Pharmaceutical Chemistry – Drug Discovery/Drug Metabolism

Approximately how long is the development and pre-clinical testing process?
6.5 Years
How long are Phase I clinical trials?
1 year
How long are Phase II clinical trials?
2 years
Which of the two (phase I or phase II) has more participants?
Phase II
How long are Phase III trials?
3 years
Approximately how many participants are in Phase III trials?
1,000 to 3,000 people
What is the average cost for the development of a drug?
$2.5 billion
How long do patents last?
20 years.
How many marketed drugs manage to recoup the cost of development?
What are some reasons drugs fail?
Clinical safety, efficacy, formulation, PK/bioavailability, commercial, toxicology, cost of goods
Generally, where is drug discovery performed?
Academic settings
Generally, where is drug development performed?
Business settings
What determines the biological activities of compounds?
Their chemistry
What chemical properties affect biological activity?
Physiochemical (lipophilicity, ionization, solubility), Electronic (resonance, inductive effects, bonding potential), and spatial (molecular dimension, stereochemistry)
How are new drugs created from old drugs?
Alteration of side chains to improve something (PK, dosage form, etc.)
Example: Captropil.
How is biological information used to create drugs?
Study of indigenous medications, study of warfare chemicals (nitrogen mustards), observations in in vivo testing (vinca alkaloids), or observations of clinical side effects (Viagra)
What is the modern approach to drug development?
Use knowledge of the biology of the disease to identify targets that can be manipulated by drugs, then use that knowledge of target protein to design new inhibitors.
What are the steps in a drug development project?
Target selection/validation, assay development, identification of a lead compound, lead optimization, preclinical evaluation, and clinical trials
What is the most critical stage of drug development?
Target selection/validation. One wants targets that are relevant and selective
What are traditional strategies for target selection?
Study disease biology, key regulatory proteins, response to previous drugs, and engineered models
Most drug targets are and ?
Receptors and enzymes
Why are other targets, like DNA or nuclear receptors, not as common?
Drugs not as selective
What is functional genomics?
Genetic analyses for disease genes and protective genes, by DNA microarray analyses. Strategy for target selection.
Methods for target validation?
Biology of disease, gene knock-out/in, constitutively-active/domiant-negative mutants, antisense, ribozymes, RNA interference
What is RNA interference?
Selectively decreases levels of expression of specific mRNA in a cell. Mimics the ‘perfect’ drug.
What is important in assay development?
Relevance, selectivity, sensitivity, reproducibility, speed, and cost.
Activities of signaling proteins are affected by what?
Subcellular localization
What are the two basic approaches to lead compound identification?
Computation modeling and screening
Rational drug design?
Substrate analogs to act as enzyme inhibitors
Computer-aided drug design downside?
Need to know structure of target
Testing collections of compounds for biological activity, don’t need to know structure. ‘Hits’ = CxD
What is “D”?
Chemical diversity of compound collection.
What are some sources of compounds for lead discovery?
Natural products, synthetic compounds
What is combination chemistry?
Assembly of molecular building blocks. Uses high-throughput synthesis. Requires high-throughput screening for lead discovery
If there are 5 sites for substitution, and 20 building blocks, how many potential compounds can be created?
20^5 > 3,200,000
What is a benefit of robotic screening systems?
Decreased time, decreased error
What are the goals of lead optimization?
Increase target selectivity (decrease adverse effects), increase potency (decrease size), simplify synthesis ( increase ease of manufacture), optimize chemical and metabolic stability, and optimize in vivo activity (therapeutic efficacy).
What do SARs define?
The pharmacophore
What is the pharmacophore?
3D arrangement of chemical determinants that are necessary for binding to the receptor
What is traditional lead optimization?
Synthesize homologous series, or use ‘chemical insight’ or bioisterism rules. Synthesis of few compounds. Low-throughput screening, high cost, slow progress
Lead optimization of combinatorial chemistry?
Synthesis of ‘focused libraries’, may/may not require high-throughput screening, high start up costs, but fast and efficient.
Lead optimization by computer-aided drug design?
