Influenza A

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General Information

The theme of SensUs 2021 is acute respiratory viruses. The current Covid-19 pandemic has made it apparent that large virus outbreaks can cause immense harm to human health and can disrupt society as a whole. The most common respiratory virus is influenza A. Therefore, the influenza virus serves as an interesting biomarker for this year’s Competition.

The influenza A virus is known to cause influenza in birds and some mammals, like humans. Different subtypes of the influenza A virus have been isolated from wild birds. Some subtypes of the influenza A virus can cause severe symptoms both in domestic poultry and (rarely) in humans and can even lead to death. Occasionally, viruses can be transmitted from wild birds to domestic animals, like chickens or pigs. This may give rise to human influenza. Influenza A viruses are negative-sense, single-stranded RNA viruses. Different subtypes of influenza A exist, these are characterized by proteins on the outermost membrane of the virus, called hemagglutinin (H or HA) and neuraminidase (N or NA). H and N are the antigens of the virus, and play an important role in the interaction between the host’s immunological response and the virus. Recently, researchers have reported the discovery of an antibody which is generally effective against all types of the influenza A virus. The subtype which will be used in SensUs 2021 is H1N1. Historically, H1N1 has been responsible for most deaths due to influenza. It is a popular influenza strain for research purposes [link, link, link] . Due to its popularity among researchers, antigens and antibodies are commercially available, making H1N1 suitable as a target for the SensUs competition. Influenza A vaccines for humans have been developed. New versions of the vaccines are developed twice per year for use all over the world, which is necessary due to rapid mutations of the influenza virus. Every year during flu season, a large part of the population is vaccinated in order to protect individuals against the virus. However, due to unforeseen mutations of the virus, it might be possible that in a certain year a vaccine will prove ineffective. In that case large portions of the population would be at risk and a pandemic could occur. The probability of a major influenza A pandemic is estimated to be around 0.5-1% each year.

History of influenza (A)

A lack of data up until 1500 AC complicates the research on influenza before that period.[206] Possibly the first influenza pandemic occurred around 6000 BC in China. The symptoms of human influenza seem to have been clearly described by Hippocrates, roughly 2,400 years ago[207][208]. Although the virus seems to have caused epidemics throughout human history, historical data on influenza is difficult to interpret, due to the fact that symptoms of influenza are similar to those found in other respiratory diseases, like RSV (respiratory syncytial virus). The most infamous and lethal outbreak was the 1918 flu pandemic (Spanish flu) (type A influenza, H1N1 subtype), which lasted into 1920. The number of deaths is unknown, but estimates range from 17 to 100 million people. This pandemic has been described as "the greatest medical holocaust in history" and may have killed as many people as the plague (Black Death). This huge death toll was caused by an extremely high infection rate of up to 50% and the severity of the symptoms, suspected to be caused by cytokine storms in which the innate immune system causes an uncontrolled and excessive release of pro-inflammatory signaling molecules called cytokines. One of the most recent outbreaks of influenza was the 2009 Swine Flu. Similar to the Spanish Flu, it was also of the subtype H1N1. The death toll of the 2009 pandemic is estimated to be around 150,000 to 575,000.

Structure of the influenza virus

The virus particle (virion) is 80–120 nanometers in diameter. The virion shape can be spherical, elliptical, or even filamentous with a length of tens of micrometers. The virion is made up of a viral envelope containing two main types of proteins, wrapped around a central core. The two large proteins found on the outside of viral particles are hemagglutinin (HA) and neuraminidase (NA). HA is a protein that mediates binding of the virion to target cells and entry of the viral genome into the target cell, and therefore plays an important role in infecting healthy cells. NA is involved in releasing the progeny viruses once a cell has been infected and has started producing the virus itself. These two proteins are a target of interest for antiviral drugs. Furthermore, they are also the antigen proteins to which a host antibodies can bind and trigger an immune response. Influenza type A viruses are categorized into different subtypes, or strains, based on which type of these two proteins is present on the surface of the virion. Currently, there are 16 subtypes of HA and 9 subtypes of NA known to exist. The most prevalent form of the different subtypes is H1N1. Single hemagglutinin-neuraminidase proteins, in which both HA and NA are found in a single protein, also exist. However, these will not be used in SensUs 2021.

