Question 1
- Explain what is meant by the terms pharmacogenomics and pharmacometabonomics
Pharmacogenomics is defined as the science concerned with the study of roles of genetic differences in individual responses to the same treatment. It explores how genes correlate with drug metabolism with respect to pharmacokinetics, such as the metabolic phenotype of drugs (Everett, 2016). Pharmacogenomics is also concerned with the development of drug therapies to minimize the differences in response to the drug caused by genetic differences. The concept may also be perceived as the study of effects of differences in genetic composition on the response to drugs.
Pharmacometabonomics is perceived as the use of pre-intervention mathematical models that utilize metabolite signatures to predict outcomes such as toxicity and efficacy of xenophobic intervention or drugs (Everett, 2016). Pharmacometonomics works in a similar manner and complements the conventional pharmacogenomics except that it gives a favorable sensitivity to both genomic and environmental factors.
- Describe how pharmacogenomics and pharmacometabonomics can help deliver personalized medicine. In particular, describe with examples the following:
- How genetic differences between people can affect the interaction between a drug and the patient
The two concepts are based on the idea that the response of an organism to drugs can be determined by its genetic makeup. Based on the concepts, scientists are now able to offer personalized medicine. In particular, the fate or disposition of drugs, as well as their toxicological and therapeutic effects on patients, is determined by a protein-dependent complex process. Depending on the codification of proteins by genes, organisms exhibit different abilities to support metabolism and transport the drug in the body. The drug may also show different action mechanisms due to unique genetic composition allowing physicians to vary treatment from person to person.
Genetic makeup also determines the ability of individuals to metabolize drugs. While some individuals can process the drugs rapidly, others take up the drugs slowly, which may lead to accumulation and toxicity (Everett, 2016). The ability to metabolize drugs in the anticipated speed maintains just enough levels for effective recovery. One enzyme involved in the metabolism of drugs is the N-acetyltransferase found in the liver. A study established that the enzyme works slowly in half all the population in the U.S. Consequently, drugs such as isoniazid tend to accumulate to higher levels in the blood and stay longer in the system of individuals with slow N-acetyltransferase (slow acetylators) compared to fast acetylators (Lynch, 2019). Similarly, individuals with lower pseudocholinesterase levels may experience prolonged muscle relaxation, which may affect treatment outcomes. Pseudocholinesterase is a blood enzyme involved in the inactivation of certain drugs, including succinylcholine administered to a patient, to help relax muscles at the time of surgical operation. As a result, physicians avoid recommending the same drugs for every patient suffering from the same condition.
Pharmacometabonomics can help physicians to decide what therapies to avoid recommending to a population or a group of patients. For instance, studies indicate that close to 10% of African American men and women have glucose-6-phosphate dehydrogenase (G6PD) enzyme deficiency (Lynch, 2019). The enzyme plays a vital role in the protection of red blood cells against several toxic chemicals. Since some drugs such as primaquine and chloroquine used for malaria treatment may result in toxicity in patients with G6PD deficiency, physicians would avoid recommending such treatment for African Americans without appropriate screening (Lynch, 2019).
Genes are believed to contain significant nucleotide sequence variation due to the influence of evolution. When the variation is located in the codifying region, the substitution of a protein with an amino acid may occur, leading to alteration of the function of the protein (Everett et al., 2013). Similarly, the occurrence of variation in the regulatory region of the gene may lead to modulation of the expression level of gene product through a translational and transcriptional mechanism.
A DNA sequence variation of more than 1% allelic frequency in the study population, polymorphism, and lower frequency, mutation, results in the development of enzymes with varying metabolic activity. The enzymes may also have different drug affinity (Mini & Nobili, 2009). Consequently, the pharmacological response to drugs by individuals is modified.