Analysis of receptor on pharmacophore. Allows for QSAR analyses and drug design without knowledge of protein structure
What is QSAR?
a. Quantitative structural activity relationships.
b. Mathematical relationship between biological activity and chemical properties and allows for prediction of activity of new compounds
The Hansch Approach?
QSAR, biological activity = f . Limits the number of compounds that are made.
Computational ADME?
Predicts solubility, oral bioavailability, biodistribution, and metabolism
What is tested in in vitro studies?
Chemical purity and stability, formulation, mechanism of action, and cellular pharmacology
What is tested in in vivo studies?
Efficacy in animals, short-term and long-term toxicity, carcinogenicity, metabolism, PK, and biodistribution
What is the final step in drug development?
Clinical trials
What is a clinical trial?
A prospective study comparing the value of intervention against a control in humans
What is required of a clinical trial?
May not be retrospective, must employ one or more intervention techniques, must contain a sufficienctly similar control group, approval for the study must be obtained from the FDA, and an Investigational New Drug application must be submitted
What must be included in the IND application?
Descriptions of the manufacture of the drug, its pharmacology and toxicology in animals, and a detailed protocol for clinical trials.
What department accepts or rejects INDs? How long does the process take?
The CDER. If, after 30 days, no rejection is received, the study has implicit approval to proceed.
What are the goals of Phase I clinical trials?
Primarily to establish maximum tolerated dose in humans. Secondarily to examine pharmacokinetics in humans. Phase I is not for treating the disease.
Who are the subjects in Phase I trials?
20-50 Mostly healthy volunteers or patients who did not respond to other treatments (especially in cancer and AIDS cases)
What is the classical procedure for determining the maximum tolerated dose in humans?
3 subjects per dose level. Starting dose is 1/10 of the LD10 for mice, or 1/3 of the toxic dose in dogs. If none of the 3 experience a toxic reaction, 3 different people are treated at a higher dose. If one of the three has a reaction, three different people are treated with the same dose. This is continued until 2 of the three have a reaction.
What are the goals of phase II clinical trials?
To determine the efficacy of the drug and to continue monitoring for toxicity.
Who are the subjects in Phase II trials?
50-300 patients with carefully matched disease
What is the procedure of a Phase II trial?
Two-stage design. Drug given at dose determined by Phase I. Blinded design. May be uncontrolled in cancer drug studies
What are the goals of Phase III?
To verify efficacy and safety
Who are the subjects in Phase III trials?
100s-1000s of patients with carefully matched disease
What is the procedure of a Phase III trial?
Often multi-arm studies in which the drug is compared with the standard drug for that disease and can involved cross-over and double-blind studies. Drug given at dose used in Phase II. Response can be increased survival, improvement of a biomarker, or improved quality of life
Who conducts the final review process? How long does it take?
The CDER. Less than one year.
What is Phase IV?
Continued monitoring of the drug post-marketing
In the past, drug discovery was essentially what kind of process?
Random, observational and relied on finding a molecule that worked and figuring out how it worked later
Who discovered penicillin?
Alexander Fleming in 1929
What are drug targets?
Biomolecules whose functions can be artificially modulated
What technologies enabled the new methods of drug discovery?
Molecular biology, in vitro assays, and 3D structural models of drugs and their receptors.
In drug design and discovery, what is the difference between high-throughput screening and computer prediction models?
a. High-throughput tests large numbers of molecules (10^6) and has a typical hit rate of <1%
b. Computer prediction tests the compounds on the computer first, thus decreasing the number of molecules actually tested and increasing the hit rate.
What forces drive states of matter and the 3D conformation of macromolecules?
Interatomic forces
What are interatomic forces?
Covalent interactions (-50 to -100 kcal/mol), and noncovalent interactions (electrostatic -80 to -120, van der walls -0.5 to -1, hydrophobic effects -0.5 to -1, and hydrogen bonding -3 to -6)
What are hydrogen bonding interactions?
It occurs when two electronegative atoms compete for the same hydrogen, and create an electron deficient hydrogen.
What are some H-bond donors? Some H-bond acceptors?
a. Donors: NH, OH
b. Acceptors: C=O, OH
Why are hydrogen bonds important in drug discovery?
Influence interactions between drug and solvent, drug and target, and drug-to-drug
What is the hydrophobic effect?