Valproate Mode of Action

The exact mode of action of valproate on molecular level is unknown, as there are many different substances that participate simultaneously in the regulation of neuronal activity. [1] VPA is connected to cortical inhibition in order to contribute to ‘neural synchrony’ and is known to provide protection from neural degradation and damage. [2] VPA inhibits histone deacetylase, which causes hyperacetylation of histones. The drug is furthermore connected to inositol depletion by preventing the gene prolyl oligopeptidase to be expressed through indirect inhibition of myo-inositoal-1-phophate-synthetase. [2] In terms of epilepsy, it is believed that VPA is affecting the activity of GABA. By increasing the activity of GABA, the epileptic attacks are prevented. This is established through inhibition of succinic semialdehyde dehydrogenase which then increases the amount of succinic semialdehyde and subsequently increases GABA neurotransmission. [3]

VPA affects the extracellular signal-regulated kinase pathway or ERK (Extracellular Receptor Kinase), which results in phosphorylation of ERK 1/2 . This has an effect on the expression of proteins that contribute to the plasticity of neurons and neuronal growth. The downside of this effect of VPA is the additional increase in GABA activity. VPA has an influence on fatty acids production, leading to lower membrane fluidity due to the presence of less sterols and glycerolipids. Subsequently, this increases the action potential threshold of the membrane and contributes to the antiepileptic effects of valproate . [4]

Pharmacokinetics of VPA

When introduced orally, VPA is found to be absorbed in a period of 4 hours. However, introducing it in other ways results in the same Cmax, AUC and Cmin in a steady state, while all the differences in absorption are neglected. When VPA is taken as extended release tablet and combined with meals, the absorption time increases from 4 to 8 hours. On the other hand, the increase in absorption time in the same situation increases from 3.3 to 4.8 hours when taken as a sprinkle tablet. There is 90% bioavailability of VPA in all oral forms. On the other hand, there is 100% bioavailability in enteric-coated forms. [5]The average half-life of VPA is 13-19 hours, the volume of distribution is 11 L/1.73 m2 . The protein binding is linear at low concentrations, but non-linear and decreased at high concentrations, which may be due to the different affinity binding sites of albumin. Parts of the drug can be metabolized in different ways. The most relevant metabolism of VPA would be glucuronide conjugates and mitochodrial-ß oxidation. [4]

Only 3% of the drug is eliminated through urine, about 30-50% is eliminated through hepatic metabolism, and about 40% being excreted through mitochondrial-beta oxidation. [5]

Efficacy and influence on hepatic function

Optimal dose and effects are achieved with upper limit of 60 mg/kg/day. If the desired effects are not achieved (in other words, the seizures are not gone or the side effects are too strong), testing for blood levels of VPA needs to be executed in order to determine whether they fall within the optimal range of total VPA 50-100 µg/mL. Otherwise, the dose is altered in agreement with the doctor according to the individual state of the patient, his/her other conditions or other medication that they take. The toxicity level of valproate in blood, although not very conclusive, is taken as 150 µg/mL[6] .

VPA impacts the hepatic drug metabolism by inhibiting it and displacing other strongly bound drugs from proteins, which implies that dosage changes need to be considered when using it in combination with other drugs. [7] The normal limits of valproate in blood of 70-100 kg person is 1000-3000 mg/day [8]

Number of patients

Epilepsy affects around 50 million people worldwide. This includes people having less than one seizure per year. The prevalence of active epilepsy (i.e. patients who have frequent seizures or use medication) is between 0.4% and 1%. On a global scale, an estimated five million people are diagnosed with epilepsy each year. Epileptic seizures can be controlled by using anti-epileptic drugs (AEDs). About 70% of epileptic patients becomes free of seizures by appropriate AEDs. Low-cost treatments are available, with daily medication that costs as little as US$ 5 per year.[9]