- how environmental factors may affect a patient’s response to a drug
Environmental factors affect patient response to various drugs. Although personalized medicine has conventionally relied on the clinical judgment in line with the medical history of the patient and the assessment of the genome of the patient. However, environmental factors have also been found to affect the efficacy and toxicity of the drug (Everett et al., 2013). Some human genomics tends to be essentially blind to some environmental factors. As such, the exclusive use of pharmacogenomics to deliver precise medication cannot yield optimal outcomes.
Environmental factors include the use of alcohol, diet, microbiome state, and exposure to other medications. The effect of environmental factors on individual response can be understood by exploring how drugs work in the body. There are several processes that drugs undergo from the time treatment is initiated to the final outcomes. These processes are summarized in the acronym ADME which stands for absorption, distribution, metabolism, and excretion. Drugs must be absorbed and distributed and metabolized to bring effect in the body and must be excreted when no longer required. At each of the stages, certain environmental factors may affect the efficacy of the drug. During absorption at the gastrointestinal tract, factors such as high-fat diet, pH, and age of the patient, cardiovascular conditions, gastric mortality, and gastric emptying rate may slow down the rate of absorption of the drug into the body system. This is the reason why some drugs are administered intravenously. At the distribution stage, the efficiency of drugs may be affected by such factors as perfusion or blood floor rate, the permeability of tissue or vascular membrane, and the nature of tissue mass. Similarly, the efficacy of drugs may be impacted at the metabolism rate where inactive drugs are converted to an active metabolite or vice versa in other cases. At this stage, environmental factors such as smoking, pregnancy, and stress may compromise the effectiveness of drugs (Everette et al., 2013). During the excretion stage, some drugs are eliminated from the body in such ways as sweating. When environmental temperatures are low, less sweating occurs, leading to prolonged duration of the potentially toxic drug in the body.
Question 2
- Answer Both parts
- Describe 2 different strategies for targeting the Epidermal Growth Factor signaling pathway in cancer. Include named examples of drugs developed from these strategies in your answer.
Two strategies with varying action mechanisms that have been used to target EGFR in treating human malignancies are the use of inhibitors such as the small molecule tyrosine kinase and monoclonal antibodies. Epidermal growth factor receptors comprise a large group of tyrosine kinases receptors found in various types of cancers such as neck, head, esophageal, lung, and breast cancers. The receptors are involved in a complex cascade that affects differentiation, signaling, migration, adhesion, and growth of the cell. Studies have indicated that the family of EGFR plays multiple roles in cancer progression makes them a target of anticancer treatment (Seshacharyulu et al., 2012).
The use of Anti-EGFR monoclonal antibodies
Monoclonal antibodies targeting EGFR are focused on the EGFR region outside the cell known for the formation of competitive ligand inhibition with the effect of interfering with rd signaling and auto-phosphorylation. The monoclonal antibodies also cause ubiquitination, prolonged downregulation, degradation, and receptor internalization (Seshacharyulu, et al., 2012). Another mechanism of the action of monoclonal antibodies include the production of cytotoxicity mediated by cell and dependent on an antibody that in turn lead to the initiation of endocytosis and a minimum contribution to mediated cytotoxicity
An example of an antibody produced based on the strategy is the Cetuximab (Erbitux and Vectibix). The antibody has been approved for use as anti-EGFR action. Cetuximab acts by binding the to the second EGFR domain (L2), prohibiting its downstream signaling by triggering encumbering the interaction between receptor and ligand as well as causing receptor internalization. Cetuximab treatment has been shown to be effective in about 60% of patients with KRAS wild-type growth. KRAS was therefore found effective biomarker in predicting the response of cancer in patients positive to the EGFR (Seshacharyulu, et al., 2012). The antibody has been adapted as a monotherapy in patients who have shown no response to therapies based on oxapliplatin and irinotecan. Additionally, the use of Cetuximab is associated with a regression rate of 10.8% and 1.5 month delay in tumor growth. Another antibody for cancer therapy is the panitumumab which is designed to address EGFR associated with colorectal cancer metastatic. The human monoclonal antibody meant specifically for EGFR is usually created by immunization of mice with transgenic capability with the ability to produce both heavy and light chains of a human immunoglobulin.