It is a major driving force in binding of drugs to receptors. Enzyme active sites are hydrophobic in nature. It occurs to maximize contact between nonpolar parts of drug and receptor and contributes to bonding.
What drives binding?
Complementarity between ligand and receptor – like a lock and key.
What are the two types of complementarity?
Shape complementarity, in which the 3D shape of the drug matches that of the receptor, and chemical complementarity, in which groups on the drug can interact with appropriate groups on the receptor.
The affinity of a drug is described as what?
The binding energy, which is the sum of the forces between the drug and the receptor
What is the equation for binding energy?
dG = dH – TdS, where H is enthalpy and S is entropy.
Where do enthalpy and entropy come from?
Enthalpy results from forces within and between molecules. Entropy comes from solvent molecules and movement and rotation of bonds
What is protein X-ray crystallography and how is it used in drug design?
X-rays shined through molecule. Diffraction finds regions with high concentrations of electrons. These regions are used to assign positions to atoms.
What is molecular modeling (computational chemistry)?
Allows one to create 3D models of molecules by calculating their properties and minimizing their energy. Can model conformations of ligands and receptors and their interactions and their binding energy
What is the pharmacophore?
The features of a drug necessary to ensure optimal interactions with a biological target and to trigger or block it’s response
How is virtual (computer) screening useful?
Matches the 3D structure of potential drug with the shape of the receptor
What is high-throughput docking?
Fitting the molecule into the receptor pocket and then scoring it based on interactions in 3D structure.
What are the goals of substrate metabolism?
Increase water solubility and polarity, deactivation and excretion, or activation.
What are some common sites of biotransformation?
The liver (main), intestinal mucosa, kidney, and lungs
What types of reactions are Phase I?
Oxidation, hydroxylation, reduction, and hydrolysis
What are the goals of Phase I reactions?
Introducing a new functional group, modifying an exisiting functional group, or exposing an existing functional group (more than one may occur).
What are the Enzymes involved in Phase I reactions?
Cytochrome P450 and Flavin-containing monooxygenases
What are Phase II reactions?
Conjugations: acetylation, glucuronidation, sulfation, and amino acid conjugation
What is the goal of Phase II reactions?
To conjugate an endogenous molecule to a functional group to increase polarity or water solubility to facilitate excretion
What is the most common reaction in xenobiotic metabolism? Which enzyme performs is?
Oxidation CYP450s
Where is the highest concentration of CYP450s?
In the liver
Where are the CYP450s located?
Most are on microsomes, some are on mitochondria.
What type of proteins are CYP450s?
Heme proteins with an iron protoporphyrin ring.
What components are required for CYP450 systems?
Oxygen, NADPH, NADPH-CYP450 reductase, and a lipid environment
Describe the catalytic pathway for CYP450.
The CYP has a Ferric Iron (Fe3+). The drug binds to the CYP. P450 reductase reducese the iron to the Ferrous (Fe2+) state. Oxygen binds to the Fe2+ iron. Radical on oxygen oxygenates the drug. Fe4+ is formed, oxygen binds to the drug and drug leaves the complex oxidized.
What are some hydroxylation mechanisms catalyzed by CYP450?
Aromatic hydroxylation, aliphatic hydroxylation, deamination, O-Dealkylation, N-dealkylation, and sulfoxidation
What does decreasing metabolism do?
It decreases toxicity. Example: in decreasing toxicity and metabolism levels: methoxyflurane, halothane, enflurane, isoflurane, desflurane.
What is particularly interesting about haloflurane?
At two doses, can cause haloflurane hepatitis
Which are the most predominant CYP genes?
CYP3A4/5, 2C, and 1A2
What does CYP2A1 generally metabolize?
Environmental compounds
What is CYP2E1 specific for?
Small molecules, ethanol, anesthetics, small organic solvents
Which isoforms metabolize most drugs?
3A4/5, 2D6, and 2C8/9
Family 1 metabolizes what?
Environmental carcinogens, in particular aromatic hydrocarbons and aryl amines
Describe CYP1A1
Aromatic hydrocarbon hydroxylase, primarily expressed in extrahepatic tissues (small intestine, lung, placenta). Inducible. Variation.