Medical use and TDM

Valproate (VPA) is usually taken as tablets of 250 mg or as a syrup with 250 mg per ml, but this may vary per product [10].The dose of VPA is at first taken in small amounts and is then gradually increased until the satisfactory dose is reached. Testing for valproate levels is important to check if the blood levels are within normal therapeutic range [7] . The test should measure the free concentration (i.e. the concentration of unbound valproate) as this fraction is pharmacologically active. The recommended levels of unbound valproate are 6-22 µg/mL in blood [7]. For epilepsy patients, the range for the treatment of total valproate should be 50-100 µg/mL. Overall, if a patient has a VPA concentration that falls within this range, does not have recurrent seizures and has minimal side effects, then the dose is said to be suitable[7] . To provide a patient their right amount of medication, therapeutic drug monitoring (TDM) is implemented in hospitals, because every patient responds differently to a certain dose of medication. VPA is a conventional drug that is used as first line monotherapy for idiopathic generalized epilepsies. Its effectiveness is not clearly conclusive and varies between different patients. The doses are not generalized and depend on patient’s age and weight. Doses are taken every day, with some patients even two times a day. It is not advised, but sometimes necessary, to take VPA in combination with other seizure drugs (e.g., ethosuximide, lamotrigine, phenytoin, rufinamide, topira mate), some antidepressants or certain antibiotics[7]. Medications based on VPA are harmful for the unborn child. If valproate is taken during pregnancy, research has shown that up to 4 in 10 babies are at risk of developmental disorders, and approximately 1 in 10 are at risk of birth defects. [11]

Safety & Lab protocols

Safety

All anti-epileptic drugs have side-effects, including VPA. The risk of hyperammonemia is nearly 40% in patients ingesting intravenous VPA in the ICU setting [12] . Other adverse effects include thrombocytopenia and endocrine effects of women. Valproate is associated with a dose-related teratogenicity rate, with risk of major malformation higher than 30% at doses greater than 1100 mg/d. In utero exposure is also linked to dose-dependent reduced verbal IQ and autism[13] . Children, who were exposed to VPA during birth, have a possible chance of a major congenital malformation (MCM) [14] . The risk of having severe consequences for these infants exposed to sodium valproate in utero has been estimated between 6% and 12%[15] . This can be prevented by reducing the dose of the drug. An example of endocrine effects might be idiosyncratic liver toxicity [16]

Lab Protocols

VPA is considered thermodynamically stable, which indicates that it is not reactive under normal environmental conditions. It should be stored in a metal can or drum and kept away from incompatible materials such as oxidizing agents, bases and strong reducing agents as ignition may result.

Working with VPA can be irritating when inhaled or when getting directly in contact with the yes. To prevent this, VPA needs to be handled in a fume hood, safety goggles, lab coats, and gloves need to worn. Direct skin contact or ingestion of VPA should be avoided. In case of contact, flush the specific body part and go to the doctor without delay [17]

State of the art

Company Product Test name Sample Volume (μL) Reportable range Dilution Precision Incubation time Measuring Technique
Beckman Coulter[18] [19] AU2700/AU5400 VALPROIC ACID EMIT® 2000 3.5 μL < 150 μg/mL 1:1 Total CV < 4.3%
Inter-assay: CV < 3.2% 		15-75 min. ELISA1
Roche COBAS[20] [21] Cobas 8000 Cobas 8000 1.5–35 3.15 – 150 mg/L [22] 1:(3-121) Inter-assay: CV < 0.7% ~ 2.9%
Total CV<3% 		9 min – 27 min ELISA1
ABBOTT LABORATORIES DIAGNOSTIC DIVISION[23]

[24]