Targeting tyrosine kinase inhibitors
The second strategy of targeting the epidermal growth factor signaling is targeting tyrosine kinase inhibitors. These are small irreversible or reversible molecules that exist as ATP or Adenosine triphosphate analogues. The molecules restrict the signaling of EGFR by competing for the active sites or binding pockets of ATP and binding with them on the catalytic kinase domain of RTKs or receptor tyrosine kinases. This process prohibits the activation and autophosphorylation of multiple pathways of downstream signaling. The various types of inhibitors have an affinity to varying types of active sites. For instance, Type II and I reversible inhibitors are in competition molecules of ATP that have the ability to recognize active conformation of kinase. Irreversible inhibitors, on the other hand, bind covalently to with the kinase active site through specific reaction with the residue of nucleophilic cysteine. Another advantage of the irreversible inhibitor is its ability to produce prolonged medical effects reducing the need for dosing the patient frequently. Although the strategy has a high efficacy level, it is associated with side effects such as cutaneous skin toxicities that may include xerosis, paronychia, trichomegaly, and acneiform eruptions.
Examples of drugs developed through this technique include gefitinib and erlotinib. Gefitinib is an inhibitor to the EGFR tyrosine kinase derived from anilinoquinazoline. The inhibitor that was originally characterized in 1996 is both low-molecular and orally active. Interestingly, the inhibitor acts on tyrosine kinase selectively but has no inhibitory action against the activity of the serine-threonine kinase. Erlotinib is also approved by the FDA and is very similar to the gefitinib in many aspects, including being low molecular weight. The drug is also orally active and has the ability to cause reversible inhibition to EGFR tyrosine kinase. Just like gefitinib, erlotinib works by acting like an anologue of ATP molecule, allowing it to compete and bind onto RTKs via the ATP binding pockets. The drug also produces anti-proliferative effects while causing apoptosis and cell-cycle arrest. Research has indicated that the drug delays cell proliferation dependent on EGF at a concentration of nanomolar range. It also prevents the progression of cell-cycle in phase G1.
- Briefly explain why screening for RAS mutations is recommended before prescribing Erbitux.
Recently the guidelines for therapies using anti-EGFR mAbs, including Cetuximab and panitumumab have been extended to include screening for RAS mutation prior to the treatment.
The reason for such screening is that some genes in the RAS type GTPase family have been shown to adversely affect response to anti-EGFR therapy. KRAS, HRAS and NRAS are the main downstream mediators in signaling of activated EGFR. Approximately 40% and 5% of colorectal cancer cases have established KRAS and NRAS as the respective main oncogenes initiating mutations. The oncogenic RAS molecules of proteins initiate processes that accelerate metastasis, progression of the tumor while acting independently of receptor kinases on the cell surface, including the EGFR (Sakthia, et al., 2018). NRAS and KRAS gene mutations have been pointed as the cause of the insensitivity of tumor to anti-EGFR treatment just as predicted by the biological mechanism.
- cis-Platin is an example of an electrophilic cross-linking anti-cancerr agent. With the aid of suitable diagrams, describe the mode of action of cis- Platin. Also, explain why trans-platin is inactive.