Describe CYP1A2
Expressed in liver, stomach, and intestine. Inducible. Variation.
Describe CYP2A6
Found in liver, lung, and nasal epithelium. Metabolizes drugs (coumarin), procarcinogens, and nicotine. Polymorphisms.
Describe CYP2C
Includes CYP2C8 (liver and skin), CYP2C9 (Liver and intestine), and CYP2C19 (liver and intestine). Metabolize 25% of clinically important drugs. Represent 20% of total CYP in liver. Polymorphisms.
Describe CYP2D6
Polymorphisms. Metabolizes 21% of clinically important drugs. 3% of total CYP in liver. Not inducible. Prefers lipophilic amines
Describe CYP2E1
Minimal drugs. Expressed in liver, kidney, intestine, and lung. Inducible by alcohol, diet, and diabetes. Polymorphism in Chinese people.
Describe CYP3A4
Most abundant CYP in liver. Found in liver, intestine, kidney, brain, uterus, and placenta. Metabolizes 33% of clinically important drugs. Inducible. Inhibited by erythromycin. Activates aflatoxin B1 and Benzo[a]pyrene. Prefers lipophilic drugs.
List the CYPs in order of drug metabolisms
What factors can affect CYP drug metabolism?
Interindividual variation, induction, inhibition, genetic polymorphisms
What is the consequence of induction of CYP450?
Altered PK and PD, increased rates of metabolism, enhanced activation of procarcinogens
What can induce CYP450?
Phenobarbital, rifampin, cigarette smoke, brussel sprouts, cabbage, cauliflower, alcohol, St. John’s Wort, omeprazole, bile acids, Vitamin D
What receptor does Omeprazole induce?
Aryl Hydrocarbon Receptor (AHR)
What does Phenobarbital induce?
a. Constitutive androstane receptor (CAR)
119) What does Rifampin induce?
a. Pregnane X Receptor (PXR)
120) What does bile acid induce?
a. Farnesoid X receptor (FXR
121) What does Vitamin D induce?
a. Vitamin D Receptor (VDR
122) What do fibrates induce?
a. Peroxisome proliferators activated receptor (PPAR)
123) What does all-trans-retinoic acid induce?
a. Retinoic acid receptor (RAR)
124) What does 9-cis-retinoic acid induce?
a. Retinoid X Receptor (RXR)
125) Which compounds cause reversible CYP inhibition?
a. Fluroquinolones, cimetidine, antifungals, and quinidine
126) What compounds cause irreversible CYP inhibition?
a. Macrolide antibiotics (trolendomycin, erythromycin, and clarithromycin), chloarmphenicol, cyclophosphamide, and spironolactone
127) What are molecular mechanisms of genetic polymorphisms?
a. Single nucleotide polymorphism substitution (SNPs) and Indels
128) What are the three types of SNPs
a. Coding nonsynoymous, coding synonymous, and noncoding
129) What is a coding nonsynoymous SNP?
a. Change in nucleotide sequence with a change in amino acid composition. Change in affinity or activity
130) What is a coding synonymous SNP?
a. Change in nucleotide sequence without a change in amino acid composition. Changes in transcript stability or splicing.
131) What is a noncoding SNP?
a. Change in nucleotide sequence, change in promoter or other regulatory regions.
132) What are Indels?
Insertion/deletion polymorphisms. Same effects as SNPs. Can be short repeats or larger deletions/insertions.
133) Which CYPs are polymorphic?
a. 2A6, 2C9, 2D6, 2C19
134) What does 2A6 do?
a. Metabolizes nicotine to conicotine. Better to be a poor metabolizer
135) What do polymorphisms in 2C19 cause?
a. Large variability in therapeutic response to mephenytoin
136) What happens to Benzo[a]pyrene?
a. It can be oxidized by many different routes. If it becomes the 4,5-dihydrolol or an ortho-quinone, it is non-toxic. However, if it becomes (+)benzo[a]pyrene-7,8-dihydrolol-9,10-epoxide, it is toxic.
137) Describe FMOs.
a. Flavin-containing monooxygenases. 5 different isozymes. NADPH and oxygen dependent. Located in smooth endoplasmic reticulum. Most xenobiotics that are substrates for CYP450 are also substrates for FMO but not vice versa.