B1P350 ARCHITECT iValproic Acid 10 2 - 150 μg/mL 1:10 Total CV≤ 7% 29 min CMIA2
SIEMENS HEALTHCARE DIAGNOSTICS INC.[25] 67070 ADVIA 1200 CHEMISTRYSYSTEM -VALPROIC ACID (VPA) ASSAY 2-3 NA 1:5 Inter-assay: CV = 0.2% - 5.3 %
Total CV= 0.4%-5.3% [26] 		3-21 min ELISA1
MICROGENICS CORPORATION[27] 62390 CEDIA TDM ASSAY -VALPROIC ACID NA 3.0-150 μg/mL NA Inter-assay: CV = 1.3% - 2.4 %
Total CV= 1.8%-3.4% 		Reagent 1 : 2-5 min
Reagent 2: 4-8 min 		ELISA1


1 ELISA: Enzyme Linked Immunosorbent Assay
Note: 2.CMIA: Chemiluminescent Microparticle Immunoassay

To measure unbound VPA, blood samples are treated by ultrafiltration, followed by an immunoassay, also referred to as ELISA. By performing the ultrafiltration, the protein-bound form of VPA is separated from its unbound form. After that, the level of the unbound fraction can be measured by an immunoassay, e.g. an ELISA kit. Alternatively, LC-MS can be used to measure VPA.[28] Steps such as solvent extraction or derivation must be executed prior to a HPLC assay, which takes significant time[24] . CMIA is a special type of ELISA [29] . Several innovations are being investigated for VPA testing. For example, 2D-LC system (two-dimensional chromatography) was studied, allowing large volume injection, reducing interfering components, and reducing the analysis time and preventing most interference components by selecting useful sections in the “heart-cut” column(1D) from entering the analysis column (2D).[30] . Another example is dried blood spot (DBS) followed by gas chromatography mass spectrometry (GC–MS), which does not require solvent extraction or elution. The limit of quantitation was 200 ng/mL. [31]