Cis-Platin works by binding to DNA, causing interference to the repair mechanism, which then leads to death of the cell. Once the cis-Platin molecule enters the cell through the cell membrane, a water molecule replaces a single chloride ion leaving the other on the original molecule. This substitution enables the remaining structure to bind onto a nitrogen ion on a nucleotide of the DNA. The next step is the replacement of the remaining chloride of cis-platin by another water molecule, allowing the platinum to bind to a second nucleotide. Studies indicate that the binding of cisplatin to DNA has a preference of the Nitrogen 7 located on two guanines located adjacent to one another on a single strand. Adenine is also preferred for binding, although to a lesser degree (Dasari & Tchoumnwou, 2014). The resulting complex of the DNA and cis-Platin has a high affinity for the High Mobility group (HMG-1). The molecules then bind with proteins involved in DNA repair in an irreversible process. This process destroys the shape of DNA, thus preventing effective repair. The inability for effective repair is due to lack of antineoplastic activity that results from the inability of the cis-Platin Trans isomer to create 1, 2 intrastrand links (Dasari & Tchounwou, 2014). Etoposide and other antineoplastic agents combine with the platinum-DNA-Protein resulting in the synergistic reinforcement of the cis-Platin activity as shown the figure below
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146684/
Figure 1: Molecular cisplatin mechanism in anticancer action
Why trans-Platin is inactive
The inactivity of trans-platin results from two maactors. First, the molecule is kinetically unstable, leading to its deactivation. Secondly, trans-platin is inactive against cancer due to the formation of DNA adducts with different stereochemistry and regioselectivity (Coluccia, 2007). High kinetic reactivity causes the trans-plating to be involved in unnecessary reactions making it unavailable for pharmacological activity. Also, the 1, 2-intrastrand formed as a result of the cross-linking of the adjacent purines or the main cytotoxic DNA lesion is not accessible to Trans isomers with respect to stereochemistry.
Question 3
- The main pathophysiological mechanisms associated with ischemic stroke are inflammation and oxidative stress. The brain is particularly vulnerable to free radicals and oxidizing species due to lack of adequate antioxidant defenses. Ischemic stroke is caused by sudden and excessive loss of blood circulating in a section of the brain. The blood loss leads to a proportionate neurological malfunction. Neurological functions refer to a group of etiologies with heterogeneous properties and may include relative hypoperfusion, embolism, and thrombosis. Ischemic stroke is mostly caused by heart embolism, intracranial, or cervical artery atherothrombosis. Shortly after the blood flow interruption in the affected section of the brain, a series of biochemical events referred to as ischemic cascade is initiated rapidly. This process ultimately leads to cell membrane disintegration and, ultimately neuronal death at the infarction center. Severe focal hypoperfusion marks the beginning of an ischemic stroke. Initially, hypoperfusion causes oxidative damage and excitotocity which then leads to dysfunction of the blood-brain barrier, and microvascular injury, triggering post-ischemic inflammation. These biochemical events aggravate the initial condition leading to possible permanent cerebral injury whose severity depends on the brain recovery capability, degree, and time of the ischemia.
The figure below demonstrates how cerebral damage occurs from the ischemic cascade. The occurrence of ischemic stroke results in hypoperfusion of a section of the brain, which in turn, triggers a series of biochemical events that include oxidative stress, excitotocity, dysfunction of the blood-brain barrier, microvascular injury and post-ischemic inflammation. Ultimately, the events leading to death of neuron, endothelial, and glia cells. The severity of cerebral damage is dependent on the duration and extent of ischemia.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2780998/
Figure 2: Biomechanical events that cause cerebral damage
- A large number of clinical studies suggest that the pathophysiology of depression is associated with a dysfunction of the glutamatergic system. Discuss this statement in reference to a shift away from the monoamine theory of depression, and how pharmacological therapy of this psychiatric disorder may be changing.
Most therapies for depression have relied on the monoamine hypothesis in the development of compounds with rational design. This approach has exhibited low rates of remission among patients. Relapses, symptoms of residual subsyndromal, and general functional impairment have also been common among patients who have received treatment based on monoamine hypothesis. Recent evidence increasingly points to the importance of the glutamatergic system in the neurobiology and major depressive disorder (MMD) treatment.