138) What types of compounds do FMOs oxygenate?
a. Nitrogen compounds and sulfur compounds
139) What are MAOs and where are they found?
a. Monoamine Oxidases, found almost all tissues. There are two forms, A and B, and most tissues contain both. Placenta contains only A and Platelets and lymphocytes contain only B. They are flavin containing.
140) What are the substrates for MAOs?
a. Primary, secondary, and tertiary amines.
141) What are two nonspecific inhibitors for MAOs? What inhibits MAO-A specifically? What inhibits MAOB specifically?
a. Nonspecific: phenelzine and tranylcypromine. Specific for A: clorgyline. Specific for B: pargyline and selegiline.
142) Describe alcohol dehydrogenase
9 subunits that form heterodimers and homodimers. NADH dependent.
143) What are the substrates for alcohol dehydrogenase?
a. Diverse primary and secondary alcohols. They convert alcohols to aldehydes.
144) Where are the highest concentrations of alcohol dehydrogenases?
a. Liver, kidney, lung, and gastric mucosa
145) What is responsible for the difficulty processing alcohol in some Asian people?
a. ADH2*2 (atypical ADH)
146) Describe aldehyde dehydrogenase
a. 12 different forms. Cystolic, mitochondria, and microsomes. NADH dependent. Reversible
147) What are the substrates for aldehyde dehydrogenase?
a. Aldehydes.
148) What are involved in hydrolysis?
a. Carboxylesterases – cholinesterase, pseudocholinesterase, arylcarboxyesterase, liver microsomal esterase.
149) What are the substrastes of the hydrolysises?
a. Carboxylic acid esters, amide, and thioesters. Note: amides more resistant to metabolism than esters.
150) What are the conjugation reactions?
a. Glucuronidation, sulfation, amino acid conjugation, acetylation, glutathione conjugation, methylation
151) What are the consequences of conjugation reactions?
a. Increase water solubility and polarity. Activation (morphine and minoxidil) and inactivation, toxicity.
152) What functional groups are conjugated and what can be done to them?
a. Hydroxyls are sulfated, glucuronidated, and methylated. Amines are acetylated, sulfated, and glucuronidated. Carboxyls are amino acid conjugated and glucuronidated.
153) What is the major and most common conjugation reaction?
a. Glucuronidation.
154) What does glucutonidation require?
a. UDP=glucuronosyl transferase and uridine diphosphate glucuronic acid.
155) Where is glucuronidation located?
a. Microsomes of liver and other tissues, near CYP450s
156) What endogenous substances are glucuronidated?
a. Thyroxine, bilirubin, steroids
157) What is involved in sulfation?
a. Sulfotransferases and phophoadenosine phosphosulfate (PAPS
158) Where is sulfation?
a. Cytolsol and other tissues
159) What are the substrates of sulfation?
a. Ones that contain hydroxyl, amines, or N-oxides
160) What endogenous substances are sulfated?
a. Steroids, catecholamine, thyroxine
161) What is SULT 2 specific for? What is SULT 1 specific for?
a. Alcohols. Phenols.
162) What is the deciding factor between glucuronidation and sulfation?
a. Sulfation has a higher affinity but lower capacity than glucuronidation.
163) What is the rate limiting step in sulfation?
164) What does amino acid conjugation do and what does it do it to?
It adds amino acids (mostly glycine, glutamine, and taurine) to substrates that contain a carboxylic acid group. Drug is conjugated to its coenzyme thioester.
165) What are the substrates of acetylation?
a. Primary aliphatic or aromatic amines on amino acids, hydrazines, sulfonamides.
166) Polymorphisms in NAT1 and NAT2 can cause what?
a. Toxicity. Examples: Isoniazid and Dapsone – peripheral neuropathy. Hydralazine and procainamine – lupus. Sulfasalazine – hematologic disorders.
167) What is glutathione?
a. Cytoprotectant tripeptide. Gamma-glutamyl-cysteinyl-glycine.