References

  1. ↑ Sodium valproate (Epilim, Epival, Episenta). (2018, June 5) , at “https://www.netdoctor.co.uk/medicines/brain-nervous-system/a6665/epilim-sodium-valproate/”.
  2. ↑ 2.0 2.1 Williams, R. S. B., Cheng, L., Mudge, A. W., & Harwood, A. J. (2002). A common mechanism of action for three mood-stabilizing drugs , at “https://www.nature.com/articles/417292a”.
  3. ↑ Flyyn, S., & Babi, M. A. (2017). Pathophysiology of Epilepsy , at “https://www.sciencedirect.com/topics/medicine-and-dentistry/pathophysiology-of-epilepsy”.
  4. ↑ 4.0 4.1 , . Drugbank, Valproic Acid Retrieved from November 25, 2019, at [https://www.drugbank.ca/drugs/DB00313 “https://www.drugbank.ca/drugs/DB00313”].
  5. ↑ 5.0 5.1 Gugler, R., & Unruh, G. E. von. (2012, December 13). Clinical Pharmacokinetics of Valproic Acid , at “https://link.springer.com/article/10.2165/00003088-198005010-00002”.
  6. ↑ valproic Acid Dosage Guide with Precautions. (2019, March 28), at “https://www.drugs.com/dosage/valproic-acid.html”.
  7. ↑ 7.0 7.1 7.2 7.3 7.4 Valproic Acid (2018, October 1) , at [1].
  8. ↑ Farmacotherapeutisch Kompas. Retrieved December 19, 2019, at [2].
  9. ↑ WHO. (2019, June 20). Epilepsy. Retrieved October 30, 2019 , at “https://www.who.int/news-room/fact-sheets/detail/epilepsy”.
  10. ↑ Valproic Acid Capsules - FDA prescribing information, side effects and uses. (2018, June 1).Retrieved November 26, 2019, at [3].
  11. ↑ gov.uk. (2018, March 23), at [4].
  12. ↑ Lind, J., & Nordlund, P. (2019, July). Intravenous use of valproic acid in status epilepticus is associated with high risk of hyperammonemia, Seizure Vol. 69. Retrieved October 23, 2019, at [5].
  13. ↑ Abou-Khalil, & Bassel, W. (2019). Update on Antiepileptic Drugs 2019. Retrieved November 6, 2019, at [ https://insights.ovid.com/crossref?an=00132979-201904000-00014“ https://insights.ovid.com/crossref?an=00132979-201904000-00014”].
  14. ↑ Morrow , J., Russell, A., Guthrie, E., Parsons, L., Robertson, I., Waddell, R., … Craig, J. (2006, January 17). Malformation risks of antiepileptic drugs in pregnancy: a prospective study from the UK Epilepsy and Pregnancy Register. Retrieved October 23, 2019, at [6].
  15. ↑ Use of Sodium Valproate in Pregnancy. (2014, December 15). Retrieved October 23, 2019, at [7].
  16. ↑ Stewart, J. D., Horvath, R., Baruffini, E., Ferrero, I., Bulst, S., Watkins, P. B., … Chinnery, P. F. (2010, November). POLG determines the risk of sodium valproate induced liver toxicity. Retrieved October 23, 2019, at [8].
  17. ↑ Chemwatch: 15242 Version No: 7.1.1.1 Safety Data Sheet (Conforms to Regulation (EU) No 2015/830)
  18. ↑ Emit 2000 Valproic Acid Assay (2010, September) Retrieved from November 7, 2019. Beckman.
  19. ↑ Beckman Coulter system Reagent, AU400/AU400e (2012, February) Retrieved from November 7, 2019. Beckman.
  20. ↑ Cobas 8000 modular analyzer series (last update: 2019, November 8).
  21. ↑ van Eckardstein, A et al. (2013). cobas 8000 Modular Analyzer Series Evaluated under Routine-like Conditions at 14 Sites in Australia, Europe, and the United States. Retrieved from October 23., at [https://pdfs.semanticscholar.org/beb9/eaea6d45ba8fec2f3049cc5e242d9d2cfb13.pdf “https://pdfs.semanticscholar.org/beb9/eaea6d45ba8fec2f3049cc5e242d9d2cfb13.pdf ”].
  22. ↑ Therapeutic drug monitoring (2011). Retrieved from November 8, 2019. R-Biopharm AG.
  23. ↑ iValproic Acid B1P350 (2009,August).
  24. ↑ 24.0 24.1 Free Valproic Acid Assay (2014, April). Retrieved from November 3, 2019, at [9].
  25. ↑ ADVIA 1900 Chemistry system
  26. ↑ American Association for Clinical Chemistry, 70th AACC Annual Scientific Meeting (July, August, 2018). Retrieved from November 7, 2019
  27. ↑ CEDIA Valproic Acid II Assay (2018, November). Retrieved from November 7, 2019
  28. ↑ ao, S., Miao, H., Tao, X., Jiang, B., Xiao, Y., Cai, F., … Chen, W. (2011, July 1). LC–MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma, Journal of Chromatography B Volume 879, Retrieved November 28, 2019, at [10].
  29. ↑ Ilyas M., Ahmad. I (2014, July 12), Chemiluminescent microparticle immunoassay based detection and prevalence of HCV infection in district Peshawar Pakistan, third alinea Background, Retrieved from November 7, 2019, at [11].
  30. ↑ Liu, W., Shang, X., Yao, S., & Wang, F. (2019, August 20). A novel and nonderivatization method for the determination of valproic acid in human serum by two‐dimensional liquid chromatography. Retrieved October 17, 2019, at [12].
  31. ↑ Guo, M., Shao, L., Chen, X., Li, H., Wang, L., Pan, Y., & Tang, D. (2019, September 13). Assay of dried blood spot from finger prick for sodium valproate via ink auxiliary headspace gas chromatography mass spectrometry, Journal of Chromatography A Vol. 1601 p. 335-339. Retrieved October 24, 2019, at [ https://doi.org/10.1016/j.chroma.2019.05.039“ https://doi.org/10.1016/j.chroma.2019.05.039”].
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