous binding, off-target effects, DDIs