Although high glutamate concentration in the brain was discovered as early as 1930, the amino acid was widely assumed to be nonspecific due to its role as a neurotransmitter. An experiment conducted in 1979 using mammalian vertebrates and utilization of various antagonist and agonist glutamate receptor identifiers in the brain changed scientists’ perception of the amino acid. Glutamate was found to have a higher concentration in both neurons and monoamines (Mathews et al., 2012). As such, glutamate is now considered as one of the most important excitatory synaptic neurotransmitters. More studies have shown that glutamate also plays a vital role in memory, learning, and neuroplasticity regulation.
The involvement of glutamate in the pathophysiology of depression has been demonstrated indirectly through postmortem and imaging studies. Fluids such as serum, brain tissue, plasma and cerebrospinal fluid (CSF) have revealed a change in levels of glutamate among suicide victims and psychic and mood disorder patients (Mathews et al., 2012). Depression has been associated with increased levels of glutamate in the serum. Even then, studying the contribution of glutamate level changes in CSF, serum, and plasma has posed a major challenge given that the condition is also affected by exposure to medication, and postmortem metabolites. Establishing the exact glutamate source poses another challenge of such study.
The total glutamatergic reservoir available for metabolic or synaptic activity in glutamine form is indicated by Glx. This relationship explains why most depression studies have focused a lot of effort in the measurement of GABA and Glx in various sections of the brain (Mathews et al., 2012). In a recent study, the levels of Glx were found to be significantly lower in the dorsoanterolateral PFC or prefrontal cortex of individuals with depression. The findings were consistent with histopathological studies of postmortem held earlier. Occipital cortexes of a patient with a mood disorder have also exhibited higher levels of glutamate, while the anterior cingulate cortex (ACC) in bipolar disorder patients showed lower levels. Another research found that Glx levels in left dorsolateral PFC, amygdala, ventromedial PFC, hippocampus, and ACC of MDD patients were lower (Lakhan et al., 2009). Glx levels were, however, higher left dorsolateral PFC, dorsomedial PFC, hippocampus, amygdala, and ventromedial PFC of depression patients.
Changing pharmacological therapy
Pharmacological treatment for a patient with depression is often not adequate in most patients, creating the need for therapies with greater tolerance, efficiency, and rapid action in comparison with the existing treatment.
As a result, more techniques such as the proton magnetic resonance spectroscopy (1H-MRS) have enabled non-invasive in-vivo methods of imaging the brain, thereby enhancing the effectiveness of the psychotropic drug action mechanism study. Thanks to the new technique, 1H-MRS resonance for various metabolites can now be accurately established even with a concentration in the range of millimolar including glutamate, Myo-inositol, creatine + phosphocreatine (Cr) and chlorine-based compounds (Cho) (Lakhan et al., 2009)
In brief, there is a lot of evidence pointing to the contribution of the glutamatergic system as the main mediator in psychiatric pathology. As scientists become more knowledgeable about the system and its role, an alternative novel treatment for depression, patients are continually being developed. Researchers hope to improve cellular resistance and neural plasticity in mental illness patients by applying information about the glutamatergic system
The action of glutamate targets on presynaptic and postsynaptic neurons as well as glia which make up the tripartite glutamate synapse whose role is to facilitate uptake, inactivation and release of glutamate through metabotropic and ionotropic glutamate receptors.
Reference
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Dasari, S., & Tchounwou, P. B. (2014). Cisplatin in cancer therapy: molecular mechanisms of action. European journal of pharmacology, 740, 364-378.
Everett J. R. (2016). From Metabonomics to Pharmacometabonomics: The Role of Metabolic Profiling in Personalized Medicine. Frontiers in pharmacology, 7, 297. https://doi.org/10.3389/fphar.2016.00297
Everett, J. R., Loo, R. L., & Pullen, F. S. (2013). Pharmacometabonomics and personalized medicine. Annals of clinical biochemistry, 50(6), 523-545.
Lakhan, S. E., Kirchgessner, A., & Hofer, M. (2009). Inflammatory mechanisms in ischemic stroke: therapeutic approaches. Journal of translational medicine, 7(1), 97.
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