168) What are the two glutathione transferases?
a. GTSM1 (anticancer drugs) and GSTT1 (small organic molecules, solvents, halocarbons, electrophiles)
169) What are the substrates in glutathione conjugation?
a. Electrophiles (like epoxides)
170) What does glutathione conjugation result in?
a. Mercapturic acid derivative
171) Describe methylation.
a. Common rxn for endogenous compounds. Minor for xenobiotics. It can increase lipophilicty and pharmacological activity. Metabolizes endogenous neurotransmitters, like norepinephrine.
172) Describe O-methylation.
a. Can decrease water solubility. Catechol-o-methyltransferases – catecholamines, steroids. Hydroxyindole-o-methyltransferase – serotonin
173) Describe N-methylation.
a. Phenyethanolamine-N-methyltransferase – norepinesphrine. Histamine-N-methyltransferase – histamine. Amine-N-methyltransferase – serotonin, dopamine, amphetamine
174) Describe S-methylation
a. Microsomal. Disulfram, propylthiouracil, captopril, penicilamine, 6-mercaptopurine.
175) What molecular weight determines if something is enteropathically cycled?
a. 300-500 MW
176) Describe intestinal metabolism
a. CYP450 (esp CYP3A4), glucuronidation, sulfation, glutathione s-transferases. The further into the GI tract, the less metabolism.
177) Describe lung metabolism.
a. CYP2E1, FMO, conjugation reactions
178) Describe nasal metabolism
a. Very active CYP450s, FMO, carboxylesterase, conjugation reactions.
179) What are the principles of toxicology?
a. Spectrum of toxic dose, duration and frequency of exposure, local versus systemic, reversible and irreversible, delayed toxicity, spectrum of undesired effects, chemical allergic reactions, and idiosyncratic reactions.
180) What are the mechanisms of toxicology?
a. High affinity binding to macromolecules, biotransformation to reactive intermediate
181) How many hospitalized and outpatients have ADRs?
a. ~15% of hospitialized patients and an estimated 15% of outpatients have ADRs
182) How many deaths per year are attributed to ADRs?
a. Approximately 106,000 deaths per year.
183) What are the predictable ADRs?
a. Overdose, known side effects, and secondary effects.
184) What are the unpredictable ADRs?
a. Intolerance, idiosyncratic, allergic, and pseudoallergic
185) Of the two, which ADRs are most troublesome?
a. The unpredictable.
186) When do drug hypersensitivity reactions NOT occur?
a. When drug dose is less than 10 mg/day
187) How heavy must compounds be to illict a response? Why do small molecules illict a response?
a. 1000-2000 Daltons. The active metabolite or the drug are binding to biomolecules and iliciting a response through haptenation.
188) What causes a protein to be recognized as foreign?
a. A change in conformation
189) What are the functional groups capable of causing immuno-hypersensitivity reactions?
a. Ring-strained beta-lactams, epoxides, alpha,beta-unsaturated carbonyl and imino compounds capable of undergoing Michael reactions, anilines, and thiols.
190) What reacts with beta lactams?
a. Nucleophiles, such as amino groups on proteins, can form a new acyclic amide bond.
191) What results in epoxides?
a. Aromatic hydroxylation reactions
191) What results in epoxides?
a. Aromatic hydroxylation reactions
192) What can epoxides react with?
a. Cellular nucleophiles such as NHR or SH
193) What is the aromatic anticonvulsive syndrome?
a. Phenytoin, Phenobarbital, and carbamazepine all undergo aromatic hydroxylation via an epoxide. A patient who has a reaction to one has a 40-60% chance of having a reaction to the other two. This is called cross-reactivity.
194) What is an example of an alpha,beta-unsaturated carbonyl or imino group capable of undergoing a Michael reaction?
a. The addition of an electrophilic SH (cysteine) or NH2 (lysine) to uroshiols that causes poison ivy.
195) What is special about anilines?
a. Almost all primary aromatic amines given at dosages of 10mg/day or more will have a significant incidence of immune mediated hypersensitivity reactions, regardless of the rest of the structure.
196) What are some drugs that are known to be mutagens?
a. Epoxides, alkylating agents, alkanesulfonates, nitroso compounds, planar polycyclic aromatic hydrocarbons (via intercalation), analogs of DNA bases, and anilines.

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